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Effects of acupuncture on angiogenesis-associated factor expression in ischemic brain tissue following cerebral infarction in rats

Effects of acupuncture on angiogenesis-associated factor expression in ischemic brain tissue... Background: We aimed to investigate changes into the levels of angiogenesis-associated factors following cerebral infarction and acupuncture intervention and reveal the underlying molecular mechanisms involved in promoting angiogenesis. Methods: Model rats with middle cerebral artery occlusion (MCAO) were randomized into electroacupuncture (EA), model control (MC), and blank control (control) groups. Changes in the degree of neurological impairment following cerebral infarction and angiogenesis in the ischemic center and peripheral area were observed using immunofluorescence double-labeling. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect changes in the Ang-1, Ang-2, PDGF-B, and bFGF levels. Moreover, the effects of EA intervention were evaluated. Results: The neurological severity score of each phase in the EA group was lower than that into the simultaneous phase in the MC group. The proliferation of vascular endothelial cells in the EA group was higher than that in the MC group at 12 hours to 7 days. The Ang-1 and Ang-2 mRNA and protein levels in the EA group were significantly higher than those in the MC group. PDGF-B levels in the EA group were significantly higher than those in the MC group at 3 to 6 hours and 3 to 12 days, and protein levels were high at 6 hours and 3 to 12 days. bFGF mRNA levels at 24 hours to 12 days and bFGF protein at 3 to 12 days were significantly elevated in the EA group than those in the MC group. Conclusions: EA at Shui Gou(DU 26) significantly improved the neurological symptoms of MCAO rats, promoted vascular endothelial cell proliferative activity around the infarct area, significantly advanced the time of proliferation of vascular endothelial cells, and upregulated the expression of angiogenesis-related factors, thereby promoting angiogenesis. Thus, EA may significantly improve the prognosis of cerebral infarction. Keywords: Angiogenesis, Cerebral infarction, DU 26, Electroacupuncture Graphical abstract: http://links.lww.com/AHM/A43. and proliferation of nerve cells require nutritional support Introduction from the blood vessels. Cerebrovascular blockage deter- The pathological process of cerebral infarction is compli- mines the fate of the brain cells in the dominant area, and [1] cated and is triggered by cerebral ischemia . The purpose their smooth functioning depends on whether the region of cerebral infarction treatment is to restore the compro- can regain compensatory blood flow. The regeneration mised function of nerve cells. However, the regeneration of cerebral microvessels and improvement of microcircu- lation following ischemia can promote the proliferation, Tianjin University of Traditional Chinese Medicine First Affiliated [2–3] differentiation, and survival of neural stem cells . The Hospital, Tianjin, China; National Center for Chinese Medicine formation of new blood vessels can be observed in the Acupuncture Clinical Medicine Research, Tianjin, China; Department ischemic penumbra area following infarction, and their of Chemical Biology, Max Planck Institute for Medical Research, [4–7] Heidelberg, Germany; Department of Internal Medicine, Mannheim density is positively correlated with patient prognosis . [8] Medical School of Heidelberg University, Mannheim, Germany In 1993, Von Kummer and Forsting indicated that *Corresponding author. Jing Li, Tianjin University of Traditional Chinese the infarct size in patients with middle cerebral artery Medicine First Affiliated Hospital, National Center for Chinese Medicine (MCA) obstruction mainly depends on collateral blood Acupuncture Clinical Medicine Research, Tianjin 300193, China, supply. An efficient collateral blood supply is crucial for E-mail: virgue1978@sina.com; Rainer Georgi, Department of Chemical limiting the extent of cerebral ischemic infarction and Biology, Max Planck Institute for Medical Research, Heidelberg 69120, spontaneous or drug-induced recanalization. Recently, Germany, E-mail: prof.dr.med.georgi@web.de. [9–10] some experts emphasized that timely restoration of Copyright © 2023 Tianjin University of Traditional Chinese Medicine. blood circulation during cerebral infarction is vital to This is an open-access article distributed under the terms of the minimizing the degree and scope of nerve cell degenera- Creative Commons Attribution-Non Commercial-No Derivatives tion caused by ischemia. Although reperfusion may cause License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be injury, its benefits outweigh its disadvantages. Improving changed in any way or used commercially without permission from the cerebral circulation is critical in the treatment of cere- journal. bral infarction. Currently, improving cerebral circulation Acupuncture and Herbal Medicine (2023) 3:1 and the compensatory function of collateral vessels in Received 11 February 2022 / Accepted 8 January 2023 the peripheral areas of cerebral infarction have gained importance in the treatment of this disease. http://dx.doi.org/10.1097/HM9.0000000000000054 46 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Preparation of the MCAO rat model The establishment of collateral circulation following cerebral infarction includes early vasodilation (collat- In the EA and MC groups, the Longa intracavitary suture [12–13] eral circulation initiation) and subsequent angiogen- method was used to block the MCA on the right esis (collateral circulation reconstruction). Cerebral side of the rat head to establish the MCAO model. Nylon infarction has been treated with acupuncture in the wire with a 0.21 mm diameter was used. In addition, a Tianjin University of Traditional Chinese Medicine 10% chloral hydrate solution (3 mL/kg, i.p.) was used for First Affiliated Hospital for more than 30 years and anesthesia, and the paraffin-impregnated nylon wire was [11] Xingnao Kaiqiao method has been established . introduced through the proximal end and bifurcation of Because acupuncture is effective, it plays an import- the common carotid artery. The length of the sutures was ant role in promoting collateral circulation. Our pre- (18 ± 0.5) mm. The suture and common carotid artery [12] vious study showed that electroacupuncture (EA) were then ligated, and the skin was sutured. at Shui Gou(DU26) could promote the proliferation of vascular endothelial cells around the infarct area in EA treatment middle cerebral artery occluded (MCAO)-model rats. Furthermore, we established for the first time that EA In the EA group, DU 26 acupoint location (as per the could significantly advance the occurrence of vascular “Atlas of Common Animal Acupoints” edited by Hua endothelial cell proliferation, which confirmed that Xingbang) was acupunctured immediately following EA at DU 26 can promote angiogenesis of the penum- the operation. A 0.5ʹʹ acupuncture needle was used to bra following cerebral infarction, thus promoting the obliquely puncture the rat’s nasal septum 2 mm upward reconstruction of collateral circulation. In addition, and to insert a needle approximately 2 mm below this we found that the function and morphology of cere- site as a reference electrode. The DU 26 and reference brovascular vessels were critically impaired following electrode were connected to the positive and negative cerebral infarction. Acupuncture at DU 26 could dilate electrodes of the EA device respectively. Electrical stim- cerebrovascular vessels, improve autonomic movement ulation of the 15 Hz/2 mA density wave was provided, and energy metabolism, promote the timely start of followed by acupuncture for 20 minutes. The 1,3,6,9,12, cerebral collateral circulation, and increase compensa- and 24-hour groups were subjected to acupuncture only tory blood flow; thus, the general goal of this study once, and the rats were sacrificed immediately follow- was to determine how acupuncture plays a positive ing the completion of treatment for sampling. The other role in angiogenesis. time-stage groups received acupuncture once a day, and Based on the above theoretical basis, this study aimed the rats were sacrificed for sampling immediately follow- to reveal the molecular mechanism of acupuncture ing the completion of treatment at 3, 7, and 12 days. The treatment of cerebral infarction from the perspective of MC and control groups were similarly fixed without any promoting angiogenesis by observing the mRNA and treatment. protein expression patterns of angiogenesis-related fac- tors Ang-1, Ang-2, PDGF-B, and bFGF in ischemic brain Neurological evaluation tissue of rats. Our findings will provide a scientific exper - [14] Neurological severity scores (NSSs) were determined imental basis for the clinical application of acupuncture for each group of rats before and after surgery. Later, the treatment for cerebral infarction. NSS for 3, 7, and 12 days was assessed only once daily. A postoperative NSSs of ≥1 is indicative of a successful operation, and thus, can be included in the experiment. Materials and methods Rats that died before the observation time point or expe- Animals rienced subarachnoid hemorrhage or internal carotid A total of 114 specific-pathogen free, healthy male Wistar artery bifurcation hemorrhage during the acquisition of rats, each weighing 180 to 200 g, were obtained from the brain samples were excluded from further studies. Experimental Animal Center of the Chinese Academy of Military Medical Sciences [Permit number: SCXK Sample processing (Beijing) 2019-0010]. All rats were housed in separate cages (five per cage) at the Animal Experiment Center of Rats were deeply anesthetized using 10% chloral hydrate the Institute of Radiation Medicine, Chinese Academy in the abdominal cavity at the specified time, and the of Medical Sciences at room temperature (25 ± 1)°C. A chest was opened rapidly. Next, the left ventricle was week’s worth of food and clean water were provided to quickly perfused with 0.9% normal saline (4°C) and 4% the rats. Before the operation, rats were fasted for 12 polyformic acid solution (4°C), and the brain was decap- hours, with ad libitum access to water. Minimal pain and itated. Considering the bifurcation of the middle and the use of experimental animals were observed during anterior cerebral arteries as a midpoint, 2 mm of brain the experiment. Rats were randomized into the model tissue was harvested just before and after this point. control (MC) and EA groups (n = 54 rats in each group) Brain samples were fixed at 4°C in 4% paraformalde- and the blank control group (control, n = 6) based on a hyde overnight, dehydrated using a sucrose gradient, and random number table method; the first two groups were embedded. Frozen sections of 10-µm thickness were pre- further divided into nine phase groups according to the pared and stored in a slide box at room temperature for time intervals: 1, 3, 6, 9, 12, and 24 hours and 3, 7, and immunofluorescence double-label staining. 12 days following the operation. Each group consisted of Tissue from the right MCA blood supply area six rats in each phase. (approximately 3 mm before and after the bregma) 47 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com was cut from the coronal region for reverse transcrip- room temperature for 2 hours. Primary diluent Ang-1 tion-polymerase chain reaction(PT-PCR) and western antibody (Abcam, 1:25,000), Ang-2 antibody (Abcam, blotting. 1:2,000), bFGF antibody (Abcam, 1:100), PDGF antibody (Abcam, 1:10,000), and GAPDH (Abcam, 1:10,000) were added. The samples were incubated at Immunofluorescence double-label staining room temperature for 1 hours and then at 4°C over- Primary antibodies [CD31 (1:100) (1 mL, Item No.: night. It was followed by washing three times with Tris MCA1334GA; AbD Serotec, UK) and Ki67 (1:1,000) Buffered Saline with Tween20 (TBST) (0.05% Tween (1 mL Item No.: ab15580; Abcam, UK)] were added 20) solution for 10 minutes. Subsequently, the samples after the samples were blocked with goat serum for 1 were immersed in the corresponding secondary anti- hour. After overnight incubation at 4°C, fluorescein-la- body diluent (Abcam, 1:5,000), incubated for 1 hour at beled secondary antibodies [goat anti-rabbit rhodamine room temperature, and washed with TBST. The electro- red-X IgG (1:100) (1 mL, Item No.: CW0149; ComWin chemiluminescence (ECL) solution was added with dark Biotech, China) and goat anti-mouse Cy2 IgG (1:100) conditions maintained, and 30 seconds later, protein (1 mL, Item No.: CW0161; ComWin Biotech)] were expression was observed using a BIO-RAD gel imaging added drop-wise. Incubation was performed for 2 hours system and gray scanning. The absorbance value of the at room temperature, and the samples were rinsed using target band and internal reference band were analyzed 0.01 M phosphate-buffered saline. The slides were using ImageJ software, with the ratio of these two values mounted with anti-fluorescence quenching tablets, and being utilized to measure relative protein expression. the images were captured using a microscope (Leica, Germany). With the Image Pro-Plus 6.0 image analysis Statistical analysis system used, three non-coincident ×400 fields were ran- domly analyzed for each tissue section. The average of Statistical tests were performed using SPSS version 16.0. the three values was used to define the number of posi- The data obtained in the experiment were normally dis- tive cells, while ≥10% was defined as positive. tributed and expressed as the mean ± standard deviation (mean±SD). One-way analysis of variance (ANOVA) was used for multiple comparisons between diverse groups. Reverse transcription-polymerase chain reaction When the variance was observed to be homogeneous, Brain tissues (50 mg) were used to extract RNA it was subjected to the least significant difference (LSD) [E.Z.N.A.HP Total RNA kit (R6812-01), Omega, analysis. However, when the variance was uneven, it was USA]. With double distilled water(ddH O) (RNase/ subjected to Dunnett’s T3 test. P < 0.05 was the signifi- Dnase free) used, micro-ultraviolet spectrophotometer cance threshold. K5500 (Kaiao Technology, China) was calibrated, and then, the absorbance of 1 μL brain RNA samples was Results measured at 260 and 280 nm. The total RNA of each sample was diluted to a final concentration of 1 μ g/μL, Neurological evaluation reverse-transcribed into cDNA, followed by RT-PCR. Each group consisted of six rats in each phase. Before The reaction was carried out in a MicroAmp Fast the operation, the NSSs of all rats were normal (NSSs = Optical 96-well reaction barcoded plate, with GAPDH 0). Additionally, the control group’s NSSs was zero after as the housekeeping gene and ddH O as the negative the procedure. The EA and MC groups were considered control. The thermocycling protocol was as follows: 1) for the score observation process from 3 hours onward Uracil-N-glycosylase (UNG) enzyme digestion at 50°C because the MCAO rats from the 1-hour group were for 10 minutes; 2) pre-denaturation at 95°C for 10 min- not fully awake. The NSSs of the EA and MC groups utes; 3) 40 cycles at 95°C for 15 seconds and 60°C for was higher than that of the control group in each phase 1 minute. The experiments were conducted in triplicate. (P < 0.01). However, the NSSs of the EA group was con- ABI 7,500 Fast Real-Time PCR System software(Roche siderably lower than that of the MC group in each phase Lightcycler480, Switzerland) was used to analyze the (P < 0.05), with the most significant difference at 3 and real-time fluorescence quantitative PCR experiment 12 days (P < 0.01), as shown in Figure 1. results and calculate the mRNA expression levels of each experimental index (Table 1). Table 1 Western immunoblotting Probe for experiment The protein levels were determined using a BCA assay TaqMan Gene Expression Assays-Ang1 ThermoFisher, USA kit (BOSTER, China). Briefly, 10 µL protein was col- (Rn01504818_m1) lected from the brain tissues of the right MCA blood TaqMan Gene Expression Assays-Ang2 ThermoFisher, USA supply area of rats in each experimental group, boiled at (Rn01756774_m1) TaqMan Gene Expression Assays-PDGF-B ThermoFisher, USA 100°C for 5 minutes, separated on a 10% SDS-PAGE gel, (Rn1502596_m1) and transferred onto a polyvinylidene difluoride mem- TaqMan Gene Expression Assays-bFGF ThermoFisher, USA brane. With standard molecular weight protein markers (Rn00570809_m1) as a reference, sample protein bands were cut accord- TaqMan Gene Expression Assays-GAPDH ThermoFisher, USA ing to their molecular weights. They were immersed (Rn01775763_g1) in an incubation box containing 5% non-fat milk at 48 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com difference at 9 to 12 hours (P < 0.01). PDGF-B mRNA expression levels in the EA group were higher than those in the MC group at 3 to 6 hours and 3 to 12 days (P < 0.05). Ang-2 and PDGF-B protein levels in each phase in the EA and MC groups were elevated relative to the controls (P < 0.01). The Ang-2 protein level in each phase in the EA group showed a higher trend than that in the MC group in a similar phase. The differ- ence was significant from 3 to 12 hours (P < 0.05), with a highly significant difference at 3 to 6 hours and 12 hours (P < 0.01). In contrast, the PDGF-B protein levels in the EA group were significantly higher than those in the MC group at 6 hours and 3 to 12 days (P < 0.05) Figure 1. Neurological severity scores. P < 0.01 vs. control group; and highly significant at 7 to 12 days (P < 0.01). From ## # P < 0.01, P < 0.05 vs. MC group. All comparisons were made at the 9 hours to 12 days, bFGF mRNA expression levels in same time point. EA: electroacupuncture; MC: Model control. the MC group were higher than those in the controls (P < 0.05, P < 0.01), whereas the expression levels in Angiogenesis in ischemic penumbra area following infarction the EA group were higher at 3 hours and 9 to 12 days Immunofluorescence double-label staining analysis of (P < 0.05, P < 0.01). In addition, compared with that cerebral arteries showed no vascular endothelial cell pro- in the MC group, the bFGF mRNA expression level in liferation surrounding the infarct area in the MC group, the EA group was significantly elevated from 24 hours for 1 to 12 hours following MCAO. Proliferation began to 12 days (P < 0.05) and was highly significant at 3 to appear at 24 hours and reached a maximum after 3 to 12 days (P < 0.01). The bFGF protein levels in the days. Later, it decreased on day 7 and disappeared on MC group were higher than those in the controls at 24 day 12. In the EA group, no proliferation of vascular hours to 3 days (P < 0.05), whereas the EA group levels endothelial cells was observed for 1 to 9 hours following were higher at 12 hours to 12 days (P < 0.05, P < 0.01). MCAO. Proliferation began at 12 hours, continued to Furthermore, the bFGF protein levels in the EA group increase at 24 hours, and reached a maximum at 3 days. were higher than those in the MC group at 3 to 12 days Later, it decreased on day 7 and disappeared on day 12. (P < 0.05) and were highly significant at 12 days (P < The proliferation trend of vascular endothelial cells in 0.01), as shown in Figures 3 and 4. the EA group following MCAO was similar to that in the MC group. However, the proliferation appeared earlier Discussion and was higher. No proliferation of vascular endothelial cells was observed on the contralateral side of the infarct Angiogenesis has vital clinical significance because it pro- area in any of the phases in the two groups. In addition, motes the repair of neural structure and function following no proliferation was observed in the controls at each cerebral infarction by stimulating the endogenous repair [15] phase, as shown in Figure 2 and Table 2. mechanism . Rapid restoration of blood supply in the ischemic penumbra area is a fundamental goal of acute [10,16] cerebral infarction treatment . Anatomical studies have Increased mRNA and protein expression of angiogenesis- indicated that the facial and trigeminal nerve branches related factors following EA are distributed around DU 26 points. Stimulation of DU [17] After the operation, the expression of angiogenesis-as- 26 may lead to activation of the terminal branches of sociated factors in the cerebral vascular region differed the trigeminal nerve and facial nerve, causing excitation of among the groups (P < 0.05, P < 0.01). The Ang-1 the sphenopalatine ganglion and modulation of cerebral mRNA expression levels in the EA and MC groups vasomotion. Electrical stimulation at 2 Hz can cause the from 12 hours to 12 days were significantly higher than parasympathetic nerve to release acetylcholine, whereas those in the controls (P < 0.05, P < 0.01). In addition, electrical stimulation at ≥10 Hz can selectively release [18] the Ang-1 mRNA expression levels in the EA group peptide neurotransmitters, such as vasoactive peptides . were significantly higher than those in the MC group Previous studies have indicated that p-Akt expression in from 3 to 12 days (P < 0.05), with a marked differ- positive cells in the hippocampal CA1 region, dentate ence at day 7 (P < 0.01). Ang-1 protein levels in the gyrus, and cortex in the moderate-intensity (2–4 mA) EA EA and MC groups from 6 hours to 12 days were sig- group was significantly higher than that in the high-inten- [19] nificantly increased compared to those in the control sity (5–7 mA) EA and control group . Based on these (P < 0.01). Moreover, the Ang-1 protein levels in the results, we chose 15 Hz/2 mA as the criterion for EA in [11] EA group were considerably higher than those in the our experiments. Our previous study showed that EA at MC group from 3 to 12 days (P < 0.05). Ang-2 and DU 26 could promote the proliferation of vascular endo- PDGF-B mRNA expression levels in each phase were thelial cells around the infarct area in MCAO model rats. significantly increased in the EA and MC groups com- It is consistent with the present results, confirming that pared to those in the controls (P < 0.05, P < 0.01). The EA at DU 26 can promote angiogenesis of the penumbra Ang-2 mRNA expression levels in each phase in the EA following cerebral infarction. group were higher than those in the MC group in a We found that with the prolongation of ischemic similar phase. However, the difference was significant time, the NSSs of the two groups showed a downward from 3 to 24 hours (P < 0.05), with a highly significant trend and there were significant differences in each phase 49 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Figure 2. EA promote the proliferation of vascular endothelial cells in ischemic penumbra area of the MCAO rat. CD31 is positively expressed by staining the membrane of vascular endothelial cells in the brain tissue in green. Ki67 is positively expressed by staining the nucleus of vascular endothelial cells in red. The number of Ki67-positive cell nuclei when CD31 and Ki67 were expressed together was used to indicate the proliferation number of vascular endothelial cells. EA: Electroacupuncture; MCAO: Middle cerebral artery occlusion. immunofluorescence double-labeled detection was per - Table 2 formed on rat brain tissue. We found that around the Semi-quantitative analysis of the proliferation of infarction area, co-expression of Ki67 and CD31 began vascular endothelial cells around the infarct zone of to appear in the EA group 12 hours following MCAO, MCAO rats (n = 6) and co-expression of Ki67 and CD31 began to appear in the MC group 24 h following MCAO. With the pro- Cerebral The MC The EA The control longation of ischemia time, co-expression gradually ischemia time group group group increased, reached a peak at day 3, and subsequently, the 1 h - - - proliferation level decreased. However, co-expression 3 h - - - was still observed on day 12 in the EA group but not in 6 h - - - the MC group. We found that the co-expression of Ki67 9 h - - - and CD31 within 24 h may be due to early endothelial 12 h - + - 24 h + ++ - cell proliferation and migration to form microvascular 3 d ++ +++ - cavities. Co-expression since 3 d may be the expression 7 d + ++ - of endothelial cells that aggregated and proliferated into 12 d - - - mature blood vessels. At this time, the stronger CD31 EA: Electroacupuncture; MC: Model control; MCAO: Middle cerebral artery occlusion. fluorescence signal may be due to the maturation and “-” indicates no positive staining; “+” indicates one to three endothelial cells; “++” indicates four stability of the endothelial cells. In addition, the prolif- to six endothelial cells; “+++” indicates seven to nine endothelial cells; “++++” indicates more than nine endothelial cells. eration and expression of endothelial cells in capillary branches were observed from day 7 to 12, and it was possible to establish branch connections of the vascu- (P < 0.05), indicating that acupuncture can signifi- lar network through endothelial cell proliferation at this cantly alleviate the neurological symptoms of MCAO time, thus forming a new capillary network. In addition, rats and slow down the trend of nerve function aggra- the newly formed capillaries in the marginal zone of the vation, with the most significant improvement at 3 and ischemic brain tissue of rats in the EA group at day 12 12 days (P < 0.01). Simultaneously, Ki67 and CD31 50 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Figure 3. Ang-1, Ang-2, PDGF-B, and bFGF mRNA in the cerebral vascular of rats by RT-PCR detection. (A) Ang-1 mRNA levels. (B) Ang-2 mRNA * ** # ## levels. (C) PDGF-B mRNA levels. (D) bFGF mRNA levels. P < 0.05, P < 0.01 vs. control group; P < 0.05, P < 0.01 vs. MC group. All comparisons were made at the same time point. MC: Model control; RT-PCR: reverse transcription-polymerase chain reaction. had complicated branches and large diameters. This may endothelium. However, the binding of Ang-2 to the Tie-2 be because EA can promote the upregulation of angio- receptor does not cause receptor phosphorylation but genesis-related factors and accelerate endothelial cell blocks Ang-1–mediated Tie-2 activation, thereby pro- [25–26] proliferation and migration to form a new blood vessel moting blood vessel lengthening . We found that the network. expression of Ang-1 mRNA in the MC group began to Angiogenesis is a complex process resulting from the upregulate at 6 hours whereas that in the EA group at 3 coordinated effects of pro-angiogenic and anti-angio- hours. The expression of Ang-2 mRNA in the MC group genic factors (vascular balance theory). Their stability was upregulated at 3 hours whereas that in the EA group determines the start time and progression of the subse- at 1 hour. The expression of Ang-1 and Ang-2 in each quent angiogenesis process. Under conditions such as phase in the EA group was consistently higher than that hypoxia, inflammation, or external agents, angiogene- in the MC group. This result shows that EA can promote sis-related factors directly or indirectly activate endothe- the early high-level expression of Ang-2 to initiate the lial cells in autocrine or paracrine forms to initiate the angiogenesis mechanism in advance, thereby prolonging [20] process of angiogenesis. Previous experiments have the angiogenesis process, whereas Ang-1 appears to be confirmed that EA at DU 26 can activate the endoge- highly expressed at 3 day to induce the maturation and nous Sonic Hedgehog signaling pathway and promote stability of new blood vessels. It can be speculated that the early and high expression of related factors. The EA induces the expression of angiogenesis-related factors Shh pathway has a very close relationship with angio- by modulating the Shh pathway and coordinating their genesis-related factors and can induce the upregulation interactions, thereby promoting the growth, maturation, [21] of bFGF and balance Ang-1 and Ang-2 expression and stability of microvessels in the neurovascular net- [27] By stimulating the expression of multiple downstream work (Figure 5). bFGF is an FGF family protein that signal transduction pathways, Ang-1 regulates angiogen- shares vital biological functions, including proliferation, esis, forms stable blood vessels, and inhibits the expres- differentiation, migration, and apoptosis. It also plays a sion of cell surface adhesion molecules, inflammatory crucial role in the early and middle stages of angiogene- [22–24] cell adhesion, and apoptosis . A dynamic balance sis and is a neurotrophic factor that can directly nourish consisting of low Ang-2 and high Ang-1 levels within neurons, accelerate neural stem cell differentiation and [28–29] the central nervous system is important for blood–brain proliferation, and promote neural remodeling . We barrier integrity. The binding of Ang-1 to Tie receptors found that the expression of bFGF mRNA in the MC contributes to the induction of phosphorylation, which group began to increase at 6 h whereas that in the EA can prevent apoptotic death of cells of the vascular group at 1 hour. The mRNA and protein expression levels 51 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Figure 4. Ang-1, Ang-2, PDGF-B, and bFGF protein in the cerebral vascular of rats by Western Blotting. (A) Ang-1 protein levels. (B) Ang-2 protein # ## levels. (C) PDGF-B protein expression. (D) bFGF protein expression. *P < 0.05, **P < 0.01 vs. control group; P < 0.05, P < 0.01 vs. MC group. All comparisons were made at the same time point. MC: Model control. [32–34] in each phase in the EA group were higher than those in PDGF-B and bFGF expression in the cortex of the the MC group, and the expression level remained higher infarct area. EA can promote the early high-level expres- [35] from 9 hours to 12 days. This result indicates that EA sion of PDGF-B and bFGF, thus protecting nerves and can stimulate the stress expression of endogenous bFGF, promoting angiogenesis during the healing and remod- thereby promoting the proliferation and migration of eling of ischemic cells and tissues. Therefore, these four endothelial cells. Shh signaling pathway inhibitors can angiogenesis-related factors, Ang-1, Ang-2, PDGF-B, and also suppress PDGF-induced vascular smooth muscle bFGF, may combinedly promote the formation of the [30] cell proliferation . PDGF-B is synthesized and secreted new blood vessel network, save dying neurons, reduce by endothelial cells. It promotes blood vessel maturation brain tissue damage, complete the vascular remodeling by recruiting pericytes and stabilizing developing blood process much earlier than in the MC group, and con- [31] vessels and is important during the late stage of angio- tinue to promote angiogenesis in the infarcted area. We genesis. Ischemia can cause transient upregulation of speculate that after cerebral infarction, angiogenesis 52 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Jing Li and Yajie Sun wrote the first draft of this manu- script. Yajie Sun conducted experiments. Rainer Georgi and Bernhard Kolberg revised the manuscript and par- ticipated in statistical analysis. Lihong Yang conducted the immunofluorescence double-label staining experi- ments. All the authors have read and approved the final manuscript. Ethical approval of studies and informed consent The rat experimental were overseen and approved by the Beijing Municipal Science & Technology Commission and the Administrative Commission of Zhongguancun Science Park [Approval number: SCXK (Beijing) 2019-0010]. Acknowledgments None. Figure 5. Electroacupuncture induces the expression of angiogene- sis-associated factors by mediating the Shh pathway. Data availability around the infarct area is not only to increase cerebral All data generated or analyzed during this study are blood perfusion and remove metabolites, but a response included in this published article. to the reorganization of nerve function in this region, that is, the high metabolic rate in the process of neu- rological reorganization requires more blood to provide References nutrients and quickly clear metabolites, and the increase [1] Jiao Y, Liu YW, Chen WG, et al. Neuroregeneration and functional recovery after stroke: advancing neural stem in demand mediates the increase of angiogenesis, which cell therapy toward clinical application. Neural Regen Res also belongs to the body’s protective mechanism against 2021;16(1):80–92. local damage. This inference needs further study, and [2] Belayev L, Hong SH, Menghani H, et al. Docosanoids promote we will design experiments to explore this mechanism neurogenesis and angiogenesis, blood-brain barrier integrity, pen- umbra protection, and neurobehavioral recovery after experimen- in depth in future studies. Moreover, EA may promote tal ischemic stroke. Mol Neurobiol 2018;55(8):7090–7106. angiogenesis by mediating the Shh signaling pathway, [3] Xing C, Hayakawa K, Lok J, et al. Injury and repair in the neuro- but its specific mechanism still needs to be studied in vascular unit. Neurol Res 2012;34(4):325–330. depth. [4] Madelaine R, Sloan SA, Huber N, et al. MicroRNA-9 couples brain neurogenesis and angiogenesis. Cell Rep 2017;20(7):1533–1542. [5] Ruan L, Wang B, ZhuGe Q, et al. Coupling of neurogenesis and Conclusions angiogenesis after ischemic stroke. Brain Res 2015;1623:166–173. [6] Huang H, Huang Q, Wang F, et al. Cerebral ischemia-induced Based on the above research results, we conclude that EA angiogenesis is dependent on tumor necrosis factor recep- at DU 26 can significantly improve neurological deficit tor 1-mediated upregulation of α5β1 and αVβ3 integrins. J symptoms in MCAO rats and promote the upregulation Neuroinflammation 2016;13(1):227. of mRNA and proteins of angiogenesis-related fac- [7] Seto SW, Chang D, Jenkins A, et al. Angiogenesis in ischemic stroke and angiogenic effects of Chinese herbal medicine. J Clin tors Ang-1, Ang-2, PDGF-B, and bFGF. Our study also Med 2016;5(6):56. indicates that EA could advance the expression phase, [8] Von Kummer R, Forsting M. Clinical research of MCAO patients thereby promoting the regeneration, maturation, and sta- treatment. Cerebrovasc Dis 1993;3(3):252–255. bilization of microvessels. Thus, EA may be an import- [9] Xu JC, Tan QJ, Sun WM, et al. Exploration on dredging collat- erals in traditional Chinese medicine and brain microcirculation ant technique for the treatment of cerebral infarction. dysfunction after cerebral infarction. Liaoning J Trad Chin Med 2021;48(1):78–80. [10] Sa-Pereira I, Brites D, Brito MA. Neurovascular unit: a focus on Conflict of interest statement pericytes. Mol Neurobiol 2012;45(2):327–347. [11] Deng S, Sang B, Li B, et al. The efficacy and safety of acupuncture The authors declare no conflict of interest. combined with language training for motor aphasia after stroke: study protocol for a multicenter randomized sham-controlled trial. Trials 2022;23(1):540. Funding [12] Yuanhao D, Lei S, Jing L, et al. 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Protein nanoparticles 082145. modified with PDGF-B as a novel therapy after acute cerebral infarc- [25] Xiong M, Feng Y, Huang S, et al. Teriparatide induces angio- tion. eNeuro 2021;8(5):ENEURO.0098ENEURO.0098-21.2021. genesis in ischemic cerebral infarction zones of rats through AC/ PKA signaling and reduces ischemia-reperfusion injury. Biomed Pharmacother 2022;148:112728. How to cite this article: Sun YJ, Li J, Georgi R, Kolberg B, Yang LH. Effects [26] Felcht M, Luck R, Schering A, et al. Angiopoietin-2 differentially of acupuncture on angiogenesis-associated factor expression in ischemic regulates angiogenesis through TIE2 and integrin signaling. J Clin brain tissue following cerebral infarction in rats. Acupunct Herb Med Invest 2012;122(6):1991–2005. 2023;3(1):46–54. doi: 10.1097/HM9.0000000000000054 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acupuncture and Herbal Medicine Wolters Kluwer Health

Effects of acupuncture on angiogenesis-associated factor expression in ischemic brain tissue following cerebral infarction in rats

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Wolters Kluwer Health
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Copyright © 2023 Tianjin University of Traditional Chinese Medicine.
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2765-8619
DOI
10.1097/hm9.0000000000000054
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Abstract

Background: We aimed to investigate changes into the levels of angiogenesis-associated factors following cerebral infarction and acupuncture intervention and reveal the underlying molecular mechanisms involved in promoting angiogenesis. Methods: Model rats with middle cerebral artery occlusion (MCAO) were randomized into electroacupuncture (EA), model control (MC), and blank control (control) groups. Changes in the degree of neurological impairment following cerebral infarction and angiogenesis in the ischemic center and peripheral area were observed using immunofluorescence double-labeling. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect changes in the Ang-1, Ang-2, PDGF-B, and bFGF levels. Moreover, the effects of EA intervention were evaluated. Results: The neurological severity score of each phase in the EA group was lower than that into the simultaneous phase in the MC group. The proliferation of vascular endothelial cells in the EA group was higher than that in the MC group at 12 hours to 7 days. The Ang-1 and Ang-2 mRNA and protein levels in the EA group were significantly higher than those in the MC group. PDGF-B levels in the EA group were significantly higher than those in the MC group at 3 to 6 hours and 3 to 12 days, and protein levels were high at 6 hours and 3 to 12 days. bFGF mRNA levels at 24 hours to 12 days and bFGF protein at 3 to 12 days were significantly elevated in the EA group than those in the MC group. Conclusions: EA at Shui Gou(DU 26) significantly improved the neurological symptoms of MCAO rats, promoted vascular endothelial cell proliferative activity around the infarct area, significantly advanced the time of proliferation of vascular endothelial cells, and upregulated the expression of angiogenesis-related factors, thereby promoting angiogenesis. Thus, EA may significantly improve the prognosis of cerebral infarction. Keywords: Angiogenesis, Cerebral infarction, DU 26, Electroacupuncture Graphical abstract: http://links.lww.com/AHM/A43. and proliferation of nerve cells require nutritional support Introduction from the blood vessels. Cerebrovascular blockage deter- The pathological process of cerebral infarction is compli- mines the fate of the brain cells in the dominant area, and [1] cated and is triggered by cerebral ischemia . The purpose their smooth functioning depends on whether the region of cerebral infarction treatment is to restore the compro- can regain compensatory blood flow. The regeneration mised function of nerve cells. However, the regeneration of cerebral microvessels and improvement of microcircu- lation following ischemia can promote the proliferation, Tianjin University of Traditional Chinese Medicine First Affiliated [2–3] differentiation, and survival of neural stem cells . The Hospital, Tianjin, China; National Center for Chinese Medicine formation of new blood vessels can be observed in the Acupuncture Clinical Medicine Research, Tianjin, China; Department ischemic penumbra area following infarction, and their of Chemical Biology, Max Planck Institute for Medical Research, [4–7] Heidelberg, Germany; Department of Internal Medicine, Mannheim density is positively correlated with patient prognosis . [8] Medical School of Heidelberg University, Mannheim, Germany In 1993, Von Kummer and Forsting indicated that *Corresponding author. Jing Li, Tianjin University of Traditional Chinese the infarct size in patients with middle cerebral artery Medicine First Affiliated Hospital, National Center for Chinese Medicine (MCA) obstruction mainly depends on collateral blood Acupuncture Clinical Medicine Research, Tianjin 300193, China, supply. An efficient collateral blood supply is crucial for E-mail: virgue1978@sina.com; Rainer Georgi, Department of Chemical limiting the extent of cerebral ischemic infarction and Biology, Max Planck Institute for Medical Research, Heidelberg 69120, spontaneous or drug-induced recanalization. Recently, Germany, E-mail: prof.dr.med.georgi@web.de. [9–10] some experts emphasized that timely restoration of Copyright © 2023 Tianjin University of Traditional Chinese Medicine. blood circulation during cerebral infarction is vital to This is an open-access article distributed under the terms of the minimizing the degree and scope of nerve cell degenera- Creative Commons Attribution-Non Commercial-No Derivatives tion caused by ischemia. Although reperfusion may cause License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be injury, its benefits outweigh its disadvantages. Improving changed in any way or used commercially without permission from the cerebral circulation is critical in the treatment of cere- journal. bral infarction. Currently, improving cerebral circulation Acupuncture and Herbal Medicine (2023) 3:1 and the compensatory function of collateral vessels in Received 11 February 2022 / Accepted 8 January 2023 the peripheral areas of cerebral infarction have gained importance in the treatment of this disease. http://dx.doi.org/10.1097/HM9.0000000000000054 46 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Preparation of the MCAO rat model The establishment of collateral circulation following cerebral infarction includes early vasodilation (collat- In the EA and MC groups, the Longa intracavitary suture [12–13] eral circulation initiation) and subsequent angiogen- method was used to block the MCA on the right esis (collateral circulation reconstruction). Cerebral side of the rat head to establish the MCAO model. Nylon infarction has been treated with acupuncture in the wire with a 0.21 mm diameter was used. In addition, a Tianjin University of Traditional Chinese Medicine 10% chloral hydrate solution (3 mL/kg, i.p.) was used for First Affiliated Hospital for more than 30 years and anesthesia, and the paraffin-impregnated nylon wire was [11] Xingnao Kaiqiao method has been established . introduced through the proximal end and bifurcation of Because acupuncture is effective, it plays an import- the common carotid artery. The length of the sutures was ant role in promoting collateral circulation. Our pre- (18 ± 0.5) mm. The suture and common carotid artery [12] vious study showed that electroacupuncture (EA) were then ligated, and the skin was sutured. at Shui Gou(DU26) could promote the proliferation of vascular endothelial cells around the infarct area in EA treatment middle cerebral artery occluded (MCAO)-model rats. Furthermore, we established for the first time that EA In the EA group, DU 26 acupoint location (as per the could significantly advance the occurrence of vascular “Atlas of Common Animal Acupoints” edited by Hua endothelial cell proliferation, which confirmed that Xingbang) was acupunctured immediately following EA at DU 26 can promote angiogenesis of the penum- the operation. A 0.5ʹʹ acupuncture needle was used to bra following cerebral infarction, thus promoting the obliquely puncture the rat’s nasal septum 2 mm upward reconstruction of collateral circulation. In addition, and to insert a needle approximately 2 mm below this we found that the function and morphology of cere- site as a reference electrode. The DU 26 and reference brovascular vessels were critically impaired following electrode were connected to the positive and negative cerebral infarction. Acupuncture at DU 26 could dilate electrodes of the EA device respectively. Electrical stim- cerebrovascular vessels, improve autonomic movement ulation of the 15 Hz/2 mA density wave was provided, and energy metabolism, promote the timely start of followed by acupuncture for 20 minutes. The 1,3,6,9,12, cerebral collateral circulation, and increase compensa- and 24-hour groups were subjected to acupuncture only tory blood flow; thus, the general goal of this study once, and the rats were sacrificed immediately follow- was to determine how acupuncture plays a positive ing the completion of treatment for sampling. The other role in angiogenesis. time-stage groups received acupuncture once a day, and Based on the above theoretical basis, this study aimed the rats were sacrificed for sampling immediately follow- to reveal the molecular mechanism of acupuncture ing the completion of treatment at 3, 7, and 12 days. The treatment of cerebral infarction from the perspective of MC and control groups were similarly fixed without any promoting angiogenesis by observing the mRNA and treatment. protein expression patterns of angiogenesis-related fac- tors Ang-1, Ang-2, PDGF-B, and bFGF in ischemic brain Neurological evaluation tissue of rats. Our findings will provide a scientific exper - [14] Neurological severity scores (NSSs) were determined imental basis for the clinical application of acupuncture for each group of rats before and after surgery. Later, the treatment for cerebral infarction. NSS for 3, 7, and 12 days was assessed only once daily. A postoperative NSSs of ≥1 is indicative of a successful operation, and thus, can be included in the experiment. Materials and methods Rats that died before the observation time point or expe- Animals rienced subarachnoid hemorrhage or internal carotid A total of 114 specific-pathogen free, healthy male Wistar artery bifurcation hemorrhage during the acquisition of rats, each weighing 180 to 200 g, were obtained from the brain samples were excluded from further studies. Experimental Animal Center of the Chinese Academy of Military Medical Sciences [Permit number: SCXK Sample processing (Beijing) 2019-0010]. All rats were housed in separate cages (five per cage) at the Animal Experiment Center of Rats were deeply anesthetized using 10% chloral hydrate the Institute of Radiation Medicine, Chinese Academy in the abdominal cavity at the specified time, and the of Medical Sciences at room temperature (25 ± 1)°C. A chest was opened rapidly. Next, the left ventricle was week’s worth of food and clean water were provided to quickly perfused with 0.9% normal saline (4°C) and 4% the rats. Before the operation, rats were fasted for 12 polyformic acid solution (4°C), and the brain was decap- hours, with ad libitum access to water. Minimal pain and itated. Considering the bifurcation of the middle and the use of experimental animals were observed during anterior cerebral arteries as a midpoint, 2 mm of brain the experiment. Rats were randomized into the model tissue was harvested just before and after this point. control (MC) and EA groups (n = 54 rats in each group) Brain samples were fixed at 4°C in 4% paraformalde- and the blank control group (control, n = 6) based on a hyde overnight, dehydrated using a sucrose gradient, and random number table method; the first two groups were embedded. Frozen sections of 10-µm thickness were pre- further divided into nine phase groups according to the pared and stored in a slide box at room temperature for time intervals: 1, 3, 6, 9, 12, and 24 hours and 3, 7, and immunofluorescence double-label staining. 12 days following the operation. Each group consisted of Tissue from the right MCA blood supply area six rats in each phase. (approximately 3 mm before and after the bregma) 47 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com was cut from the coronal region for reverse transcrip- room temperature for 2 hours. Primary diluent Ang-1 tion-polymerase chain reaction(PT-PCR) and western antibody (Abcam, 1:25,000), Ang-2 antibody (Abcam, blotting. 1:2,000), bFGF antibody (Abcam, 1:100), PDGF antibody (Abcam, 1:10,000), and GAPDH (Abcam, 1:10,000) were added. The samples were incubated at Immunofluorescence double-label staining room temperature for 1 hours and then at 4°C over- Primary antibodies [CD31 (1:100) (1 mL, Item No.: night. It was followed by washing three times with Tris MCA1334GA; AbD Serotec, UK) and Ki67 (1:1,000) Buffered Saline with Tween20 (TBST) (0.05% Tween (1 mL Item No.: ab15580; Abcam, UK)] were added 20) solution for 10 minutes. Subsequently, the samples after the samples were blocked with goat serum for 1 were immersed in the corresponding secondary anti- hour. After overnight incubation at 4°C, fluorescein-la- body diluent (Abcam, 1:5,000), incubated for 1 hour at beled secondary antibodies [goat anti-rabbit rhodamine room temperature, and washed with TBST. The electro- red-X IgG (1:100) (1 mL, Item No.: CW0149; ComWin chemiluminescence (ECL) solution was added with dark Biotech, China) and goat anti-mouse Cy2 IgG (1:100) conditions maintained, and 30 seconds later, protein (1 mL, Item No.: CW0161; ComWin Biotech)] were expression was observed using a BIO-RAD gel imaging added drop-wise. Incubation was performed for 2 hours system and gray scanning. The absorbance value of the at room temperature, and the samples were rinsed using target band and internal reference band were analyzed 0.01 M phosphate-buffered saline. The slides were using ImageJ software, with the ratio of these two values mounted with anti-fluorescence quenching tablets, and being utilized to measure relative protein expression. the images were captured using a microscope (Leica, Germany). With the Image Pro-Plus 6.0 image analysis Statistical analysis system used, three non-coincident ×400 fields were ran- domly analyzed for each tissue section. The average of Statistical tests were performed using SPSS version 16.0. the three values was used to define the number of posi- The data obtained in the experiment were normally dis- tive cells, while ≥10% was defined as positive. tributed and expressed as the mean ± standard deviation (mean±SD). One-way analysis of variance (ANOVA) was used for multiple comparisons between diverse groups. Reverse transcription-polymerase chain reaction When the variance was observed to be homogeneous, Brain tissues (50 mg) were used to extract RNA it was subjected to the least significant difference (LSD) [E.Z.N.A.HP Total RNA kit (R6812-01), Omega, analysis. However, when the variance was uneven, it was USA]. With double distilled water(ddH O) (RNase/ subjected to Dunnett’s T3 test. P < 0.05 was the signifi- Dnase free) used, micro-ultraviolet spectrophotometer cance threshold. K5500 (Kaiao Technology, China) was calibrated, and then, the absorbance of 1 μL brain RNA samples was Results measured at 260 and 280 nm. The total RNA of each sample was diluted to a final concentration of 1 μ g/μL, Neurological evaluation reverse-transcribed into cDNA, followed by RT-PCR. Each group consisted of six rats in each phase. Before The reaction was carried out in a MicroAmp Fast the operation, the NSSs of all rats were normal (NSSs = Optical 96-well reaction barcoded plate, with GAPDH 0). Additionally, the control group’s NSSs was zero after as the housekeeping gene and ddH O as the negative the procedure. The EA and MC groups were considered control. The thermocycling protocol was as follows: 1) for the score observation process from 3 hours onward Uracil-N-glycosylase (UNG) enzyme digestion at 50°C because the MCAO rats from the 1-hour group were for 10 minutes; 2) pre-denaturation at 95°C for 10 min- not fully awake. The NSSs of the EA and MC groups utes; 3) 40 cycles at 95°C for 15 seconds and 60°C for was higher than that of the control group in each phase 1 minute. The experiments were conducted in triplicate. (P < 0.01). However, the NSSs of the EA group was con- ABI 7,500 Fast Real-Time PCR System software(Roche siderably lower than that of the MC group in each phase Lightcycler480, Switzerland) was used to analyze the (P < 0.05), with the most significant difference at 3 and real-time fluorescence quantitative PCR experiment 12 days (P < 0.01), as shown in Figure 1. results and calculate the mRNA expression levels of each experimental index (Table 1). Table 1 Western immunoblotting Probe for experiment The protein levels were determined using a BCA assay TaqMan Gene Expression Assays-Ang1 ThermoFisher, USA kit (BOSTER, China). Briefly, 10 µL protein was col- (Rn01504818_m1) lected from the brain tissues of the right MCA blood TaqMan Gene Expression Assays-Ang2 ThermoFisher, USA supply area of rats in each experimental group, boiled at (Rn01756774_m1) TaqMan Gene Expression Assays-PDGF-B ThermoFisher, USA 100°C for 5 minutes, separated on a 10% SDS-PAGE gel, (Rn1502596_m1) and transferred onto a polyvinylidene difluoride mem- TaqMan Gene Expression Assays-bFGF ThermoFisher, USA brane. With standard molecular weight protein markers (Rn00570809_m1) as a reference, sample protein bands were cut accord- TaqMan Gene Expression Assays-GAPDH ThermoFisher, USA ing to their molecular weights. They were immersed (Rn01775763_g1) in an incubation box containing 5% non-fat milk at 48 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com difference at 9 to 12 hours (P < 0.01). PDGF-B mRNA expression levels in the EA group were higher than those in the MC group at 3 to 6 hours and 3 to 12 days (P < 0.05). Ang-2 and PDGF-B protein levels in each phase in the EA and MC groups were elevated relative to the controls (P < 0.01). The Ang-2 protein level in each phase in the EA group showed a higher trend than that in the MC group in a similar phase. The differ- ence was significant from 3 to 12 hours (P < 0.05), with a highly significant difference at 3 to 6 hours and 12 hours (P < 0.01). In contrast, the PDGF-B protein levels in the EA group were significantly higher than those in the MC group at 6 hours and 3 to 12 days (P < 0.05) Figure 1. Neurological severity scores. P < 0.01 vs. control group; and highly significant at 7 to 12 days (P < 0.01). From ## # P < 0.01, P < 0.05 vs. MC group. All comparisons were made at the 9 hours to 12 days, bFGF mRNA expression levels in same time point. EA: electroacupuncture; MC: Model control. the MC group were higher than those in the controls (P < 0.05, P < 0.01), whereas the expression levels in Angiogenesis in ischemic penumbra area following infarction the EA group were higher at 3 hours and 9 to 12 days Immunofluorescence double-label staining analysis of (P < 0.05, P < 0.01). In addition, compared with that cerebral arteries showed no vascular endothelial cell pro- in the MC group, the bFGF mRNA expression level in liferation surrounding the infarct area in the MC group, the EA group was significantly elevated from 24 hours for 1 to 12 hours following MCAO. Proliferation began to 12 days (P < 0.05) and was highly significant at 3 to appear at 24 hours and reached a maximum after 3 to 12 days (P < 0.01). The bFGF protein levels in the days. Later, it decreased on day 7 and disappeared on MC group were higher than those in the controls at 24 day 12. In the EA group, no proliferation of vascular hours to 3 days (P < 0.05), whereas the EA group levels endothelial cells was observed for 1 to 9 hours following were higher at 12 hours to 12 days (P < 0.05, P < 0.01). MCAO. Proliferation began at 12 hours, continued to Furthermore, the bFGF protein levels in the EA group increase at 24 hours, and reached a maximum at 3 days. were higher than those in the MC group at 3 to 12 days Later, it decreased on day 7 and disappeared on day 12. (P < 0.05) and were highly significant at 12 days (P < The proliferation trend of vascular endothelial cells in 0.01), as shown in Figures 3 and 4. the EA group following MCAO was similar to that in the MC group. However, the proliferation appeared earlier Discussion and was higher. No proliferation of vascular endothelial cells was observed on the contralateral side of the infarct Angiogenesis has vital clinical significance because it pro- area in any of the phases in the two groups. In addition, motes the repair of neural structure and function following no proliferation was observed in the controls at each cerebral infarction by stimulating the endogenous repair [15] phase, as shown in Figure 2 and Table 2. mechanism . Rapid restoration of blood supply in the ischemic penumbra area is a fundamental goal of acute [10,16] cerebral infarction treatment . Anatomical studies have Increased mRNA and protein expression of angiogenesis- indicated that the facial and trigeminal nerve branches related factors following EA are distributed around DU 26 points. Stimulation of DU [17] After the operation, the expression of angiogenesis-as- 26 may lead to activation of the terminal branches of sociated factors in the cerebral vascular region differed the trigeminal nerve and facial nerve, causing excitation of among the groups (P < 0.05, P < 0.01). The Ang-1 the sphenopalatine ganglion and modulation of cerebral mRNA expression levels in the EA and MC groups vasomotion. Electrical stimulation at 2 Hz can cause the from 12 hours to 12 days were significantly higher than parasympathetic nerve to release acetylcholine, whereas those in the controls (P < 0.05, P < 0.01). In addition, electrical stimulation at ≥10 Hz can selectively release [18] the Ang-1 mRNA expression levels in the EA group peptide neurotransmitters, such as vasoactive peptides . were significantly higher than those in the MC group Previous studies have indicated that p-Akt expression in from 3 to 12 days (P < 0.05), with a marked differ- positive cells in the hippocampal CA1 region, dentate ence at day 7 (P < 0.01). Ang-1 protein levels in the gyrus, and cortex in the moderate-intensity (2–4 mA) EA EA and MC groups from 6 hours to 12 days were sig- group was significantly higher than that in the high-inten- [19] nificantly increased compared to those in the control sity (5–7 mA) EA and control group . Based on these (P < 0.01). Moreover, the Ang-1 protein levels in the results, we chose 15 Hz/2 mA as the criterion for EA in [11] EA group were considerably higher than those in the our experiments. Our previous study showed that EA at MC group from 3 to 12 days (P < 0.05). Ang-2 and DU 26 could promote the proliferation of vascular endo- PDGF-B mRNA expression levels in each phase were thelial cells around the infarct area in MCAO model rats. significantly increased in the EA and MC groups com- It is consistent with the present results, confirming that pared to those in the controls (P < 0.05, P < 0.01). The EA at DU 26 can promote angiogenesis of the penumbra Ang-2 mRNA expression levels in each phase in the EA following cerebral infarction. group were higher than those in the MC group in a We found that with the prolongation of ischemic similar phase. However, the difference was significant time, the NSSs of the two groups showed a downward from 3 to 24 hours (P < 0.05), with a highly significant trend and there were significant differences in each phase 49 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Figure 2. EA promote the proliferation of vascular endothelial cells in ischemic penumbra area of the MCAO rat. CD31 is positively expressed by staining the membrane of vascular endothelial cells in the brain tissue in green. Ki67 is positively expressed by staining the nucleus of vascular endothelial cells in red. The number of Ki67-positive cell nuclei when CD31 and Ki67 were expressed together was used to indicate the proliferation number of vascular endothelial cells. EA: Electroacupuncture; MCAO: Middle cerebral artery occlusion. immunofluorescence double-labeled detection was per - Table 2 formed on rat brain tissue. We found that around the Semi-quantitative analysis of the proliferation of infarction area, co-expression of Ki67 and CD31 began vascular endothelial cells around the infarct zone of to appear in the EA group 12 hours following MCAO, MCAO rats (n = 6) and co-expression of Ki67 and CD31 began to appear in the MC group 24 h following MCAO. With the pro- Cerebral The MC The EA The control longation of ischemia time, co-expression gradually ischemia time group group group increased, reached a peak at day 3, and subsequently, the 1 h - - - proliferation level decreased. However, co-expression 3 h - - - was still observed on day 12 in the EA group but not in 6 h - - - the MC group. We found that the co-expression of Ki67 9 h - - - and CD31 within 24 h may be due to early endothelial 12 h - + - 24 h + ++ - cell proliferation and migration to form microvascular 3 d ++ +++ - cavities. Co-expression since 3 d may be the expression 7 d + ++ - of endothelial cells that aggregated and proliferated into 12 d - - - mature blood vessels. At this time, the stronger CD31 EA: Electroacupuncture; MC: Model control; MCAO: Middle cerebral artery occlusion. fluorescence signal may be due to the maturation and “-” indicates no positive staining; “+” indicates one to three endothelial cells; “++” indicates four stability of the endothelial cells. In addition, the prolif- to six endothelial cells; “+++” indicates seven to nine endothelial cells; “++++” indicates more than nine endothelial cells. eration and expression of endothelial cells in capillary branches were observed from day 7 to 12, and it was possible to establish branch connections of the vascu- (P < 0.05), indicating that acupuncture can signifi- lar network through endothelial cell proliferation at this cantly alleviate the neurological symptoms of MCAO time, thus forming a new capillary network. In addition, rats and slow down the trend of nerve function aggra- the newly formed capillaries in the marginal zone of the vation, with the most significant improvement at 3 and ischemic brain tissue of rats in the EA group at day 12 12 days (P < 0.01). Simultaneously, Ki67 and CD31 50 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Figure 3. Ang-1, Ang-2, PDGF-B, and bFGF mRNA in the cerebral vascular of rats by RT-PCR detection. (A) Ang-1 mRNA levels. (B) Ang-2 mRNA * ** # ## levels. (C) PDGF-B mRNA levels. (D) bFGF mRNA levels. P < 0.05, P < 0.01 vs. control group; P < 0.05, P < 0.01 vs. MC group. All comparisons were made at the same time point. MC: Model control; RT-PCR: reverse transcription-polymerase chain reaction. had complicated branches and large diameters. This may endothelium. However, the binding of Ang-2 to the Tie-2 be because EA can promote the upregulation of angio- receptor does not cause receptor phosphorylation but genesis-related factors and accelerate endothelial cell blocks Ang-1–mediated Tie-2 activation, thereby pro- [25–26] proliferation and migration to form a new blood vessel moting blood vessel lengthening . We found that the network. expression of Ang-1 mRNA in the MC group began to Angiogenesis is a complex process resulting from the upregulate at 6 hours whereas that in the EA group at 3 coordinated effects of pro-angiogenic and anti-angio- hours. The expression of Ang-2 mRNA in the MC group genic factors (vascular balance theory). Their stability was upregulated at 3 hours whereas that in the EA group determines the start time and progression of the subse- at 1 hour. The expression of Ang-1 and Ang-2 in each quent angiogenesis process. Under conditions such as phase in the EA group was consistently higher than that hypoxia, inflammation, or external agents, angiogene- in the MC group. This result shows that EA can promote sis-related factors directly or indirectly activate endothe- the early high-level expression of Ang-2 to initiate the lial cells in autocrine or paracrine forms to initiate the angiogenesis mechanism in advance, thereby prolonging [20] process of angiogenesis. Previous experiments have the angiogenesis process, whereas Ang-1 appears to be confirmed that EA at DU 26 can activate the endoge- highly expressed at 3 day to induce the maturation and nous Sonic Hedgehog signaling pathway and promote stability of new blood vessels. It can be speculated that the early and high expression of related factors. The EA induces the expression of angiogenesis-related factors Shh pathway has a very close relationship with angio- by modulating the Shh pathway and coordinating their genesis-related factors and can induce the upregulation interactions, thereby promoting the growth, maturation, [21] of bFGF and balance Ang-1 and Ang-2 expression and stability of microvessels in the neurovascular net- [27] By stimulating the expression of multiple downstream work (Figure 5). bFGF is an FGF family protein that signal transduction pathways, Ang-1 regulates angiogen- shares vital biological functions, including proliferation, esis, forms stable blood vessels, and inhibits the expres- differentiation, migration, and apoptosis. It also plays a sion of cell surface adhesion molecules, inflammatory crucial role in the early and middle stages of angiogene- [22–24] cell adhesion, and apoptosis . A dynamic balance sis and is a neurotrophic factor that can directly nourish consisting of low Ang-2 and high Ang-1 levels within neurons, accelerate neural stem cell differentiation and [28–29] the central nervous system is important for blood–brain proliferation, and promote neural remodeling . We barrier integrity. The binding of Ang-1 to Tie receptors found that the expression of bFGF mRNA in the MC contributes to the induction of phosphorylation, which group began to increase at 6 h whereas that in the EA can prevent apoptotic death of cells of the vascular group at 1 hour. The mRNA and protein expression levels 51 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Figure 4. Ang-1, Ang-2, PDGF-B, and bFGF protein in the cerebral vascular of rats by Western Blotting. (A) Ang-1 protein levels. (B) Ang-2 protein # ## levels. (C) PDGF-B protein expression. (D) bFGF protein expression. *P < 0.05, **P < 0.01 vs. control group; P < 0.05, P < 0.01 vs. MC group. All comparisons were made at the same time point. MC: Model control. [32–34] in each phase in the EA group were higher than those in PDGF-B and bFGF expression in the cortex of the the MC group, and the expression level remained higher infarct area. EA can promote the early high-level expres- [35] from 9 hours to 12 days. This result indicates that EA sion of PDGF-B and bFGF, thus protecting nerves and can stimulate the stress expression of endogenous bFGF, promoting angiogenesis during the healing and remod- thereby promoting the proliferation and migration of eling of ischemic cells and tissues. Therefore, these four endothelial cells. Shh signaling pathway inhibitors can angiogenesis-related factors, Ang-1, Ang-2, PDGF-B, and also suppress PDGF-induced vascular smooth muscle bFGF, may combinedly promote the formation of the [30] cell proliferation . PDGF-B is synthesized and secreted new blood vessel network, save dying neurons, reduce by endothelial cells. It promotes blood vessel maturation brain tissue damage, complete the vascular remodeling by recruiting pericytes and stabilizing developing blood process much earlier than in the MC group, and con- [31] vessels and is important during the late stage of angio- tinue to promote angiogenesis in the infarcted area. We genesis. Ischemia can cause transient upregulation of speculate that after cerebral infarction, angiogenesis 52 Sun et al. • Volume 3 • Number 1 • 2023 www.ahmedjournal.com Jing Li and Yajie Sun wrote the first draft of this manu- script. Yajie Sun conducted experiments. Rainer Georgi and Bernhard Kolberg revised the manuscript and par- ticipated in statistical analysis. Lihong Yang conducted the immunofluorescence double-label staining experi- ments. All the authors have read and approved the final manuscript. Ethical approval of studies and informed consent The rat experimental were overseen and approved by the Beijing Municipal Science & Technology Commission and the Administrative Commission of Zhongguancun Science Park [Approval number: SCXK (Beijing) 2019-0010]. Acknowledgments None. Figure 5. Electroacupuncture induces the expression of angiogene- sis-associated factors by mediating the Shh pathway. Data availability around the infarct area is not only to increase cerebral All data generated or analyzed during this study are blood perfusion and remove metabolites, but a response included in this published article. to the reorganization of nerve function in this region, that is, the high metabolic rate in the process of neu- rological reorganization requires more blood to provide References nutrients and quickly clear metabolites, and the increase [1] Jiao Y, Liu YW, Chen WG, et al. 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Acupuncture and Herbal MedicineWolters Kluwer Health

Published: Mar 12, 2023

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