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Lactobacillus plantarum A3 attenuates ulcerative colitis by modulating gut microbiota and metabolism

Lactobacillus plantarum A3 attenuates ulcerative colitis by modulating gut microbiota and metabolism Antibiotics are widely used to treat various diseases. However, growing evidence indicates that antibiotic therapy in human life increases the incidence of inflammatory bowel disease (IBD). Therefore, we need appropriate methods to reduce the incidence or symptoms of IBD. In this study, we used lincomycin hydrochloride to construct a gut micro- bial dysbiosis model in mice, and then, constructed an ulcerative colitis (UC) model. Meanwhile, we used Lactobacillus plantarum A3 from equine to treat UC in mice with gut microbial dysbiosis. The results showed that lincomycin hydro- chloride had little effect on the small gut microbiota in mice, but had a more destructive effect on the large intestin. Lactobacillus plantarum A3 alleviated the symptoms of UC in mice, which was reflected in its significantly reduced spleen index and disease activity index (DAI) (p < 0.05), inhibited the shortening of colon and alleviated the invasion of inflammatory cells in the colon. Moreover, we found that it played a mitigatory role by inhibiting oxidative stress and regulating inflammatory cytokines in mice. At the same time, it restored the diversity and composition of the colonic microbiota and significantly increased the abundance of beneficial bacteria such as Blautia and Akkermansia (p < 0.05); Notably, it significantly increased the concentrations of arachidonoyl ethanolamide phosphate (AEA-P) and cortisone (p < 0.05) which have analgesic and anti-inflammatory effects. In conclusion, our study found that Lactobacillus plan- tarum A3 has the potential to regulate UC in mice with gut microbial dysbiosis. Keywords Antibiotics, Gut microbiota, Lactobacillus plantarum, DSS, Ulcerative colitis, Untargeted metabolomics Introduction Inflammatory bowel disease (IBD) is a nonspecific chronic intestinal inflammatory disease, that includes ulcerative colitis (UC) and Crohn’s disease (CD). UC mainly occurs in the colon and rectum, and spreads to the cecum as the *Correspondence: Yaoqin Shen disease progresses, while CD can occur anywhere in the yshen@mail.hzau.edu.cn intestine, generally from superficial ulcers to deep spread College of Veterinary Medicine, Huazhong Agricultural University, (Imhann et  al. 2018). On the one hand, IBD is difficult to Wuhan 430070, People’s Republic of China Faculty of Veterinary and Animal Sciences, The Islamia University cure, prone to recurrent attacks and has a risk of carcino- of Bahawalpur, Bahawalpur 63100, Pakistan genesis at a later stage; on the other hand, IBD has grad- Key Laboratory of Environment Correlative Dietology, Interdisciplinary ually become a disease with an increasing incidence in Sciences Institute, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, China modern society. Not only are there more cases in devel- State Key Laboratory of Agricultural Microbiology, Huazhong oped countries such as Europe and the United States, Agricultural University, Wuhan, China but the incidence is also increasing in regions with faster © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Qin et al. Animal Diseases (2023) 3:16 Page 2 of 18 economic development such as Asia, Africa and South jejunum, ileum, cecum, colon and rectum. Compared with America (Glassner et  al. 2020). Therefore, IBD has gained the C_LCM group, the number of OTUs in the duode- increasing attention. Unfortunately, the real cause of IBD num, jejunum and ileum did not change much, and even has not been explored. the number of OTUs in the duodenum increased (Fig. S1 Recently, it has been demonstrated that antibiot- A-C). However, the rectum decreased significantly after ics may be one of the causes of IBD. A study involving exposure to lincomycin hydrochloride in the LCM group 107,2426 research projects and nearly 660,000 follow-up (p < 0.01) (Fig.  1A-C). Regarding the alpha diversity, there results revealed that the incidence of IBD in children who was no significant change in Chao 1, Shannon and Simp - were  unexposed and exposed to antibiotic treatment was son indexes of duodenum, jejunum and ileum in the LCM 0.83‰ and 1.52‰, respectively, indicating that children group, cecal, colonic and rectal microbiota were signifi - exposed to antibiotic treatment had an 84% increased risk of cantly decreased (p < 0.05) (Fig.  1D-F, Fig. S1 d-f ). These IBD in later stages. In addtiton, it has been proven that the results indicated that lincomycin hydrochloride had a occurrence of IBD is negatively correlated with age (Kro- greater effect on the number and diversity of microbiota nman et al. 2012). Consistent with studies in children, adults in the large intestine of mice than in the small intestine of exposed to antibiotic therapy also have an increased risk of mice. IBD. An investigation covering 23,982 IBD patients (15,951 Based on the results of OTUs and alpha diversity analy- with UC, 7898 with CD, and 133 with unnamed IBD) span- sis, we focused our analysis on beta diversity of cecal, ning 10 years indicated that patients who used antibiotics colonic and rectal microbiota in mice. PCoA and ANO- for more than 1 year had 88, 74, and 127% increased risk of SIM analysis were performed by calculating Bray-Curtis, IBD, UC and CD compared with patients who did not use weighted UniFrac and unweighted UniFrac distances. antibiotics (p < 0.001) (Nguyen et al. 2020). In addition, after The results showed that there were significant differ - mice were intervened by antibiotics, their gut microbiota ences in beta diversity between the LCM group and the was changed, and their sensitivity to dextran sulfate sodium C_LCM group (p < 0.01) (Fig. 1G-I, Table S3), which indi- (DSS) was increased, which means that they were more cated that lincomycin hydrochloride significantly altered likely to induce UC (Ozkul et al. 2020). Therefore, based on the microbiota structure of the mouse cecum, colon and gut microbial dysbiosis, it is very important to use appro- rectum. The above results showed that lincomycin hydro - priate drugs and preparations to restore gut microbiota, and chloride had little effect on the microbiota of the small reduce the incidence or symptoms of IBD. intestine (duodenum, jejunum and ileum), but had more Studies have shown that probiotics can not only alter destructive effect on the large intestine (cecum, colon the expression of inflammatory cytokines in the body and and rectum) after intervention in mice. reduce the severity of inflammation (Yu et  al. 2020), but also change the gut microbiota such as the number, activity, and microbiota composition (Ma et al. 2020; Li et al. 2021a, Lactobacillus plantarum A3 can alleviate the symptoms 2021b, 2021c). Currently, probiotics have been used as IBD of ulcerative colitis in mice therapy to modulate the gut microbiota of the host (Biagioli To further study the effect of equine Lactobacillus plan - et al. 2020; Oka and Sartor 2020). This suggests that probiot - tarum A3 on ulcerative colitis in mice with gut micro- ics may reduce the incidence of IBD or alleviate symptoms bial dysbiosis, clinical indexes of mice  were analyzed of IBD when used in IBD patients with gut microbial dys- (Fig.  2A). During the 7-day period of DSS intervention, biosis. Lactobacillus plantarum A3 is a probiotic with good body weight changes and DAI index of mice in each properties  that has been isolated and screened in healthy group were monitored, and the results showed that adult horses, and has a significant recovery effect on DSS- except for the control group, which showed a stable induced UC in mice (Qin et al. 2022). Therefore, the aim of and slow upward trend, the body weight of mice in the this study was to investigate the regulatory effect of Lacto - other three groups showed a downward trend with DSS bacillus plantarum A3 from equine on UC in mice with gut intervention, and more obvious over time, and eventu- microbial dysbiosis and provide a reference for subsequent ally, body weight decreased to the lowest level on the last screening of potential mitigatory agents. day (Fig.  2B). In addition, except for the control group, the DAI index of mice tended to be stable, and  the DAI Results index of mice in the other three groups showed upward Lincomycin had a more destructive effect on the large gut trend over time. Notably, the increasing trend of DAI microbiota in mice in the DSS group was the fastest, and at the end of the To establish a mouse model of gut microbial dysbiosis, experiment, the DAI index in the DSS group was signifi - we treated the mice with lincomycin hydrochloride and cantly higher than that in other groups (p < 0.05) (Fig. 2C, analyzed the changes in gut microbiota in the duodenum, D). These results also showed that both Lactobacillus Qin  et al. Animal Diseases (2023) 3:16 Page 3 of 18 A B C G H bray_curtis 1.0 bray_curtis 1.0 C_LCM LCM C_LCM LCM C_LCM LCM 0.5 0.5 92 28 23 2 LCM04 LCM04 322 33 23 LCM06 336 23 17 LCM05 LCM06 LCM05 C03 C01 C02 0.0 C01 0.0 C03 C02 C05 C04 LCM03 C04 C06 LCM01 C05 LCM01 C06 LCM03 LCM02 LCM02 -0.5 -0.5 -1.0 6 ** 1.0 250 ** ** -0.5 0.0 0.5 -0.5 0.0 0.5 0.8 200 Axis.1 [62.1%] 0.6 Axis.1 [56.9%] 0.4 weighted_unifrac weighted_unifrac 50 0.2 0.4 0.5 0 0 0.0 C04 C01 LCM01 C02 LCM03 -1.0 C01 C04 LCM02 -0.5 0.0 0.5 LCM02 Axis.1 [56.9%] LCM03 C06 LCM01 C06 0.0 0.0 C05 C03 C02 C05 C03 LCM05 LCM05 LCM04 LCM04 -0.4 LCM06 LCM06 -0.5 250 6 1.0 ** ** * 200 0.8 -0.8 -1.0 150 0.6 -0.5 0.0 0.5 1.0 0.0 0.5 Axis.1 [71.9%] Axis.1 [64.9%] 100 0.4 50 0.2 unweighted_unifrac unweighted_unifrac 1.0 0 0 0.0 0.5 LCM03 0.5 F LCM06 200 ** 5 ** 1.0 * LCM01 LCM02 LCM05 C02 LCM04 C03 0.0 C01 C05 C04 4 0.8 C06 0.0 C05 C02 C06 LCM05 LCM03 C03 C04 3 0.6 C01 LCM06 LCM02 LCM04 100 -0.5 LCM01 -0.5 2 0.4 1 0.2 -1.0 -1.0 0 0 0.0 -0.50 0.50 0.0 0.5 0.00 0.25 0.5 -0.25 Axis.1 [62.5%] Axis.1 [57.3%] 1.0 bray_curtis weighted_unifrac 0.6 unweighted_unifrac 0.5 0.5 LCM05 LCM04 LCM06 LCM05 0.3 LCM06 LCM04 LCM06 LCM04 C_LCM LCM05 C02 C06 C04 LCM 0.0 C03 0.0 a C01 LCM02 C01 C05 C05 LCM01 C03 C04 0.0 C05 LCM03 LCM03 LCM03 C02 C06 LCM02 LCM01 LCM01 C02 LCM02 -0.5 C03 C01 -0.5 -0.3 C06 C04 -1.0 -0.5 0.0 0.5 0.0 -0.8 -0.4 0.0 0.4 -0.50 -0.25 0.00 0.25 0.50 Axis.1 [59.9%] Axis.1 [54.7%] Axis.1 [65.8%] Fig. 1 Lincomycin had a more destructive effect on the large gut microbiota in mice. A-C Venn diagram (A: Cecum; B: Colon; C: Rectum); D-F Alpha ns diversity (D: Cecum; E: Colon; F: Rectum); p > 0.05, *p < 0.05, **p < 0.01; G-I Based on PCoA, beta diversity of different intestinal segment contents in mice (G: Cecum; H: Colon; I: Rectum) plantarum A3 and butyrate slowed the increasing trend mice in the LP_DSS group were similar to the control of the  DAI index. After the experiment, the mice were group. This showed that Lactobacillus plantarum A3 euthanized and dissected. The results showed that com - can prevent the occurrence of UC, and  more evidence pared with the control group, the spleen index of mice appeared in the longer colon length and lighter color of in the DSS group was significantly increased (p < 0.05) colonic contents in the LP_DSS group (Fig. 2F). (Fig.  2E), but the colon length was significantly shorter After mouse colon tissue was stained by H&E, the (p < 0.05) (Fig.  2F). The spleen index and colon length of colonic injury  were  further observed. Compared with LCM LCM _LCM LCM _LCM LCM _LCM _LCM LCM C_LCM LCM LCM C_LCM C_LCM LCM LCM C_LCM LCM Chao1 Chao1 Chao1 Axis.2 [23.2%] Shannon Shannon Shannon Axis.2 [39.3%] Simpson Simpson Simpson Axis.2 [23.6%] Axis.2 [16.3%] Axis.2 [20.9%] Axis.2 [21%] Axis.2 [19.6%] Axis.2 [26.4%] Axis.2 [21.2%] Qin et al. Animal Diseases (2023) 3:16 Page 4 of 18 Fig. 2 Lactobacillus plantarum A3 can alleviate the clinical symptoms of ulcerative colitis in mice with gut microbial dysbiosis. A Mouse model of ulcerative colitis in mice with gut microbial dysbiosis; B Weight changes; C Changes in DAI score; D DAI index of mice in each group after 7 days of DSS intervention; E Spleen index changes; F Colon length changes among the four groups, and the colon morphology of DSS group and DSS group mice; G H&E staining results and histology scores, the red arrow points to the infiltration of inflammatory cells, the blue arrow points to the ns shedding of mucosal epithelial cells; p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; n = 10 Qin  et al. Animal Diseases (2023) 3:16 Page 5 of 18 the control group, a large number of inflammatory Similarly, Lactobacillus plantarum A3 can inhibit the cells invaded the mucosal and submucosal layers of the inflammatory response at gene and protein levels. colonic tissue, and many colonic mucosal epithelial cells To further explore the expression of Foxp3 at the detached in the DSS group. After treatment with Lacto- protein level, immunohistochemical analysis was  per- bacillus plantarum A3 or butyrate, no colonic mucosal formed  (Fig.  4F). Distribution of the brownish yel- epithelial cell detachment was observed in colonic tissue, low positive signals indicated that Foxp3 was mostly but some colonic mucosal and submucosal layers still had expressed in the mucosa and submucosa of the  colon. inflammatory cell invasion, and the pathological score After DSS inducing, the mean optical density (MOD) of was also significantly decreased (p < 0.001) (Fig.  2G), sug- Foxp3 in colon tissue increased significantly (p < 0.05), gesting that Lactobacillus plantarum A3 can reduce DSS which means that its expression increased. When mice damage to colonic tissue. were treated with Lactobacillus plantarum A3, the expression of Foxp3 in their colonic tissue was further Lactobacillus plantarum A3 can regulate the serum increased (p < 0.001). These results  showed that DSS biochemical indexes of mice induced a feedback regulatory immune response in mice, Serological parameters can often more intuitively illus- while Lactobacillus plantarum A3 further enhanced the trate the degree of damage to body cells. Thus, we studied immune response. the serum biochemical indexes in each group. The results showed that, compared with the control group, both Lactobacillus plantarum A3 regulates the colonic MPO and MDA were significantly upregulated (p < 0.001), microbiota in mice but SOD was significantly downregulated (p < 0.001) in To investigate the regulatory effect of Lactobacillus plan - the DSS group. Compared with the DSS group, MPO and tarum A3 on UC in mice with gut microbial dysbiosis, a MDA in the LP_DSS group were significantly decreased high-throughput gene sequencing of 16S rDNA was per- (p < 0.05), while SOD was significantly increased (p < 0.05) formed in the colonic content microbiota in each group (Fig. 3A-C), and the concentration was closer to the con- of mice. For the alpha diversity, compared with the  DSS trol mice. This indicated that Lactobacillus plantarum A3 group, the Shannon and Simpson indexes of colonic con- alleviated the degree of cell damage in mice. tent microbiota increased significantly in the LP_DSS group (p < 0.001), but the  Chao 1 index did not increase Lactobacillus plantarum A3 can regulate cytokines significantly (p > 0.05) (Fig.  5A). These results  showed expressed in mouse colon tissue that A3 can significantly restore the community diversity Inflammation in the body is often accompanied by of intestinal microorganisms in mice, but its restoration changes in inflammatory cytokines. In DSS-induced effect on community richness is limited (Dill-McFarland mice, the expression of anti-inflammatory cytokine et  al. 2019; He et  al. 2017). For the beta diversity, com- such as IL-1β, IL-6, TNF-α and IFN-γ were significantly pared with the DSS group, Bray-curtis, unweighted Uni- increased at the gene level (p < 0.001) (Fig.  3D-G), while Frac and weighted UniFrac distances were significantly anti-inflammatory cytokine like IL-10 were signifi - changed in the LP_DSS group (p < 0.01) (Fig.  5B, Table cantly decreased (p < 0.001) (Fig. 3H). In addition, Foxp3, S3), which indicated that Lactobacillus plantarum A3 the main regulator of Treg cells, was also significantly could significantly affect intestinal bacteria in the colon increased (p < 0.05) (Fig.  3I). These showed that DSS of mice. promotes the inflammatory response at the gene level. To further investigate which bacterium was specifi - When Lactobacillus plantarum A3 was involved in the cally regulated by Lactobacillus plantarum A3, we con- regulation, IL-1β, IL-6, TNF-α and IFN-γ were signifi - ducted an analysis between the DSS group and LP_DSS cantly decreased at the gene level (p < 0.05) (Fig.  3D-G), group  were conducted. At the phylum level, the four while IL-10 increased significantly, rather than decreased phyla with the highest abundance of colonic contents (p < 0.001) (Fig. 3H). Besides, A3 significantly upregulated in the two groups were Bacteroides, Firmicutes, Proteo- the expression of Foxp3 (p < 0.001) (Fig.  3). In summary, bacteria and Verrucomicrobia. The relative abundance Lactobacillus plantarum A3 can inhibit the progression of each phylum was 63.80, 14.40, 15.77 and 6.03% in the of inflammatory responses at the gene level. DSS group, and 50.38, 22.84, 16.64 and 10.14% in the The expression of these cytokines at the protein LP_DSS group (Fig.5, C). The results showed that A3 was level  were also interrogated  by western blot, which was involved in regulating each phylum. At the genus level, consistent with their trend at the gene level (Fig.  4A- Bacteroides (63.80%), Enterobacter (9.32%) and Blau- D), indicating that DSS can promote the inflammatory tia (8.26%) were the top three microbiota in the DSS response of mice at both the gene and protein levels. group, Bacteroides (50.38%), Enterobacter (15.33%) and Qin et al. Animal Diseases (2023) 3:16 Page 6 of 18 Fig. 3 Lactobacillus plantarum A3 can regulate the biochemical indexes and cytokine gene expression of ulcerative colitis in mice with gut microbial dysbiosis. A-C Serum biochemical indexes (A: MPO; B: SOD; C: MDA); D-I Cytokine gene expression level in colonic tissue (D: IL-1β; E: IL-6; F: TNF-α; G: IFN-γ; H: IL-10; I: Foxp3); *p < 0.05, **p < 0.01, ***p < 0.001; n = 10 Akkermansia (10.14%) were the top three microbiota in compared with the DSS group, but Bacteroidetes was sig- the LP_DSS group which meant that Lactobacillus plan- nificantly downregulated (p < 0.05) (Fig. 5E). At the genus tarum A3 changed the microbiota with high abundance, level, the abundance of Blautia, Akkermansia, Citrobac- which was consistent with the beta diversity analysis ter and Flexispira was significantly upregulated (p < 0.05) (Fig. 5D). in the LP_DSS group, but Bacteroides was significantly LEfSe confirm the specific bacteria that had significant downregulated (p < 0.05) (Fig.  5F). In general, Lactobacil- changes between the two groups. At the phylum level, lus plantarum A3 treatment significantly transformed the the abundance of Firmicutes and Verrucomicrobia was diversity and composition of gut microbiota. significantly upregulated in the LP_DSS group (p < 0.05) Qin  et al. Animal Diseases (2023) 3:16 Page 7 of 18 Fig. 4 Lactobacillus plantarum A3 can regulate the biochemical indexes and cytokine expression of ulcerative colitis in mice with gut microbial dysbiosis. A-E Expression of cytokines at protein level in colonic tissue was measured by western blot in each group (A: IL-1β; B: IL-6; C: TNF-α; D: IFN-γ; E: IL-10); F Foxp3 in colonic tissue was measured by immunohistochemistry, and the mean optical density was calculated in each group. *p < 0.05, **p < 0.01, ***p < 0.001 Lactobacillus plantarum A3 can regulate changes of colonic metabolites with significant differences between the two metabolites in mice groups. Untargeted metabolomic analysis can detect small Then, according to the above discriminant analy- molecule metabolites with a relative molecular weight sis model, the metabolites that play an important role of less than 1000 in the sample, from which we can in the discrimination process are analyzed. These obtain differential metabolites and metabolic pathways metabolites have the potential to be used as biomark- between the DSS group and the LP_DSS group. We ers between the two groups. Value importance in pro- used PCA to model each sample and found that there jection (VIP) > 1 was as the standard and screened 383 were no outlier points in the intragroup samples under metabolites in positive polarity mode and 285 metabo- both positive and negative polarity modes, which indi- lites in negative polarity mode. To further explore the cated that there was little difference in the intragroup differential metabolites, univariate analysis  were  also samples and could be used for the analysis of differential performed. p < 0.05 and fold change (FC) > 2 were metabolites (Fig. 6A-D). The factors that can distinguish used  as the screening criteria. The results showed sample grouping to the greatest extent can be found that, compared with the DSS group, there were 23 using PLS-DA, and the overfitting caused by the PLS- metabolites with more than 2 FC in the LP_DSS group DA model can be improved using OPLS-DA. There - (p < 0.05) in the positive polarity mode, of which ara- fore, we plotted the scatter diagram with the two best chidonoyl ethanolamide phosphate guanosine (AEA- discriminating factors and found that the sample point P) had the highest fold upregulation, reaching 28.70 clouds of the DSS group and the LP_DSS group were FC (p < 0.001), and four metabolites including seri- distributed in different regions, which indicated that lin and Cortisone were also upregulated by more the PLS-DA model and the OPLS-DA model had better than 10 FC (p < 0.001) (Fig .  6I, Table  1). In the nega- discrimination effects (Fig.  6E-H), and there should be tive polarity mode, there were nine metabolites with Qin et al. Animal Diseases (2023) 3:16 Page 8 of 18 Fig. 5 Lactobacillus plantarum A3 can regulate the gut microbiota of ulcerative colitis in mice with gut microbial dysbiosis. A Alpha diversity of ns colon contents in mice, p > 0.05, ***p < 0.001. B Beta diversity of colon contents in mice. C Bar plots of the phylum taxonomic levels in the DSS and LP_DSS groups; D Bar plots of the genus taxonomic levels in the DSS and LP_DSS groups; E-F Difference in dominant microorganisms of colon contents between the DSS group and LP_DSS group via distribution histogram based on LDA. The criteria for feature selection was log LDA score > 2.0 (E: at the phylum level; F: at the genus level) Qin  et al. Animal Diseases (2023) 3:16 Page 9 of 18 Fig. 6 Lactobacillus plantarum A3 can regulate the metabolite changes of colon contents in ulcerative colitis in mice with gut microbial dysbiosis. A-D PCA score diagram (A-B: under positive polarity mode; C-D: under negative polarity mode). E-F Scatter diagram of PLS-DA models (E: under positive polarity mode; F: under negative polarity mode); G-H Scatter diagram of OPLS-DA models and OPLS-DA models (G: under positive polarity mode; H: under negative polarity mode). I-J Univariate analysis of differential metabolites in mouse colonic contents between the DSS group and LP_DSS group (I: under positive polarity mode; J: under negative polarity mode), log2(FC) > 0 represents metabolites with high abundance in the LP_DSS group. K-L The metabolic pathways of differential metabolite enrichment by ORA (K: under positive polarity mode; L: under negative polarity mode) Qin et al. Animal Diseases (2023) 3:16 Page 10 of 18 Fig. 6 continued more than 2 FC upregulation in the LP_DSS group, metabolites that were significantly different between and the two metabolites with the highest upregula- the DSS group and the LP_DSS group (t  test, p < 0.05), tion were O-LPL-carnitine (p < 0.05, 5.712-fold) and we looked for the metabolic pathways in which these N2, N2-dimyl (p < 0.001, 5.06-fold) (Fig .  6J,  Table  2). metabolites appeared and calculated the p value and Enrichment analysis can help us find pathways with fold enrichment of ORA for these metabolic path- key roles and thus can predict the molecular mecha- ways (Fig.  6K-L). Impact  can be  calculated by topo- nisms of the entire regulatory process. Based on the logical analysis to judge the magnitude of the effect Qin  et al. Animal Diseases (2023) 3:16 Page 11 of 18 Table 1 Differential metabolites in mouse colonic contents of metabolites in metabolic pathways. Combined between the DSS group and LP_DSS group under positive enrichment and topological analysis, using p < 0.05 polarity mode (screening criteria: VIP > 1; p < 0.05; FC > 2) and Impact >0.2 as criteria, metabolic pathways sig- nificantly affected by differential metabolites from Differential metabolites VIP p FC the above metabolic pathways  were  selected: ubiqui- Arachidonoyl ethanolamide phos- 2.810 <0.001 28.70 none and other terpenoid-biosynthesisu (impact = 0.5, phate p < 0.05, fold enrichment = 26.24), phenylalanine, PS 170/171 1.945 0.0190 23.80 tyrosine and tryptophan (impact = 0.25, p < 0.05, fold PE 203/204 1.808 0.0220 13.77 enrichment = 19.68). The above results showed that Stercobilin 2.652 <0.001 11.09 Lactobacillus plantarum A3 affected  not only the Cortisone 2.779 <0.001 10.77 changes of metabolites in the  mouse colon, but also Nicotinamide 1.792 0.0360 6.486 the metabolic pathway. TAG 161-181-181 1.893 0.0136 5.250 Oxohongdenafil 2.377 <0.001 5.087 Discussion Pantethine 2.499 <0.001 4.827 Recent studies have found that early gut microbiota Abscisic acid 2.189 0.0041 4.060 imbalance can increase the incidence of diseases such as 5,8-dihydroxy-10-methyl-5,8,9,10- 1.922 0.0194 3.866 asthma (Arrieta et  al. 2015), IBD (Hviid et  al. 2011) and tetrahydro-2H-oxecin-2-one immune diseases (Round and Mazmanian 2009). How- 4-acetyl-4-ethoxycarbonylheptan- 2.591 <0.001 3.677 edioic acid ever, antibiotics are not only inevitably used in clinical Methyl EudesMate 1.787 0.0360 3.423 treatment, but have also become one of the most widely Gamma-Glu-Leu 1.803 0.0340 2.657 used drugs (Adriaenssens et  al. 2011; Vaz et  al. 2014). Therefore, restoring antibiotic-induced gut microbiota L-Tyrosine 1.761 0.0320 2.490 imbalance and preventing related diseases is a  key prob- methyl 3-1H-indol-3-yl-2-phenyl- 2.465 <0.001 2.476 methyleneaminopropanoate lems. UC is an idiopathic inflammatory bowel disease. gamma-Glutamylglutamic acid 1.807 0.0327 2.456 Its high incidence and lack of specific drugs are the rea - IQH 1.677 0.0392 2.240 sons why it has become a problem in modern medicine Genistein 4′-O-glucuronide 1.812 0.0240 2.219 (Fan et  al. 2019). Although the etiology is unknown, the 2.350 0.0013 2.192 N, N′-di4-2,6- phenomenon of gut microbial dysbiosis has been proven dimethylmorpholinophenylthiourea to appear before UC (Wu et al. 2020). In addition, when LMH 2.205 0.0046 2.128 mice were first treated with antibiotics, the stability of 4-2,3-dihydro-1,4-benzodioxin-6-ylb- 1.932 0.0157 2.060 gut microbiota was destroyed, their sensitivity to DSS utanoic acid increased, and finally, the symptoms of ulcerative coli - Muramic acid 2.004 0.0140 2.030 tis were more serious (Ozkul et  al. 2020). This suggests Add notes: VIP value importance in projection, FC fold change that timely treatment and restoration of the stability of gut microbiota after gut microbial dysbiosis may reduce IBD’s incidence or symptoms. As a kind of probiotic, Lactobacillus has been proven to maintain and restore the homeostasis and stable heredity of gut microbiota (Ma et al. 2020), and it also has a certain Table 2 Differential metabolites in mouse colonic contents therapeutic effect on IBD (including UC and CD) (Guo between the DSS group and LP_DSS group under negative et al. 2019; Wang et al. 2020). Lactobacillus plantarum A3 polarity mode (screening criteria: VIP > 1; p < 0.05; FC > 2) is a lactobacillus that has good probiotic characteristics Differential metabolites VIP p FC isolated and screened from healthy adult horses, and it has a certain alleviating effect on UC in mice (Qin et al. 2022), O-Acetyl-L-carnitine 2.800 0.0319 5.712 Therefore, in this study, we used Lactobacillus plantarum N2, N2-dimethylguanosine 2.658 <0.001 5.057 A3 as the main therapeutic drug to explore its regula- 20-Carboxy-Leukotriene B4 2.226 0.0340 3.386 tory effect on colitis in mice with gut microbial dysbiosis. 3,4-Dihydroxyphenylglycol 2.010 <0.001 3.233 In addition, as a short chain fatty acid (SCFA), butyrate Pyrogallol 2.176 0.0111 2.843 can not only be the main energy source of colonic epi- W-Lactose 2.187 0.0402 2.513 thelial cells, but also regulate epithelial stem cells (Kaiko 8-iso-15-keto Prostaglandin E2 2.109 0.0476 2.259 et  al. 2016), and promote epithelial homeostasis together Mono2-ethylhexyl phthalate MEHP 2.100 0.0194 2.163 with other SCFAs (Macia et  al. 2015). Therefore, when Hexadecanedioic acid 1.998 0.0262 2.150 Add notes: VIP value importance in projection, FC foldchange Qin et al. Animal Diseases (2023) 3:16 Page 12 of 18 constructing the mouse UC model, we used butyrate as a of lipid peroxidation in the body. SOD is an antioxidant positive control of treatment. enzyme, that  can catalyze free radicals into oxygen and In the first stage, lincomycin hydrochloride was used hydrogen peroxide (Yu et al. 2020; Li et al. 2023). There - to establishe a mouse model of gut microbial dysbiosis. fore, the contents of MDA and SOD in the body are often In clinical practice, lincomycin hydrochloride is com- negatively correlated. Our study showed that Lactobacil- monly used to treat various bacterial infections. It can lus plantarum A3 could significantly upregulate the level inhibit the growth of bacteria by inhibiting the synthe- of SOD and downregulate the level of MDA (p < 0.05), sis of proteins in bacteria, and it has strong antibacte- indicating that they can play a regulatory role through rial and bactericidal effects on Gram-positive bacteria, antioxidation. especially anaerobic bacteria (Geddes et al. 1964). Recent At present, it is generally believed that patients with studies have shown that short-term overdose of linco- UC have intestinal mucosal inflammation and immune mycin hydrochloride can lead to microbiota imbalance, abnormalities (Medzhitov 2008). MPO exists in neutro- intestinal tissue damage and immune function decline in phils, and its activity is directly proportional to the popu- mice (Li et al. 2019; Lv et al. 2017). Therefore, lincomycin lation of neutrophils in the inflammatory area (Bastaki hydrochloride was used as an intervention drug, and its et al. 2018). Therefore, the significantly increased content effect on each gut microbiota of mice was explored. The of MPO in the DSS group also suggested the presence of results showed that lincomycin hydrochloride had no sig- excessive neutrophil invasion in mice, which was consist- nificant effect on the microbiota of the small intestine in ent with the results of H&E staining in colon tissue. The mice, but it had a significant damage on the microbiota aggravation of oxidative stress and the increase of neu- of the large intestine, not only the number, but also the trophils in the body can further stimulate the release of structure. immune cytokines associated with UC (Leppkes and Based on the constructed mouse gut microbial dysbio- Neurath 2020). For example, oxidative stress can not only sis model, we induced UC in mice with DSS and explored stimulate macrophages to release IFN-γ, but also induce the regulatory effect of Lactobacillus plantarum A3. the release of TNF-α, IL-6, IL-1β and other inflamma - UC often requires some specific indicators to evaluate, tory factors, further causing intestinal mucosal metabolic such as spleen index, colon length and DAI index. Sple- disorders and intestinal mucosal inflammatory immune nomegaly is one of the common symptoms in UC mice disorders, which can lead to systemic inflammatory (Zhang et al. 2017). Colon length and DAI index are often response syndrome and multiple organ dysfunction (Li used to evaluate the severity of ulcerative colitis in clini- et  al. 2021a, 2021b, 2021c; Vlahakos et  al. 2012). In our cal diagnosis and treatment (El Sayed and Sayed 2019). In study, the expression levels of IFN-γ, TNF-α, IL-6, and addition, pathological tests are often used as one of the IL-1β in the DSS group were significantly higher than gold standards for diagnosing the symptoms of ulcera- those of the control group at both gene and protein lev- tive colitis (Allen et  al. 2012; Meira et  al. 2008). After els (p < 0.001), which indirectly indicated abnormalities in DSS induction, the spleen index and DAI index of mice intestinal mucosal inflammation and immune response were significantly increased (p < 0.05), and the colon was in mice. As an anti-inflammatory cytokine, IL-10 can significantly shortened (p < 0.05). H&E staining showed inhibit the secretion of proinflammatory cytokines such that there were a large number of inflammatory cells in as IFN-γ, TNF-α, IL-6 and IL-1β, regulate the differen - the mucosal layer and submucosal layer in the colon tis- tiation and proliferation of macrophages, T cells and B sue, accompanied by a large number of mucosal epithe- cells, inhibit Th1/Th17 responses, which are essential lial cells falling off. The above results showed that DSS for intestinal immune homeostasis (Bárcena et al. 2019). successfully induced ulcerative colitis in mice with gut Lactobacillus plantarum A3 promoted the expression microbial dysbiosis. When mice were treated with Lac- of IL-10 and inhibited IL-1β, IL-6, TNF-α and IFN-γ, tobacillus plantarum A3, the spleen index and DAI index which further proved that it can effectively regulate intes - of mice decreased significantly (p < 0.05), the colon length tinal inflammation and intestinal immune response in was closer to that of  the control group, and H&E stain- mice. Foxp3 is considered to be a specific transcriptional + + + + ing showed that the degree of inflammatory cell invasion marker of CD4 CD25 regulatory T cells (C D4 CD25 decreased significantly. The above results showed that Treg), which plays an important role in the proliferation, Lactobacillus plantarum A3 could alleviate the clinical differentiation and immune function of Treg cells (Jia symptoms of ulcerative colitis in mice with gut microbial et  al. 2020; Zhou et  al. 2020). Moreover, recent studies dysbiosis. have also shown that the expression of Foxp3 is positively Oxidative stress is closely related to ulcerative colitis. correlated with the expression of programmed cell death MDA is the end product of the peroxidation reaction ligand (PD-L1), and PD-L1 mediated signal transduction between free radicals and lipids. It reflects the degree pathway is one of the main processes regulating intestinal Qin  et al. Animal Diseases (2023) 3:16 Page 13 of 18 local inflammatory response (Yamamoto et  al. 2022). upregulating the abundance of beneficial bacteria such as This illustrated that Lactobacillus plantarum A3 can reg - Blautia and Akkermansia. ulate the immune response and inflammatory response Intestinal metabolites can regulate the intestinal envi- of mouse intestine by upregulating Foxp3. ronment together with gut microbiota. Compared with The severity of UC is related to the decline in the diver - the DSS group, the colonic metabolites in the LP_DSS sity of intestinal microorganisms, such as the relative group were significantly changed, with the highest up- decrease in the abundance of Clostridium and Akker- regulation of arachidonoyl ethanolamide phosphate mansia (Pittayanon et  al. 2020; Zhou et  al. 2018), which (AEA-P), reaching 28.70-fold (p < 0.001). AEA-P is a is consistent with the changes in the DSS group in our newly discovered endogenous lipid, a precursor of ara- study. Lactobacillus plantarum A3 significantly restored chidonoyl ethanolamide (AEA), and its function is simi- the alpha diversity and beta diversity of intestinal micro- lar to AEA. As an “endocannabinoid” (which has similar organisms in mice (p < 0.05), regulated the microbiota pharmacological effects to the active ingredient of can - structure, and increased the relative abundance of Blau- nabis: Δ -tetrahydro-cannabinol), AEA is often released tia, Akkermansia, Citrobacter and Flexispira. Blautia is from depolarized neurons, endothelial cells, and mac- an anaerobic bacterium with probiotic characteristics. rophages in the body, and then participates in cellular According to the analysis of phenotype and phylogeny, activities as an endogenous ligand for cannabinoid recep- some species of Clostridium and Ruminococcus have tors in the central nervous system (CB1 subtype) and been reclassified to this genus. Blautia plays an impor- peripheral immune cells (CB2 subtype). Besides, it can be tant role in regulating the growth of gut microbiota and selectively absorbed by cells, and then degraded by fatty inflammation. This is mainly because Blautia has the acid amide hydrolase (FAAH) to ethanolamine and ara- ability to produce bacteriocin, which makes it have the chidonic acid (AA), further playing an anti-inflammatory potential to inhibit the colonization of pathogenic bac- role. It is worth mentioning that AEA can also reduce teria in the intestine, especially Clostridium perfringens pain at the site of tissue injury (Maccarrone et al. 2000). and vancomycin-resistant enterococci (Liu et  al. 2021; Li These may be the reasons for the significant upregulation et  al. 2021a, 2021b, 2021c). In addition, Blautia plays an of AEA-P concentration in group A mice. In addition, important role in obesity related diseases. It is the only Lactobacillus plantarum A3 also increased the concen- bacterium negatively related to visceral fat accumulation trations of cortisone in the colon of mice by 10.77-fold (Ozato et al. 2019; Song et al. 2014). As a mucin decom- (p < 0.001). Cortisone is a type of glucocorticoid, that posing bacterium in the body, Akkermansia widely exists was used to treat IBD as early as 1955 (Singh 2018). It in the intestinal tract of animals. It obtains a competitive can regulate cytokines in the body, such as inhibiting the advantage by improving the utilization rate of mucin in release of pro-inflammatory cytokines IL-6, IFN-γ and the body, so it can be highly colonized in the intestinal TNF-α, while stimulating the production of anti-inflam - tract, and it can alleviate the inflammatory reaction by matory cytokine IL-10, which shows that the increase in reducing the permeability of the intestinal tract and the cortisone concentration is also one of the reasons for the infiltration derived pro-inflammatory lipopolysaccharide changes of cytokine in mice. Today, there are still studies (Bárcena et  al. 2019; Depommier et  al. 2019; Kim et  al. on synthetic cortisone analoges for improving the stabil- 2022). Citrobacter and Flexispira are common microbi- ity of drugs and optimizing their efficacy in treating IBD ota in the intestine. There are few studies on their rela - (Rol et al. 2021). Therefore, the reason why Lactobacillus tionship with UC. Some studies have intervened with plantarum A3 induced a significant increase in intes - Citrobacter rodentium to induce colitis in mice and found tinal cortisone might be one of the priorities in further that it can reduce the weight of mice and cause diarrhea research. Through enrichment analysis and topological (Bhinder et  al. 2013). However, in our study, it was not analysis, it was also found that Lactobacillus plantarum found that after the intervention of Lactobacillus plan- A3 significantly affects ubiquinone and other terpenoid tarum A3, the weight of mice was less than that of the quinone biosynthesis, phenylalanine, tyrosine and tryp- DSS group and there was no diarrhea, which indicated tophan biosynthesis. These pathways predict the intes - that not all Citrobacter can induce disease. The limita - tinal metabolic activities that Lactobacillus plantarum tions of the genus level made it impossible for us to fur- A3 may have participated in. In further studies, we can ther analyze it, which also provided a new idea  for our explore the specific mechanism by which Lactobacillus future research. In general, Lactobacillus plantarum A3 plantarum A3 regulats UC in mice with gut microbial had the ability to regulate gut microbiota in mice. It not dysbiosis by verifying these metabolic pathways. only increased the diversity of gut microbiota, but also further regulated the inflammatory response in  vivo by Qin et al. Animal Diseases (2023) 3:16 Page 14 of 18 Conclusion bodyweight), and mice in the DSS group were intragas- In this study, we successfully constructed a gut microbial trically administered an equal volume of normal saline. dysbiosis model in mice by lincomycin hydrochloride and Notably, on the last 7 d of the experiment, except for the found that it had a more significant destructive effect on control group, which maintained normal drinking water, the microbiota of the large intestines. Subsequently, we the other three groups of mice were given drinking water constructed a UC model on the basis of gut microbial containing 3% (w/v) DSS, in which freshly prepared DSS dysbiosis in mice and found that Lactobacillus plantarum solution was changed every 2 d (Fig.  2A). At the end of A3 could alleviate the clinical symptoms of mice and the experiment, mice were euthanized, and serum, spleen achieve a mitigatory effect by regulating the expression of tissue and colon tissue, colon contents were collected to cytokines, affecting the structure of gut microbiota and subsequent studies. regulating the level of intestinal metabolites. Intestinal contents genomic DNA extraction and 16S-rRNA Methods sequencing Animals The intestinal contents of mice in each group were sub - Five-week-old male C57BL/6 mice were  obtained from jected to total DNA extraction by the  E.Z.N.A. soil kit the animal center of Huazhong Agricultural Univer- (Omega Biotek, Norcross, GA, U.S.). After ensuring the sity (Wuhan, China). They were housed under specific eligibility of the DNA, the V3-V4 variable region was pathogen-free conditions and had access to food and amplified by PCR with 338F (5′-ACT CCT ACG GGA water ad  libitum at a temperature of 25±2°C, relative GGC AGC AG-3′) and 806R (5′-GGA CTA CHVGGG humidity of 40-60% (12:12-hour light/dark cycle). All TWT CTA AT-3′) primers, and the amplification pro - animal experimental protocols (HZAUMO-2021-0068 gram was: 95°C for 3 min, 27 cycles (95°C for 30 s, 55°C and HZAUMO-2021-0138) were approved by the Eth- for 30 s, 72°C for 30 s), and 72°C for 10 min. The result - ics Committee of Huazhong Agricultural University ing PCR products were purified using the AxyPrep DNA (Wuhan, China) and performed following the guidelines Gel Extraction Kit (Axygen Biosciences, Union City, CA, of the National Institutes of Health Guide for the care USA). Standard operating procedures for generating and Use of Laboratory Animals. sequencing libraries from the purified amplified frag - ments. Then, purified amplicons were pooled in equi - Mouse model of gut microbial dysbiosis molar  amounts and paired-end sequenced (2 × 300) on C57BL/6 mice were randomly divided into two groups: an Illumina MiSeq platform (Illumina, San Diego, USA). the  control-antibiotic group (C_LCM group) and Quality control and species annotation were conducted the  antibiotic intervention group (LCM group). The by previous methods (Bokulich et al. 2018; Callahan et al. first 3 d, mice were adaptive feeding, and then, mice in 2016). Any contaminating mitochondrial and chloroplast the LCM group were given lincomycin hydrochloride sequences were filtered using the QIIME2 feature-table (30 mg/10 g body weight) for 5 d. Mice in the C_LCM plugin. Venn diagram was drawn to analyze the unique group were given normal saline as  a control. After that, or common OTUs between different groups. Alpha the mice were euthanized and the intestinal contents of diversity, including the  Chao1, Shannon and Simpson duodenum, jejunum, ileum, cecum, colon and rectum indexes, was calculated to estimate the microbial diver- were taken. sity within an individual sample. Beta diversity, including BrayCurtis, weighted UniFrac and unweighted UniFrac Mouse model of ulcerative colitis was determined to investigate the structural variation in C57BL/6 mice were randomly divided into four groups microbial communities across samples and then visual- (n = 15): Control-DSS group (Control group), DSS group, ized via principal coordinate analysis (PCoA) (Vázquez- Lactobacillus plantarum A3-DSS group (LP_DSS group) Baeza et al. 2013). Linear discriminant analysis effect size and butyrate-DSS group (BA_DSS group, as a positive (LEfSe) was employed to identify the bacteria with differ - control group). The experimental period was 22 d, in ent abundances among samples and groups (Segata et al. which the first 3 d were adaptive feeding. In the follow- 2011). ing 5 d, mice in the DSS group and LP_DSS group were intragastrically administered lincomycin hydrochloride Clinical indicator analysis (30 mg/10 g bodyweight). In the remaining 14 d, mice During the experiment, mental status, weight, fecal shape in the LP_DSS group were intragastrically administered and fecal occult blood for mice were recorded every day. Lactobacillus plantarum A3 (5 × 10 CFU/10 g body- The disease activity index (DAI) was scored based on the weight), mice in the BA_DSS group were intragastrically weight loss rate, fecal score and occult blood score of administered an equal volume of butyrate (30 mg/10 g Qin  et al. Animal Diseases (2023) 3:16 Page 15 of 18 mice. The score range for each parameter is 0-4 points, membrane was rinsed with blocking solution contain- and the sum for the parameters is the final score (up to 12 ing 5% skimmed milk for 2 h at room temperature and points) (Table  S1) (Liu et  al. 2020). After the mice were incubated with primary antibody overnight at 4°C. After euthanized, the colon length and the weight of the mice the membranes were incubated with secondary antibody in each group were measured, and the spleen index was for 1 h at room temperature, the proteins were visualized calculated. using an enhanced chemiluminescence (ECL) system (Thermo Scientific, USA) and analyzed with ImageJ soft - Histology ware. Antibodies are listed in Supplementary materials. The colonic tissue sections of mice in each group were taken and the feces were washed with 0.9% sterile nor- Immunohistochemistry mal saline, fixed in 4% paraformaldehyde, dehydrated After the dewaxing of paraffin sections, the antigens were with ethanol, and processed into a paraffin-embedded repaired by microwave heating and endogenous per- block, cut into 5 μm sections, and stained with hematoxy- oxidase was blocked by 3% hydrogen peroxide solution. lin and eosin (H&E). Pathobiological slices were evalu- The colon tissue sections were sealed at room tempera - ated in a blinded manner, and the pathological scores ture for 30 min with 3% BSA, and incubated at 4°C over- were assessed based on the following parameters: inflam - night with rabbit anti-Foxp3 (A12051, ABclonal, Wuhan, mation, epithelial defects, crypt pathology, dysplasia/ PRC, 1:100 dilution). After that, the colon tissue sections neoplasia and the area of dysplasia/neoplasia (scoring were washed three times and incubated with goat anti- criteria in supplementary materials) (Allen et  al. 2012; rabbit IgG (K5007, Dako, Shanghai, PRC, 1:200 dilution) Meira et al. 2008). at room temperature for 50 min. Sections of negative control were treated in the same manner, but the pri- Serum biochemical detection mary antibody was omitted. Three 400 visual fields were Serum samples from mice were collected for biochemical randomly selected for each slice in each group. ImageJ index analysis. The activities of myeloperoxidase (MPO), software was used to analyze each image to obtain the and  superoxide dismutase (SOD), along with the con- positive integrated optical density value (IOD) and the centrations of malonaldehyde (MDA), were measured area of visual fields for each image, and then, calculate by specific assay kits (Nanjing Jiancheng Bioengineering the mean optical density (MOD). Institute, Nanjing, PRC). MOD = IOD/area Gene expression analysis Total RNA from colon tissue was extracted with a Steady- Colon contents untargeted metabolomic analysis Pure Universal RNA Extraction Kit (AG21017, Accurate Metabolite extraction and UHPLC-MS/MS analyses were Biotechnology, Hunan, PRC). RNA was reverse tran- performed according to previous protocols (Want et  al. scribed into complementary DNA (cDNA) (Evo M-MLV 2013). The raw data files were processed using the Com - RT Mix Kit with gDNA Clean for qPCR; AG11728, Accu- pound Discoverer 3.1 (CD 3.1, ThermoFisher, USA). After rate Biotechnology, Hunan, PRC). Real-time PCR (SYBR that, these metabolites were annotated using the KEGG, Green premix Pro Taq HS qPCR kit; AG11701, Accurate HMDB and LIPIDMaps database. Data normalization, Biotechnology, Hunan, PRC) was used to detect gene partial least squares discriminant Aanalysis (PLS-DA), -ΔΔCt expression and the 2 method was used for calcula- orthogonal partial least squares discriminant analysis tion and analysis. The primers are shown in Table S2. (OPLS-DA) were performed with R package MetaboAn- alystR (Chong and Xia 2018). To make the data close to Western blot a normal distribution, the Normalization function in IL-1β, IL-6, TNF-α, IFN-γ and IL-10 in colonic tissue in the  MetaboAnalystR package (with arguments Medi- each group were determined. After the colon tissue was anNorm, LogNorm, and AutoNorm) was adopted. We ground into powder by liquid nitrogen, it was added into applied a univariate analysis (t test) to calculate the statis- ice cold analysis buffer containing a cocktail of protec - tical significance (p value). The metabolites with VIP > 1, tion inhibitors. The mixture was lysed on ice for 30 min p < 0.05 and | log2 (fold Change) | > 1 were considered to (mixed by repeated shaking with a vortex during this be differential metabolites. The metabolites with p < 0.05 time to ensure complete cell lysis), and then the super- (t  test) were used to conduct an over representation natant fluid of the lysate was collected (4°C, 12,000 rpm, analysis (ORA), and the resulting KEGG pathways with 10 min). After electrophoresis, the proteins were elec- p < 0.05 (ORA) were considered as statistically significant trotransferred onto a polyvinylidene fluoride (PVDF) enrichment. membrane (Millipore, Boston, MA, USA). Then, the Qin et al. Animal Diseases (2023) 3:16 Page 16 of 18 Data analysis Availability of data and materials The materials and data not presented in this manuscript are available from The normality of the data was tested using the Shapiro- the corresponding author upon request. The datasets generated during the Wilk normality test. The data that conformed to a normal current study are available in the NCBI repository, [https:// www. ncbi. nlm. nih. distribution were analyzed by one-way ANOVA or t tests gov/, PRJNA841483]. using the Graphpad Prism 6.0 software (GraphPad, La Jolla, CA, USA) and were presented as means±standard Declarations deviation (mean±SD). In addition, differential analysis of Ethics approval and consent to participate alpha diversity was performed using Wilcoxon rank sum This study was approved by the Ethics Committee of Huazhong Agricul- test, beta diversity index was performed using the analy- tural U-niversity ( Wuhan, China) (Permit No. HZAUMO-2021-0068 and HZAUMO-2021-0138) and performed based on the state guidelines from the sis of similarities (ANOSIM), and microbiota between Laboratory Animal Research Center of Hubei province in China. different groups was performed using LEfSe. The signifi - cance level declared at ns p > 0.05, *p < 0.05, **p < 0.01 and Consent for publication Not applicable. ***p < 0.001. Competing interests The authors declare that they have no competing interests. Abbreviations IBD inflammat ory bowel disease UC ulcerative colitis Received: 11 January 2023 Accepted: 28 February 2023 AEA-P arachidonoyl ethanolamide phosphate CD Crohn’s disease PCoA pr incipal coordinate analysis LEfSe Linear discriminant analysis Eec ff t Size DAI disease activity index References MPO myeloperoxidase Adriaenssens, N., S. Coenen, A. Versporten, A. Muller, V. Vankerckhoven, H. SOD superoxide dismutase Goossens, and ESAC Project Group. 2011. European surveillance of MDA malonaldehyde antimicrobial consumption (ESAC): quality appraisal of antibiotic use in IOD integrated optical density Europe. The Journal of Antimicrobial Chemotherapy. 66: vi71–vi77. https:// MOD mean optical density doi. org/ 10. 1093/ jac/ dkr459. IL-1β Interleukin-1 beta Allen, I.C., J.E. 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Regula- tory effect of Zuojin Pill on correlation with gut microbiota an-d Treg cells in DSS-induced colitis. Journal of Ethnopharmacology 262: 113211. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : https:// doi. org/ 10. 1016/j. jep. 2020. 113211. fast, convenient online submission Publisher’s Note thorough peer review by experienced researchers in your field Springer Nature remains neutral with regard to jurisdictional claims in pub- rapid publication on acceptance lished maps and institutional affiliations. support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Animal Diseases Springer Journals

Lactobacillus plantarum A3 attenuates ulcerative colitis by modulating gut microbiota and metabolism

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Abstract

Antibiotics are widely used to treat various diseases. However, growing evidence indicates that antibiotic therapy in human life increases the incidence of inflammatory bowel disease (IBD). Therefore, we need appropriate methods to reduce the incidence or symptoms of IBD. In this study, we used lincomycin hydrochloride to construct a gut micro- bial dysbiosis model in mice, and then, constructed an ulcerative colitis (UC) model. Meanwhile, we used Lactobacillus plantarum A3 from equine to treat UC in mice with gut microbial dysbiosis. The results showed that lincomycin hydro- chloride had little effect on the small gut microbiota in mice, but had a more destructive effect on the large intestin. Lactobacillus plantarum A3 alleviated the symptoms of UC in mice, which was reflected in its significantly reduced spleen index and disease activity index (DAI) (p < 0.05), inhibited the shortening of colon and alleviated the invasion of inflammatory cells in the colon. Moreover, we found that it played a mitigatory role by inhibiting oxidative stress and regulating inflammatory cytokines in mice. At the same time, it restored the diversity and composition of the colonic microbiota and significantly increased the abundance of beneficial bacteria such as Blautia and Akkermansia (p < 0.05); Notably, it significantly increased the concentrations of arachidonoyl ethanolamide phosphate (AEA-P) and cortisone (p < 0.05) which have analgesic and anti-inflammatory effects. In conclusion, our study found that Lactobacillus plan- tarum A3 has the potential to regulate UC in mice with gut microbial dysbiosis. Keywords Antibiotics, Gut microbiota, Lactobacillus plantarum, DSS, Ulcerative colitis, Untargeted metabolomics Introduction Inflammatory bowel disease (IBD) is a nonspecific chronic intestinal inflammatory disease, that includes ulcerative colitis (UC) and Crohn’s disease (CD). UC mainly occurs in the colon and rectum, and spreads to the cecum as the *Correspondence: Yaoqin Shen disease progresses, while CD can occur anywhere in the yshen@mail.hzau.edu.cn intestine, generally from superficial ulcers to deep spread College of Veterinary Medicine, Huazhong Agricultural University, (Imhann et  al. 2018). On the one hand, IBD is difficult to Wuhan 430070, People’s Republic of China Faculty of Veterinary and Animal Sciences, The Islamia University cure, prone to recurrent attacks and has a risk of carcino- of Bahawalpur, Bahawalpur 63100, Pakistan genesis at a later stage; on the other hand, IBD has grad- Key Laboratory of Environment Correlative Dietology, Interdisciplinary ually become a disease with an increasing incidence in Sciences Institute, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, China modern society. Not only are there more cases in devel- State Key Laboratory of Agricultural Microbiology, Huazhong oped countries such as Europe and the United States, Agricultural University, Wuhan, China but the incidence is also increasing in regions with faster © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Qin et al. Animal Diseases (2023) 3:16 Page 2 of 18 economic development such as Asia, Africa and South jejunum, ileum, cecum, colon and rectum. Compared with America (Glassner et  al. 2020). Therefore, IBD has gained the C_LCM group, the number of OTUs in the duode- increasing attention. Unfortunately, the real cause of IBD num, jejunum and ileum did not change much, and even has not been explored. the number of OTUs in the duodenum increased (Fig. S1 Recently, it has been demonstrated that antibiot- A-C). However, the rectum decreased significantly after ics may be one of the causes of IBD. A study involving exposure to lincomycin hydrochloride in the LCM group 107,2426 research projects and nearly 660,000 follow-up (p < 0.01) (Fig.  1A-C). Regarding the alpha diversity, there results revealed that the incidence of IBD in children who was no significant change in Chao 1, Shannon and Simp - were  unexposed and exposed to antibiotic treatment was son indexes of duodenum, jejunum and ileum in the LCM 0.83‰ and 1.52‰, respectively, indicating that children group, cecal, colonic and rectal microbiota were signifi - exposed to antibiotic treatment had an 84% increased risk of cantly decreased (p < 0.05) (Fig.  1D-F, Fig. S1 d-f ). These IBD in later stages. In addtiton, it has been proven that the results indicated that lincomycin hydrochloride had a occurrence of IBD is negatively correlated with age (Kro- greater effect on the number and diversity of microbiota nman et al. 2012). Consistent with studies in children, adults in the large intestine of mice than in the small intestine of exposed to antibiotic therapy also have an increased risk of mice. IBD. An investigation covering 23,982 IBD patients (15,951 Based on the results of OTUs and alpha diversity analy- with UC, 7898 with CD, and 133 with unnamed IBD) span- sis, we focused our analysis on beta diversity of cecal, ning 10 years indicated that patients who used antibiotics colonic and rectal microbiota in mice. PCoA and ANO- for more than 1 year had 88, 74, and 127% increased risk of SIM analysis were performed by calculating Bray-Curtis, IBD, UC and CD compared with patients who did not use weighted UniFrac and unweighted UniFrac distances. antibiotics (p < 0.001) (Nguyen et al. 2020). In addition, after The results showed that there were significant differ - mice were intervened by antibiotics, their gut microbiota ences in beta diversity between the LCM group and the was changed, and their sensitivity to dextran sulfate sodium C_LCM group (p < 0.01) (Fig. 1G-I, Table S3), which indi- (DSS) was increased, which means that they were more cated that lincomycin hydrochloride significantly altered likely to induce UC (Ozkul et al. 2020). Therefore, based on the microbiota structure of the mouse cecum, colon and gut microbial dysbiosis, it is very important to use appro- rectum. The above results showed that lincomycin hydro - priate drugs and preparations to restore gut microbiota, and chloride had little effect on the microbiota of the small reduce the incidence or symptoms of IBD. intestine (duodenum, jejunum and ileum), but had more Studies have shown that probiotics can not only alter destructive effect on the large intestine (cecum, colon the expression of inflammatory cytokines in the body and and rectum) after intervention in mice. reduce the severity of inflammation (Yu et  al. 2020), but also change the gut microbiota such as the number, activity, and microbiota composition (Ma et al. 2020; Li et al. 2021a, Lactobacillus plantarum A3 can alleviate the symptoms 2021b, 2021c). Currently, probiotics have been used as IBD of ulcerative colitis in mice therapy to modulate the gut microbiota of the host (Biagioli To further study the effect of equine Lactobacillus plan - et al. 2020; Oka and Sartor 2020). This suggests that probiot - tarum A3 on ulcerative colitis in mice with gut micro- ics may reduce the incidence of IBD or alleviate symptoms bial dysbiosis, clinical indexes of mice  were analyzed of IBD when used in IBD patients with gut microbial dys- (Fig.  2A). During the 7-day period of DSS intervention, biosis. Lactobacillus plantarum A3 is a probiotic with good body weight changes and DAI index of mice in each properties  that has been isolated and screened in healthy group were monitored, and the results showed that adult horses, and has a significant recovery effect on DSS- except for the control group, which showed a stable induced UC in mice (Qin et al. 2022). Therefore, the aim of and slow upward trend, the body weight of mice in the this study was to investigate the regulatory effect of Lacto - other three groups showed a downward trend with DSS bacillus plantarum A3 from equine on UC in mice with gut intervention, and more obvious over time, and eventu- microbial dysbiosis and provide a reference for subsequent ally, body weight decreased to the lowest level on the last screening of potential mitigatory agents. day (Fig.  2B). In addition, except for the control group, the DAI index of mice tended to be stable, and  the DAI Results index of mice in the other three groups showed upward Lincomycin had a more destructive effect on the large gut trend over time. Notably, the increasing trend of DAI microbiota in mice in the DSS group was the fastest, and at the end of the To establish a mouse model of gut microbial dysbiosis, experiment, the DAI index in the DSS group was signifi - we treated the mice with lincomycin hydrochloride and cantly higher than that in other groups (p < 0.05) (Fig. 2C, analyzed the changes in gut microbiota in the duodenum, D). These results also showed that both Lactobacillus Qin  et al. Animal Diseases (2023) 3:16 Page 3 of 18 A B C G H bray_curtis 1.0 bray_curtis 1.0 C_LCM LCM C_LCM LCM C_LCM LCM 0.5 0.5 92 28 23 2 LCM04 LCM04 322 33 23 LCM06 336 23 17 LCM05 LCM06 LCM05 C03 C01 C02 0.0 C01 0.0 C03 C02 C05 C04 LCM03 C04 C06 LCM01 C05 LCM01 C06 LCM03 LCM02 LCM02 -0.5 -0.5 -1.0 6 ** 1.0 250 ** ** -0.5 0.0 0.5 -0.5 0.0 0.5 0.8 200 Axis.1 [62.1%] 0.6 Axis.1 [56.9%] 0.4 weighted_unifrac weighted_unifrac 50 0.2 0.4 0.5 0 0 0.0 C04 C01 LCM01 C02 LCM03 -1.0 C01 C04 LCM02 -0.5 0.0 0.5 LCM02 Axis.1 [56.9%] LCM03 C06 LCM01 C06 0.0 0.0 C05 C03 C02 C05 C03 LCM05 LCM05 LCM04 LCM04 -0.4 LCM06 LCM06 -0.5 250 6 1.0 ** ** * 200 0.8 -0.8 -1.0 150 0.6 -0.5 0.0 0.5 1.0 0.0 0.5 Axis.1 [71.9%] Axis.1 [64.9%] 100 0.4 50 0.2 unweighted_unifrac unweighted_unifrac 1.0 0 0 0.0 0.5 LCM03 0.5 F LCM06 200 ** 5 ** 1.0 * LCM01 LCM02 LCM05 C02 LCM04 C03 0.0 C01 C05 C04 4 0.8 C06 0.0 C05 C02 C06 LCM05 LCM03 C03 C04 3 0.6 C01 LCM06 LCM02 LCM04 100 -0.5 LCM01 -0.5 2 0.4 1 0.2 -1.0 -1.0 0 0 0.0 -0.50 0.50 0.0 0.5 0.00 0.25 0.5 -0.25 Axis.1 [62.5%] Axis.1 [57.3%] 1.0 bray_curtis weighted_unifrac 0.6 unweighted_unifrac 0.5 0.5 LCM05 LCM04 LCM06 LCM05 0.3 LCM06 LCM04 LCM06 LCM04 C_LCM LCM05 C02 C06 C04 LCM 0.0 C03 0.0 a C01 LCM02 C01 C05 C05 LCM01 C03 C04 0.0 C05 LCM03 LCM03 LCM03 C02 C06 LCM02 LCM01 LCM01 C02 LCM02 -0.5 C03 C01 -0.5 -0.3 C06 C04 -1.0 -0.5 0.0 0.5 0.0 -0.8 -0.4 0.0 0.4 -0.50 -0.25 0.00 0.25 0.50 Axis.1 [59.9%] Axis.1 [54.7%] Axis.1 [65.8%] Fig. 1 Lincomycin had a more destructive effect on the large gut microbiota in mice. A-C Venn diagram (A: Cecum; B: Colon; C: Rectum); D-F Alpha ns diversity (D: Cecum; E: Colon; F: Rectum); p > 0.05, *p < 0.05, **p < 0.01; G-I Based on PCoA, beta diversity of different intestinal segment contents in mice (G: Cecum; H: Colon; I: Rectum) plantarum A3 and butyrate slowed the increasing trend mice in the LP_DSS group were similar to the control of the  DAI index. After the experiment, the mice were group. This showed that Lactobacillus plantarum A3 euthanized and dissected. The results showed that com - can prevent the occurrence of UC, and  more evidence pared with the control group, the spleen index of mice appeared in the longer colon length and lighter color of in the DSS group was significantly increased (p < 0.05) colonic contents in the LP_DSS group (Fig. 2F). (Fig.  2E), but the colon length was significantly shorter After mouse colon tissue was stained by H&E, the (p < 0.05) (Fig.  2F). The spleen index and colon length of colonic injury  were  further observed. Compared with LCM LCM _LCM LCM _LCM LCM _LCM _LCM LCM C_LCM LCM LCM C_LCM C_LCM LCM LCM C_LCM LCM Chao1 Chao1 Chao1 Axis.2 [23.2%] Shannon Shannon Shannon Axis.2 [39.3%] Simpson Simpson Simpson Axis.2 [23.6%] Axis.2 [16.3%] Axis.2 [20.9%] Axis.2 [21%] Axis.2 [19.6%] Axis.2 [26.4%] Axis.2 [21.2%] Qin et al. Animal Diseases (2023) 3:16 Page 4 of 18 Fig. 2 Lactobacillus plantarum A3 can alleviate the clinical symptoms of ulcerative colitis in mice with gut microbial dysbiosis. A Mouse model of ulcerative colitis in mice with gut microbial dysbiosis; B Weight changes; C Changes in DAI score; D DAI index of mice in each group after 7 days of DSS intervention; E Spleen index changes; F Colon length changes among the four groups, and the colon morphology of DSS group and DSS group mice; G H&E staining results and histology scores, the red arrow points to the infiltration of inflammatory cells, the blue arrow points to the ns shedding of mucosal epithelial cells; p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; n = 10 Qin  et al. Animal Diseases (2023) 3:16 Page 5 of 18 the control group, a large number of inflammatory Similarly, Lactobacillus plantarum A3 can inhibit the cells invaded the mucosal and submucosal layers of the inflammatory response at gene and protein levels. colonic tissue, and many colonic mucosal epithelial cells To further explore the expression of Foxp3 at the detached in the DSS group. After treatment with Lacto- protein level, immunohistochemical analysis was  per- bacillus plantarum A3 or butyrate, no colonic mucosal formed  (Fig.  4F). Distribution of the brownish yel- epithelial cell detachment was observed in colonic tissue, low positive signals indicated that Foxp3 was mostly but some colonic mucosal and submucosal layers still had expressed in the mucosa and submucosa of the  colon. inflammatory cell invasion, and the pathological score After DSS inducing, the mean optical density (MOD) of was also significantly decreased (p < 0.001) (Fig.  2G), sug- Foxp3 in colon tissue increased significantly (p < 0.05), gesting that Lactobacillus plantarum A3 can reduce DSS which means that its expression increased. When mice damage to colonic tissue. were treated with Lactobacillus plantarum A3, the expression of Foxp3 in their colonic tissue was further Lactobacillus plantarum A3 can regulate the serum increased (p < 0.001). These results  showed that DSS biochemical indexes of mice induced a feedback regulatory immune response in mice, Serological parameters can often more intuitively illus- while Lactobacillus plantarum A3 further enhanced the trate the degree of damage to body cells. Thus, we studied immune response. the serum biochemical indexes in each group. The results showed that, compared with the control group, both Lactobacillus plantarum A3 regulates the colonic MPO and MDA were significantly upregulated (p < 0.001), microbiota in mice but SOD was significantly downregulated (p < 0.001) in To investigate the regulatory effect of Lactobacillus plan - the DSS group. Compared with the DSS group, MPO and tarum A3 on UC in mice with gut microbial dysbiosis, a MDA in the LP_DSS group were significantly decreased high-throughput gene sequencing of 16S rDNA was per- (p < 0.05), while SOD was significantly increased (p < 0.05) formed in the colonic content microbiota in each group (Fig. 3A-C), and the concentration was closer to the con- of mice. For the alpha diversity, compared with the  DSS trol mice. This indicated that Lactobacillus plantarum A3 group, the Shannon and Simpson indexes of colonic con- alleviated the degree of cell damage in mice. tent microbiota increased significantly in the LP_DSS group (p < 0.001), but the  Chao 1 index did not increase Lactobacillus plantarum A3 can regulate cytokines significantly (p > 0.05) (Fig.  5A). These results  showed expressed in mouse colon tissue that A3 can significantly restore the community diversity Inflammation in the body is often accompanied by of intestinal microorganisms in mice, but its restoration changes in inflammatory cytokines. In DSS-induced effect on community richness is limited (Dill-McFarland mice, the expression of anti-inflammatory cytokine et  al. 2019; He et  al. 2017). For the beta diversity, com- such as IL-1β, IL-6, TNF-α and IFN-γ were significantly pared with the DSS group, Bray-curtis, unweighted Uni- increased at the gene level (p < 0.001) (Fig.  3D-G), while Frac and weighted UniFrac distances were significantly anti-inflammatory cytokine like IL-10 were signifi - changed in the LP_DSS group (p < 0.01) (Fig.  5B, Table cantly decreased (p < 0.001) (Fig. 3H). In addition, Foxp3, S3), which indicated that Lactobacillus plantarum A3 the main regulator of Treg cells, was also significantly could significantly affect intestinal bacteria in the colon increased (p < 0.05) (Fig.  3I). These showed that DSS of mice. promotes the inflammatory response at the gene level. To further investigate which bacterium was specifi - When Lactobacillus plantarum A3 was involved in the cally regulated by Lactobacillus plantarum A3, we con- regulation, IL-1β, IL-6, TNF-α and IFN-γ were signifi - ducted an analysis between the DSS group and LP_DSS cantly decreased at the gene level (p < 0.05) (Fig.  3D-G), group  were conducted. At the phylum level, the four while IL-10 increased significantly, rather than decreased phyla with the highest abundance of colonic contents (p < 0.001) (Fig. 3H). Besides, A3 significantly upregulated in the two groups were Bacteroides, Firmicutes, Proteo- the expression of Foxp3 (p < 0.001) (Fig.  3). In summary, bacteria and Verrucomicrobia. The relative abundance Lactobacillus plantarum A3 can inhibit the progression of each phylum was 63.80, 14.40, 15.77 and 6.03% in the of inflammatory responses at the gene level. DSS group, and 50.38, 22.84, 16.64 and 10.14% in the The expression of these cytokines at the protein LP_DSS group (Fig.5, C). The results showed that A3 was level  were also interrogated  by western blot, which was involved in regulating each phylum. At the genus level, consistent with their trend at the gene level (Fig.  4A- Bacteroides (63.80%), Enterobacter (9.32%) and Blau- D), indicating that DSS can promote the inflammatory tia (8.26%) were the top three microbiota in the DSS response of mice at both the gene and protein levels. group, Bacteroides (50.38%), Enterobacter (15.33%) and Qin et al. Animal Diseases (2023) 3:16 Page 6 of 18 Fig. 3 Lactobacillus plantarum A3 can regulate the biochemical indexes and cytokine gene expression of ulcerative colitis in mice with gut microbial dysbiosis. A-C Serum biochemical indexes (A: MPO; B: SOD; C: MDA); D-I Cytokine gene expression level in colonic tissue (D: IL-1β; E: IL-6; F: TNF-α; G: IFN-γ; H: IL-10; I: Foxp3); *p < 0.05, **p < 0.01, ***p < 0.001; n = 10 Akkermansia (10.14%) were the top three microbiota in compared with the DSS group, but Bacteroidetes was sig- the LP_DSS group which meant that Lactobacillus plan- nificantly downregulated (p < 0.05) (Fig. 5E). At the genus tarum A3 changed the microbiota with high abundance, level, the abundance of Blautia, Akkermansia, Citrobac- which was consistent with the beta diversity analysis ter and Flexispira was significantly upregulated (p < 0.05) (Fig. 5D). in the LP_DSS group, but Bacteroides was significantly LEfSe confirm the specific bacteria that had significant downregulated (p < 0.05) (Fig.  5F). In general, Lactobacil- changes between the two groups. At the phylum level, lus plantarum A3 treatment significantly transformed the the abundance of Firmicutes and Verrucomicrobia was diversity and composition of gut microbiota. significantly upregulated in the LP_DSS group (p < 0.05) Qin  et al. Animal Diseases (2023) 3:16 Page 7 of 18 Fig. 4 Lactobacillus plantarum A3 can regulate the biochemical indexes and cytokine expression of ulcerative colitis in mice with gut microbial dysbiosis. A-E Expression of cytokines at protein level in colonic tissue was measured by western blot in each group (A: IL-1β; B: IL-6; C: TNF-α; D: IFN-γ; E: IL-10); F Foxp3 in colonic tissue was measured by immunohistochemistry, and the mean optical density was calculated in each group. *p < 0.05, **p < 0.01, ***p < 0.001 Lactobacillus plantarum A3 can regulate changes of colonic metabolites with significant differences between the two metabolites in mice groups. Untargeted metabolomic analysis can detect small Then, according to the above discriminant analy- molecule metabolites with a relative molecular weight sis model, the metabolites that play an important role of less than 1000 in the sample, from which we can in the discrimination process are analyzed. These obtain differential metabolites and metabolic pathways metabolites have the potential to be used as biomark- between the DSS group and the LP_DSS group. We ers between the two groups. Value importance in pro- used PCA to model each sample and found that there jection (VIP) > 1 was as the standard and screened 383 were no outlier points in the intragroup samples under metabolites in positive polarity mode and 285 metabo- both positive and negative polarity modes, which indi- lites in negative polarity mode. To further explore the cated that there was little difference in the intragroup differential metabolites, univariate analysis  were  also samples and could be used for the analysis of differential performed. p < 0.05 and fold change (FC) > 2 were metabolites (Fig. 6A-D). The factors that can distinguish used  as the screening criteria. The results showed sample grouping to the greatest extent can be found that, compared with the DSS group, there were 23 using PLS-DA, and the overfitting caused by the PLS- metabolites with more than 2 FC in the LP_DSS group DA model can be improved using OPLS-DA. There - (p < 0.05) in the positive polarity mode, of which ara- fore, we plotted the scatter diagram with the two best chidonoyl ethanolamide phosphate guanosine (AEA- discriminating factors and found that the sample point P) had the highest fold upregulation, reaching 28.70 clouds of the DSS group and the LP_DSS group were FC (p < 0.001), and four metabolites including seri- distributed in different regions, which indicated that lin and Cortisone were also upregulated by more the PLS-DA model and the OPLS-DA model had better than 10 FC (p < 0.001) (Fig .  6I, Table  1). In the nega- discrimination effects (Fig.  6E-H), and there should be tive polarity mode, there were nine metabolites with Qin et al. Animal Diseases (2023) 3:16 Page 8 of 18 Fig. 5 Lactobacillus plantarum A3 can regulate the gut microbiota of ulcerative colitis in mice with gut microbial dysbiosis. A Alpha diversity of ns colon contents in mice, p > 0.05, ***p < 0.001. B Beta diversity of colon contents in mice. C Bar plots of the phylum taxonomic levels in the DSS and LP_DSS groups; D Bar plots of the genus taxonomic levels in the DSS and LP_DSS groups; E-F Difference in dominant microorganisms of colon contents between the DSS group and LP_DSS group via distribution histogram based on LDA. The criteria for feature selection was log LDA score > 2.0 (E: at the phylum level; F: at the genus level) Qin  et al. Animal Diseases (2023) 3:16 Page 9 of 18 Fig. 6 Lactobacillus plantarum A3 can regulate the metabolite changes of colon contents in ulcerative colitis in mice with gut microbial dysbiosis. A-D PCA score diagram (A-B: under positive polarity mode; C-D: under negative polarity mode). E-F Scatter diagram of PLS-DA models (E: under positive polarity mode; F: under negative polarity mode); G-H Scatter diagram of OPLS-DA models and OPLS-DA models (G: under positive polarity mode; H: under negative polarity mode). I-J Univariate analysis of differential metabolites in mouse colonic contents between the DSS group and LP_DSS group (I: under positive polarity mode; J: under negative polarity mode), log2(FC) > 0 represents metabolites with high abundance in the LP_DSS group. K-L The metabolic pathways of differential metabolite enrichment by ORA (K: under positive polarity mode; L: under negative polarity mode) Qin et al. Animal Diseases (2023) 3:16 Page 10 of 18 Fig. 6 continued more than 2 FC upregulation in the LP_DSS group, metabolites that were significantly different between and the two metabolites with the highest upregula- the DSS group and the LP_DSS group (t  test, p < 0.05), tion were O-LPL-carnitine (p < 0.05, 5.712-fold) and we looked for the metabolic pathways in which these N2, N2-dimyl (p < 0.001, 5.06-fold) (Fig .  6J,  Table  2). metabolites appeared and calculated the p value and Enrichment analysis can help us find pathways with fold enrichment of ORA for these metabolic path- key roles and thus can predict the molecular mecha- ways (Fig.  6K-L). Impact  can be  calculated by topo- nisms of the entire regulatory process. Based on the logical analysis to judge the magnitude of the effect Qin  et al. Animal Diseases (2023) 3:16 Page 11 of 18 Table 1 Differential metabolites in mouse colonic contents of metabolites in metabolic pathways. Combined between the DSS group and LP_DSS group under positive enrichment and topological analysis, using p < 0.05 polarity mode (screening criteria: VIP > 1; p < 0.05; FC > 2) and Impact >0.2 as criteria, metabolic pathways sig- nificantly affected by differential metabolites from Differential metabolites VIP p FC the above metabolic pathways  were  selected: ubiqui- Arachidonoyl ethanolamide phos- 2.810 <0.001 28.70 none and other terpenoid-biosynthesisu (impact = 0.5, phate p < 0.05, fold enrichment = 26.24), phenylalanine, PS 170/171 1.945 0.0190 23.80 tyrosine and tryptophan (impact = 0.25, p < 0.05, fold PE 203/204 1.808 0.0220 13.77 enrichment = 19.68). The above results showed that Stercobilin 2.652 <0.001 11.09 Lactobacillus plantarum A3 affected  not only the Cortisone 2.779 <0.001 10.77 changes of metabolites in the  mouse colon, but also Nicotinamide 1.792 0.0360 6.486 the metabolic pathway. TAG 161-181-181 1.893 0.0136 5.250 Oxohongdenafil 2.377 <0.001 5.087 Discussion Pantethine 2.499 <0.001 4.827 Recent studies have found that early gut microbiota Abscisic acid 2.189 0.0041 4.060 imbalance can increase the incidence of diseases such as 5,8-dihydroxy-10-methyl-5,8,9,10- 1.922 0.0194 3.866 asthma (Arrieta et  al. 2015), IBD (Hviid et  al. 2011) and tetrahydro-2H-oxecin-2-one immune diseases (Round and Mazmanian 2009). How- 4-acetyl-4-ethoxycarbonylheptan- 2.591 <0.001 3.677 edioic acid ever, antibiotics are not only inevitably used in clinical Methyl EudesMate 1.787 0.0360 3.423 treatment, but have also become one of the most widely Gamma-Glu-Leu 1.803 0.0340 2.657 used drugs (Adriaenssens et  al. 2011; Vaz et  al. 2014). Therefore, restoring antibiotic-induced gut microbiota L-Tyrosine 1.761 0.0320 2.490 imbalance and preventing related diseases is a  key prob- methyl 3-1H-indol-3-yl-2-phenyl- 2.465 <0.001 2.476 methyleneaminopropanoate lems. UC is an idiopathic inflammatory bowel disease. gamma-Glutamylglutamic acid 1.807 0.0327 2.456 Its high incidence and lack of specific drugs are the rea - IQH 1.677 0.0392 2.240 sons why it has become a problem in modern medicine Genistein 4′-O-glucuronide 1.812 0.0240 2.219 (Fan et  al. 2019). Although the etiology is unknown, the 2.350 0.0013 2.192 N, N′-di4-2,6- phenomenon of gut microbial dysbiosis has been proven dimethylmorpholinophenylthiourea to appear before UC (Wu et al. 2020). In addition, when LMH 2.205 0.0046 2.128 mice were first treated with antibiotics, the stability of 4-2,3-dihydro-1,4-benzodioxin-6-ylb- 1.932 0.0157 2.060 gut microbiota was destroyed, their sensitivity to DSS utanoic acid increased, and finally, the symptoms of ulcerative coli - Muramic acid 2.004 0.0140 2.030 tis were more serious (Ozkul et  al. 2020). This suggests Add notes: VIP value importance in projection, FC fold change that timely treatment and restoration of the stability of gut microbiota after gut microbial dysbiosis may reduce IBD’s incidence or symptoms. As a kind of probiotic, Lactobacillus has been proven to maintain and restore the homeostasis and stable heredity of gut microbiota (Ma et al. 2020), and it also has a certain Table 2 Differential metabolites in mouse colonic contents therapeutic effect on IBD (including UC and CD) (Guo between the DSS group and LP_DSS group under negative et al. 2019; Wang et al. 2020). Lactobacillus plantarum A3 polarity mode (screening criteria: VIP > 1; p < 0.05; FC > 2) is a lactobacillus that has good probiotic characteristics Differential metabolites VIP p FC isolated and screened from healthy adult horses, and it has a certain alleviating effect on UC in mice (Qin et al. 2022), O-Acetyl-L-carnitine 2.800 0.0319 5.712 Therefore, in this study, we used Lactobacillus plantarum N2, N2-dimethylguanosine 2.658 <0.001 5.057 A3 as the main therapeutic drug to explore its regula- 20-Carboxy-Leukotriene B4 2.226 0.0340 3.386 tory effect on colitis in mice with gut microbial dysbiosis. 3,4-Dihydroxyphenylglycol 2.010 <0.001 3.233 In addition, as a short chain fatty acid (SCFA), butyrate Pyrogallol 2.176 0.0111 2.843 can not only be the main energy source of colonic epi- W-Lactose 2.187 0.0402 2.513 thelial cells, but also regulate epithelial stem cells (Kaiko 8-iso-15-keto Prostaglandin E2 2.109 0.0476 2.259 et  al. 2016), and promote epithelial homeostasis together Mono2-ethylhexyl phthalate MEHP 2.100 0.0194 2.163 with other SCFAs (Macia et  al. 2015). Therefore, when Hexadecanedioic acid 1.998 0.0262 2.150 Add notes: VIP value importance in projection, FC foldchange Qin et al. Animal Diseases (2023) 3:16 Page 12 of 18 constructing the mouse UC model, we used butyrate as a of lipid peroxidation in the body. SOD is an antioxidant positive control of treatment. enzyme, that  can catalyze free radicals into oxygen and In the first stage, lincomycin hydrochloride was used hydrogen peroxide (Yu et al. 2020; Li et al. 2023). There - to establishe a mouse model of gut microbial dysbiosis. fore, the contents of MDA and SOD in the body are often In clinical practice, lincomycin hydrochloride is com- negatively correlated. Our study showed that Lactobacil- monly used to treat various bacterial infections. It can lus plantarum A3 could significantly upregulate the level inhibit the growth of bacteria by inhibiting the synthe- of SOD and downregulate the level of MDA (p < 0.05), sis of proteins in bacteria, and it has strong antibacte- indicating that they can play a regulatory role through rial and bactericidal effects on Gram-positive bacteria, antioxidation. especially anaerobic bacteria (Geddes et al. 1964). Recent At present, it is generally believed that patients with studies have shown that short-term overdose of linco- UC have intestinal mucosal inflammation and immune mycin hydrochloride can lead to microbiota imbalance, abnormalities (Medzhitov 2008). MPO exists in neutro- intestinal tissue damage and immune function decline in phils, and its activity is directly proportional to the popu- mice (Li et al. 2019; Lv et al. 2017). Therefore, lincomycin lation of neutrophils in the inflammatory area (Bastaki hydrochloride was used as an intervention drug, and its et al. 2018). Therefore, the significantly increased content effect on each gut microbiota of mice was explored. The of MPO in the DSS group also suggested the presence of results showed that lincomycin hydrochloride had no sig- excessive neutrophil invasion in mice, which was consist- nificant effect on the microbiota of the small intestine in ent with the results of H&E staining in colon tissue. The mice, but it had a significant damage on the microbiota aggravation of oxidative stress and the increase of neu- of the large intestine, not only the number, but also the trophils in the body can further stimulate the release of structure. immune cytokines associated with UC (Leppkes and Based on the constructed mouse gut microbial dysbio- Neurath 2020). For example, oxidative stress can not only sis model, we induced UC in mice with DSS and explored stimulate macrophages to release IFN-γ, but also induce the regulatory effect of Lactobacillus plantarum A3. the release of TNF-α, IL-6, IL-1β and other inflamma - UC often requires some specific indicators to evaluate, tory factors, further causing intestinal mucosal metabolic such as spleen index, colon length and DAI index. Sple- disorders and intestinal mucosal inflammatory immune nomegaly is one of the common symptoms in UC mice disorders, which can lead to systemic inflammatory (Zhang et al. 2017). Colon length and DAI index are often response syndrome and multiple organ dysfunction (Li used to evaluate the severity of ulcerative colitis in clini- et  al. 2021a, 2021b, 2021c; Vlahakos et  al. 2012). In our cal diagnosis and treatment (El Sayed and Sayed 2019). In study, the expression levels of IFN-γ, TNF-α, IL-6, and addition, pathological tests are often used as one of the IL-1β in the DSS group were significantly higher than gold standards for diagnosing the symptoms of ulcera- those of the control group at both gene and protein lev- tive colitis (Allen et  al. 2012; Meira et  al. 2008). After els (p < 0.001), which indirectly indicated abnormalities in DSS induction, the spleen index and DAI index of mice intestinal mucosal inflammation and immune response were significantly increased (p < 0.05), and the colon was in mice. As an anti-inflammatory cytokine, IL-10 can significantly shortened (p < 0.05). H&E staining showed inhibit the secretion of proinflammatory cytokines such that there were a large number of inflammatory cells in as IFN-γ, TNF-α, IL-6 and IL-1β, regulate the differen - the mucosal layer and submucosal layer in the colon tis- tiation and proliferation of macrophages, T cells and B sue, accompanied by a large number of mucosal epithe- cells, inhibit Th1/Th17 responses, which are essential lial cells falling off. The above results showed that DSS for intestinal immune homeostasis (Bárcena et al. 2019). successfully induced ulcerative colitis in mice with gut Lactobacillus plantarum A3 promoted the expression microbial dysbiosis. When mice were treated with Lac- of IL-10 and inhibited IL-1β, IL-6, TNF-α and IFN-γ, tobacillus plantarum A3, the spleen index and DAI index which further proved that it can effectively regulate intes - of mice decreased significantly (p < 0.05), the colon length tinal inflammation and intestinal immune response in was closer to that of  the control group, and H&E stain- mice. Foxp3 is considered to be a specific transcriptional + + + + ing showed that the degree of inflammatory cell invasion marker of CD4 CD25 regulatory T cells (C D4 CD25 decreased significantly. The above results showed that Treg), which plays an important role in the proliferation, Lactobacillus plantarum A3 could alleviate the clinical differentiation and immune function of Treg cells (Jia symptoms of ulcerative colitis in mice with gut microbial et  al. 2020; Zhou et  al. 2020). Moreover, recent studies dysbiosis. have also shown that the expression of Foxp3 is positively Oxidative stress is closely related to ulcerative colitis. correlated with the expression of programmed cell death MDA is the end product of the peroxidation reaction ligand (PD-L1), and PD-L1 mediated signal transduction between free radicals and lipids. It reflects the degree pathway is one of the main processes regulating intestinal Qin  et al. Animal Diseases (2023) 3:16 Page 13 of 18 local inflammatory response (Yamamoto et  al. 2022). upregulating the abundance of beneficial bacteria such as This illustrated that Lactobacillus plantarum A3 can reg - Blautia and Akkermansia. ulate the immune response and inflammatory response Intestinal metabolites can regulate the intestinal envi- of mouse intestine by upregulating Foxp3. ronment together with gut microbiota. Compared with The severity of UC is related to the decline in the diver - the DSS group, the colonic metabolites in the LP_DSS sity of intestinal microorganisms, such as the relative group were significantly changed, with the highest up- decrease in the abundance of Clostridium and Akker- regulation of arachidonoyl ethanolamide phosphate mansia (Pittayanon et  al. 2020; Zhou et  al. 2018), which (AEA-P), reaching 28.70-fold (p < 0.001). AEA-P is a is consistent with the changes in the DSS group in our newly discovered endogenous lipid, a precursor of ara- study. Lactobacillus plantarum A3 significantly restored chidonoyl ethanolamide (AEA), and its function is simi- the alpha diversity and beta diversity of intestinal micro- lar to AEA. As an “endocannabinoid” (which has similar organisms in mice (p < 0.05), regulated the microbiota pharmacological effects to the active ingredient of can - structure, and increased the relative abundance of Blau- nabis: Δ -tetrahydro-cannabinol), AEA is often released tia, Akkermansia, Citrobacter and Flexispira. Blautia is from depolarized neurons, endothelial cells, and mac- an anaerobic bacterium with probiotic characteristics. rophages in the body, and then participates in cellular According to the analysis of phenotype and phylogeny, activities as an endogenous ligand for cannabinoid recep- some species of Clostridium and Ruminococcus have tors in the central nervous system (CB1 subtype) and been reclassified to this genus. Blautia plays an impor- peripheral immune cells (CB2 subtype). Besides, it can be tant role in regulating the growth of gut microbiota and selectively absorbed by cells, and then degraded by fatty inflammation. This is mainly because Blautia has the acid amide hydrolase (FAAH) to ethanolamine and ara- ability to produce bacteriocin, which makes it have the chidonic acid (AA), further playing an anti-inflammatory potential to inhibit the colonization of pathogenic bac- role. It is worth mentioning that AEA can also reduce teria in the intestine, especially Clostridium perfringens pain at the site of tissue injury (Maccarrone et al. 2000). and vancomycin-resistant enterococci (Liu et  al. 2021; Li These may be the reasons for the significant upregulation et  al. 2021a, 2021b, 2021c). In addition, Blautia plays an of AEA-P concentration in group A mice. In addition, important role in obesity related diseases. It is the only Lactobacillus plantarum A3 also increased the concen- bacterium negatively related to visceral fat accumulation trations of cortisone in the colon of mice by 10.77-fold (Ozato et al. 2019; Song et al. 2014). As a mucin decom- (p < 0.001). Cortisone is a type of glucocorticoid, that posing bacterium in the body, Akkermansia widely exists was used to treat IBD as early as 1955 (Singh 2018). It in the intestinal tract of animals. It obtains a competitive can regulate cytokines in the body, such as inhibiting the advantage by improving the utilization rate of mucin in release of pro-inflammatory cytokines IL-6, IFN-γ and the body, so it can be highly colonized in the intestinal TNF-α, while stimulating the production of anti-inflam - tract, and it can alleviate the inflammatory reaction by matory cytokine IL-10, which shows that the increase in reducing the permeability of the intestinal tract and the cortisone concentration is also one of the reasons for the infiltration derived pro-inflammatory lipopolysaccharide changes of cytokine in mice. Today, there are still studies (Bárcena et  al. 2019; Depommier et  al. 2019; Kim et  al. on synthetic cortisone analoges for improving the stabil- 2022). Citrobacter and Flexispira are common microbi- ity of drugs and optimizing their efficacy in treating IBD ota in the intestine. There are few studies on their rela - (Rol et al. 2021). Therefore, the reason why Lactobacillus tionship with UC. Some studies have intervened with plantarum A3 induced a significant increase in intes - Citrobacter rodentium to induce colitis in mice and found tinal cortisone might be one of the priorities in further that it can reduce the weight of mice and cause diarrhea research. Through enrichment analysis and topological (Bhinder et  al. 2013). However, in our study, it was not analysis, it was also found that Lactobacillus plantarum found that after the intervention of Lactobacillus plan- A3 significantly affects ubiquinone and other terpenoid tarum A3, the weight of mice was less than that of the quinone biosynthesis, phenylalanine, tyrosine and tryp- DSS group and there was no diarrhea, which indicated tophan biosynthesis. These pathways predict the intes - that not all Citrobacter can induce disease. The limita - tinal metabolic activities that Lactobacillus plantarum tions of the genus level made it impossible for us to fur- A3 may have participated in. In further studies, we can ther analyze it, which also provided a new idea  for our explore the specific mechanism by which Lactobacillus future research. In general, Lactobacillus plantarum A3 plantarum A3 regulats UC in mice with gut microbial had the ability to regulate gut microbiota in mice. It not dysbiosis by verifying these metabolic pathways. only increased the diversity of gut microbiota, but also further regulated the inflammatory response in  vivo by Qin et al. Animal Diseases (2023) 3:16 Page 14 of 18 Conclusion bodyweight), and mice in the DSS group were intragas- In this study, we successfully constructed a gut microbial trically administered an equal volume of normal saline. dysbiosis model in mice by lincomycin hydrochloride and Notably, on the last 7 d of the experiment, except for the found that it had a more significant destructive effect on control group, which maintained normal drinking water, the microbiota of the large intestines. Subsequently, we the other three groups of mice were given drinking water constructed a UC model on the basis of gut microbial containing 3% (w/v) DSS, in which freshly prepared DSS dysbiosis in mice and found that Lactobacillus plantarum solution was changed every 2 d (Fig.  2A). At the end of A3 could alleviate the clinical symptoms of mice and the experiment, mice were euthanized, and serum, spleen achieve a mitigatory effect by regulating the expression of tissue and colon tissue, colon contents were collected to cytokines, affecting the structure of gut microbiota and subsequent studies. regulating the level of intestinal metabolites. Intestinal contents genomic DNA extraction and 16S-rRNA Methods sequencing Animals The intestinal contents of mice in each group were sub - Five-week-old male C57BL/6 mice were  obtained from jected to total DNA extraction by the  E.Z.N.A. soil kit the animal center of Huazhong Agricultural Univer- (Omega Biotek, Norcross, GA, U.S.). After ensuring the sity (Wuhan, China). They were housed under specific eligibility of the DNA, the V3-V4 variable region was pathogen-free conditions and had access to food and amplified by PCR with 338F (5′-ACT CCT ACG GGA water ad  libitum at a temperature of 25±2°C, relative GGC AGC AG-3′) and 806R (5′-GGA CTA CHVGGG humidity of 40-60% (12:12-hour light/dark cycle). All TWT CTA AT-3′) primers, and the amplification pro - animal experimental protocols (HZAUMO-2021-0068 gram was: 95°C for 3 min, 27 cycles (95°C for 30 s, 55°C and HZAUMO-2021-0138) were approved by the Eth- for 30 s, 72°C for 30 s), and 72°C for 10 min. The result - ics Committee of Huazhong Agricultural University ing PCR products were purified using the AxyPrep DNA (Wuhan, China) and performed following the guidelines Gel Extraction Kit (Axygen Biosciences, Union City, CA, of the National Institutes of Health Guide for the care USA). Standard operating procedures for generating and Use of Laboratory Animals. sequencing libraries from the purified amplified frag - ments. Then, purified amplicons were pooled in equi - Mouse model of gut microbial dysbiosis molar  amounts and paired-end sequenced (2 × 300) on C57BL/6 mice were randomly divided into two groups: an Illumina MiSeq platform (Illumina, San Diego, USA). the  control-antibiotic group (C_LCM group) and Quality control and species annotation were conducted the  antibiotic intervention group (LCM group). The by previous methods (Bokulich et al. 2018; Callahan et al. first 3 d, mice were adaptive feeding, and then, mice in 2016). Any contaminating mitochondrial and chloroplast the LCM group were given lincomycin hydrochloride sequences were filtered using the QIIME2 feature-table (30 mg/10 g body weight) for 5 d. Mice in the C_LCM plugin. Venn diagram was drawn to analyze the unique group were given normal saline as  a control. After that, or common OTUs between different groups. Alpha the mice were euthanized and the intestinal contents of diversity, including the  Chao1, Shannon and Simpson duodenum, jejunum, ileum, cecum, colon and rectum indexes, was calculated to estimate the microbial diver- were taken. sity within an individual sample. Beta diversity, including BrayCurtis, weighted UniFrac and unweighted UniFrac Mouse model of ulcerative colitis was determined to investigate the structural variation in C57BL/6 mice were randomly divided into four groups microbial communities across samples and then visual- (n = 15): Control-DSS group (Control group), DSS group, ized via principal coordinate analysis (PCoA) (Vázquez- Lactobacillus plantarum A3-DSS group (LP_DSS group) Baeza et al. 2013). Linear discriminant analysis effect size and butyrate-DSS group (BA_DSS group, as a positive (LEfSe) was employed to identify the bacteria with differ - control group). The experimental period was 22 d, in ent abundances among samples and groups (Segata et al. which the first 3 d were adaptive feeding. In the follow- 2011). ing 5 d, mice in the DSS group and LP_DSS group were intragastrically administered lincomycin hydrochloride Clinical indicator analysis (30 mg/10 g bodyweight). In the remaining 14 d, mice During the experiment, mental status, weight, fecal shape in the LP_DSS group were intragastrically administered and fecal occult blood for mice were recorded every day. Lactobacillus plantarum A3 (5 × 10 CFU/10 g body- The disease activity index (DAI) was scored based on the weight), mice in the BA_DSS group were intragastrically weight loss rate, fecal score and occult blood score of administered an equal volume of butyrate (30 mg/10 g Qin  et al. Animal Diseases (2023) 3:16 Page 15 of 18 mice. The score range for each parameter is 0-4 points, membrane was rinsed with blocking solution contain- and the sum for the parameters is the final score (up to 12 ing 5% skimmed milk for 2 h at room temperature and points) (Table  S1) (Liu et  al. 2020). After the mice were incubated with primary antibody overnight at 4°C. After euthanized, the colon length and the weight of the mice the membranes were incubated with secondary antibody in each group were measured, and the spleen index was for 1 h at room temperature, the proteins were visualized calculated. using an enhanced chemiluminescence (ECL) system (Thermo Scientific, USA) and analyzed with ImageJ soft - Histology ware. Antibodies are listed in Supplementary materials. The colonic tissue sections of mice in each group were taken and the feces were washed with 0.9% sterile nor- Immunohistochemistry mal saline, fixed in 4% paraformaldehyde, dehydrated After the dewaxing of paraffin sections, the antigens were with ethanol, and processed into a paraffin-embedded repaired by microwave heating and endogenous per- block, cut into 5 μm sections, and stained with hematoxy- oxidase was blocked by 3% hydrogen peroxide solution. lin and eosin (H&E). Pathobiological slices were evalu- The colon tissue sections were sealed at room tempera - ated in a blinded manner, and the pathological scores ture for 30 min with 3% BSA, and incubated at 4°C over- were assessed based on the following parameters: inflam - night with rabbit anti-Foxp3 (A12051, ABclonal, Wuhan, mation, epithelial defects, crypt pathology, dysplasia/ PRC, 1:100 dilution). After that, the colon tissue sections neoplasia and the area of dysplasia/neoplasia (scoring were washed three times and incubated with goat anti- criteria in supplementary materials) (Allen et  al. 2012; rabbit IgG (K5007, Dako, Shanghai, PRC, 1:200 dilution) Meira et al. 2008). at room temperature for 50 min. Sections of negative control were treated in the same manner, but the pri- Serum biochemical detection mary antibody was omitted. Three 400 visual fields were Serum samples from mice were collected for biochemical randomly selected for each slice in each group. ImageJ index analysis. The activities of myeloperoxidase (MPO), software was used to analyze each image to obtain the and  superoxide dismutase (SOD), along with the con- positive integrated optical density value (IOD) and the centrations of malonaldehyde (MDA), were measured area of visual fields for each image, and then, calculate by specific assay kits (Nanjing Jiancheng Bioengineering the mean optical density (MOD). Institute, Nanjing, PRC). MOD = IOD/area Gene expression analysis Total RNA from colon tissue was extracted with a Steady- Colon contents untargeted metabolomic analysis Pure Universal RNA Extraction Kit (AG21017, Accurate Metabolite extraction and UHPLC-MS/MS analyses were Biotechnology, Hunan, PRC). RNA was reverse tran- performed according to previous protocols (Want et  al. scribed into complementary DNA (cDNA) (Evo M-MLV 2013). The raw data files were processed using the Com - RT Mix Kit with gDNA Clean for qPCR; AG11728, Accu- pound Discoverer 3.1 (CD 3.1, ThermoFisher, USA). After rate Biotechnology, Hunan, PRC). Real-time PCR (SYBR that, these metabolites were annotated using the KEGG, Green premix Pro Taq HS qPCR kit; AG11701, Accurate HMDB and LIPIDMaps database. Data normalization, Biotechnology, Hunan, PRC) was used to detect gene partial least squares discriminant Aanalysis (PLS-DA), -ΔΔCt expression and the 2 method was used for calcula- orthogonal partial least squares discriminant analysis tion and analysis. The primers are shown in Table S2. (OPLS-DA) were performed with R package MetaboAn- alystR (Chong and Xia 2018). To make the data close to Western blot a normal distribution, the Normalization function in IL-1β, IL-6, TNF-α, IFN-γ and IL-10 in colonic tissue in the  MetaboAnalystR package (with arguments Medi- each group were determined. After the colon tissue was anNorm, LogNorm, and AutoNorm) was adopted. We ground into powder by liquid nitrogen, it was added into applied a univariate analysis (t test) to calculate the statis- ice cold analysis buffer containing a cocktail of protec - tical significance (p value). The metabolites with VIP > 1, tion inhibitors. The mixture was lysed on ice for 30 min p < 0.05 and | log2 (fold Change) | > 1 were considered to (mixed by repeated shaking with a vortex during this be differential metabolites. The metabolites with p < 0.05 time to ensure complete cell lysis), and then the super- (t  test) were used to conduct an over representation natant fluid of the lysate was collected (4°C, 12,000 rpm, analysis (ORA), and the resulting KEGG pathways with 10 min). After electrophoresis, the proteins were elec- p < 0.05 (ORA) were considered as statistically significant trotransferred onto a polyvinylidene fluoride (PVDF) enrichment. membrane (Millipore, Boston, MA, USA). Then, the Qin et al. Animal Diseases (2023) 3:16 Page 16 of 18 Data analysis Availability of data and materials The materials and data not presented in this manuscript are available from The normality of the data was tested using the Shapiro- the corresponding author upon request. The datasets generated during the Wilk normality test. The data that conformed to a normal current study are available in the NCBI repository, [https:// www. ncbi. nlm. nih. distribution were analyzed by one-way ANOVA or t tests gov/, PRJNA841483]. using the Graphpad Prism 6.0 software (GraphPad, La Jolla, CA, USA) and were presented as means±standard Declarations deviation (mean±SD). In addition, differential analysis of Ethics approval and consent to participate alpha diversity was performed using Wilcoxon rank sum This study was approved by the Ethics Committee of Huazhong Agricul- test, beta diversity index was performed using the analy- tural U-niversity ( Wuhan, China) (Permit No. HZAUMO-2021-0068 and HZAUMO-2021-0138) and performed based on the state guidelines from the sis of similarities (ANOSIM), and microbiota between Laboratory Animal Research Center of Hubei province in China. different groups was performed using LEfSe. The signifi - cance level declared at ns p > 0.05, *p < 0.05, **p < 0.01 and Consent for publication Not applicable. ***p < 0.001. Competing interests The authors declare that they have no competing interests. Abbreviations IBD inflammat ory bowel disease UC ulcerative colitis Received: 11 January 2023 Accepted: 28 February 2023 AEA-P arachidonoyl ethanolamide phosphate CD Crohn’s disease PCoA pr incipal coordinate analysis LEfSe Linear discriminant analysis Eec ff t Size DAI disease activity index References MPO myeloperoxidase Adriaenssens, N., S. Coenen, A. Versporten, A. Muller, V. Vankerckhoven, H. SOD superoxide dismutase Goossens, and ESAC Project Group. 2011. European surveillance of MDA malonaldehyde antimicrobial consumption (ESAC): quality appraisal of antibiotic use in IOD integrated optical density Europe. The Journal of Antimicrobial Chemotherapy. 66: vi71–vi77. https:// MOD mean optical density doi. org/ 10. 1093/ jac/ dkr459. IL-1β Interleukin-1 beta Allen, I.C., J.E. 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Regula- tory effect of Zuojin Pill on correlation with gut microbiota an-d Treg cells in DSS-induced colitis. Journal of Ethnopharmacology 262: 113211. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : https:// doi. org/ 10. 1016/j. jep. 2020. 113211. fast, convenient online submission Publisher’s Note thorough peer review by experienced researchers in your field Springer Nature remains neutral with regard to jurisdictional claims in pub- rapid publication on acceptance lished maps and institutional affiliations. support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

Journal

Animal DiseasesSpringer Journals

Published: May 24, 2023

Keywords: Antibiotics; Gut microbiota; Lactobacillus plantarum; DSS; Ulcerative colitis; Untargeted metabolomics

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