AIMS:This study aimed to elucidate the autonomic pathways underlying the cardioprotective effect of electroacupuncture (EA) at Neiguan (PC6) after myocardial infarction (MI). Specifically, we investigated whether these protective effects are mediated through a sympathetic efferent anti-inflammatory pathway, rather than the classical vagal efferent anti-inflammatory pathways. METHODS AND RESULTS:In a mouse MI model, EA intervention was combined with surgical and pharmacological manipulations, including right cervical vagotomy, splenectomy, adrenalectomy, and peripheral sympathetic blockade. Unilateral (left or right) and bilateral EA stimulations at PC6 were applied to characterize the side-specific involvement of vagal afferent fibers. Cardiac function was assessed via M-mode echocardiography, infarct size was measured using TTC staining, and macrophage infiltration and M2 polarization in infarcted myocardium were analyzed by flow cytometry. EA markedly reduced infarct size and improved cardiac function, as evidenced by increased left ventricular ejection fraction and fractional shortening. Concurrently, EA alleviated excessive macrophage accumulation in the infarcted myocardium and promoted M2 macrophage polarization, accompanied by elevated IL-10 levels that positively correlated with the proportion of M2 macrophages. Right cervical vagotomy completely abrogated the cardioprotective effects of right-sided EA (due to total blockade of afferent input), while left sided and bilateral EA remained effective, indicating that the vagal afferent signaling is the primary vagal component mediating EA's effects. Furthermore, neither splenectomy nor adrenalectomy weakened EA's beneficial effects on cardiac function or its promotion of M2 polarization, ruling out the classical vagal-splenic and vagal-adrenal reflexes as the principal mediating pathways. In contrast, chemical blockade of peripheral sympathetic activity abolished these cardioprotective and immunomodulatory benefits of EA, demonstrating that intact sympathetic signaling is essential for EA-mediated cardioprotection and macrophage polarization after MI. CONCLUSION:Our findings demonstrate that EA at PC6 improves cardiac function after MI by promoting M2 polarization of cardiac macrophages, predominantly through a vagal afferent-sympathetic efferent neuroimmune pathway, whereas the classical vagal-splenic and vagal-adrenal pathways are not required.

Myocardial infarction (MI) triggers maladaptive sympathetic overactivation, which acts as a key driver of adverse cardiac remodeling and progression to heart failure. Electroacupuncture (EA) at the Neiguan (PC6) acupoint exhibits reliable cardioprotective effects, yet its central neural mechanisms remain incompletely clarified. This study investigated whether EA at PC6 improves post-MI outcomes by modulating the hypothalamic paraventricular nucleus-superior cervical ganglion (PVN-SCG) sympathetic axis. MI was surgically induced by permanent ligation of the left anterior descending coronary artery in mice, followed by consecutive EA treatment at PC6. Cardiac function was assessed by echocardiography, while sympathetic activity was evaluated via heart rate variability (HRV) analysis and norepinephrine levels of serum and heart. Myocardial fibrosis and key molecular markers within the PVN-SCG axis were systematically analyzed. In this mice MI model, EA at PC6 significantly improved cardiac function as indicated by ejection fraction and attenuated ventricular dilation. Mechanistically, EA suppressed systemic sympathetic activity, as evidenced by decreased serum and cardiac norepinephrine levels as well as normalized HRV parameters. Notably, EA markedly inhibited neuronal hyperactivity (c-fos expression) in both the PVN and SCG. Peripherally, EA mitigated cardiac sympathetic remodeling by downregulating tyrosine hydroxylase (TH) and synaptophysin (SYN) expression, accompanied by reduced myocardial fibrosis and collagen deposition. These findings demonstrate that EA at PC6 exerts s potent cardioprotection against post-MI injury by specifically inhibiting sympathetic hyperactivation at both central (PVN-SCG axis) and peripheral levels. This study identifies a novel neuromodulatory mechanism underlying EA therapy and supports PC6 EA as a promising non-pharmacological strategy for post-MI cardiac protection.

BACKGROUND:Acupuncture at different traditional acupuncture point locations activates different autonomic pathways and modulates systemic inflammation. Electroacupuncture preconditioning (EAP) at PC6 or ST36 has been demonstrated to be cardioprotective during acute myocardial ischemia (MI) injury, but the mechanism remains unclear. As inflammation is crucial for repair post-MI, we aimed to investigate whether the protective effect of EAP at PC6 or ST36 is related to the suppression of inflammation and whether autonomic pathways are involved. METHODS:Mice underwent ligation of the left anterior descending (LAD) coronary artery to generate model MI after 3 days of EAP at PC6 or ST36. Echocardiography and triphenyltetrazolium chloride (TTC) staining were used to evaluate cardiac function and myocardial infarction area. Cardiac troponin (cTn)I and norepinephrine (NE) were measured by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was employed to determine macrophage M1/M2 phenotypic polarization, and the levels of pro- and anti-inflammatory cytokines in cardiac tissue were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, unilateral cervical vagotomy and chemical sympathetic blockade were employed to clarify specific autonomic pathways. RESULTS:EAP at PC6 or ST36 preserved cardiac function, reduced myocardial infarct size, decreased serum cTnI levels, promoted M2 macrophage polarization and decreased the cardiac pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-18 and nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3). Compared to EAP at ST36, EAP at PC6 yielded a better cardioprotective effect and regulated the anti-inflammatory cytokine transforming growth factor (TGF)-β. Moreover, sympathetic nerves were involved in the protective effect of EAP at PC6, while the vagus nerve was necessary for EAP at ST36. CONCLUSION:Our findings suggest that EAP at PC6 or ST36 activates different autonomic pathways and modulates M2 macrophage polarization, resulting in the suppression of myocardial inflammation.

OBJECTIVE:Electroacupuncture (EA) at Neiguan (PC6) acupoint confers protection against myocardial injury, though its precise neuroimmune mechanisms are not well understood. This study investigates whether EA-mediated attenuation of cardiac inflammation occurs through localized sympathetic and splenic regulation. METHODS:Acute myocardial infarction was induced in mice by coronary artery ligation. Preoperative EA was administered bilaterally at PC6 (2/15 Hz, 1 mA, 20 min/d for 3 d). Cardiac function was assessed by echocardiography. Myocardial inflammation was evaluated by hematoxylin and eosin staining, and infarct size was measured by 2,3,5-triphenyltetrazolium chloride staining. Macrophage recruitment and polarization (toward M2) were analyzed using flow cytometry. Serum norepinephrine, acetylcholine and pro-inflammatory cytokines were measured using enzyme-linked immunosorbent assay, whereas myocardial cytokines were evaluated using immunohistochemistry staining. Splenic sympathetic activity was indexed through tyrosine hydroxylase expression via immunofluorescence staining. The neural pathways were dissected through chemical sympathectomy, splenic denervation, and adoptive splenocyte transfer. RESULTS:EA at PC6 significantly attenuated myocardial inflammation by suppressing cardiac sympathetic activity, downregulating pro-inflammatory cytokines interleukin-6, interleukin-1β and tumor necrosis factor-α, and promoting M2 macrophage polarization. These effects correlated with reduced infarct size, diminished immune cell infiltration and improved cardiac function. Chemical sympathectomy mimicked EA's cardioprotective profile. Splenic denervation exacerbated systemic inflammation but did not impair EA-driven macrophage phenotypic switching. Adoptive splenocyte transfer, regardless of donor EA pretreatment, facilitated cardiac repair, arguing against a splenocyte-mediated mechanism for EA-induced cardioprotection. CONCLUSION:EA at PC6 exerts its cardioprotective effect primarily through modulating local cardiac sympathetic hyperactivity, which rebalances macrophage polarization toward the M2 phenotype and mitigates inflammation-induced injury. While the spleen contributes to systemic immunity, it plays a minimal role in EA-driven myocardial repair. Please cite this article as: Jiang MJ, Peng R, Lu XH, Yan YH, Qian DY, Zou LY, Liu XE, Chen LY, Bai H, Zhuang Y, Yu ML, Lu SF. Cardiac-specific sympathetic modulation, not splenic innervation, mediates PC6 electroacupuncture-induced cardioprotection via M2 macrophage polarization in mice with acute myocardial infarction. J Integr Med. 2026; 24(4):573-584.

Macrophages orchestrate the immune microenvironment during skin wound healing. While acupuncture’s efficacy in accelerating wound healing is established, its underlying mechanisms, particularly those related to macrophage modulation, remain poorly characterized. This study aimed to investigate how acupuncture modulates macrophage phenotype and inflammatory responses to facilitate skin repair. We established an 8-mm full-thickness dorsal skin defect model in C57BL/6 J mice, randomizing them into control and acupuncture groups. To investigate the role of the calcitonin gene-related peptide (CGRP) pathway, the CGRP receptor antagonist BIBN4096 was administered intradermally before each acupuncture treatment. For the acupuncture group, we performed a daily 20-min intervention for 10 days, which consisted of oblique manual needling at four predefined locations around the wound. Wound repair quality, inflammatory cytokine levels, and macrophage polarization were assessed using histological analysis (H E and Masson's staining), flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Acupuncture significantly facilitated wound closure, enhanced collagen deposition, and improved tissue repair quality. These benefits were associated with an immunomodulatory effect, characterized by enhanced M2 macrophage polarization within the wounds, a reduction in systemic macrophage load in the spleen, and reduced local and systemic levels of IL-1β and IL-6. Mechanistically, the activation of the CGRP-RAMP1-TSP-1 pathway was critical, as its inhibition with BIBN4096 abrogated the effects of acupuncture on macrophage polarization and wound healing. Notably, the suppression of inflammatory cytokines by acupuncture was only partially dependent on CGRP signaling. Our findings indicate that acupuncture promotes wound healing and inflammation resolution, at least in part, by activating the CGRP-RAMP1-TSP-1 pathway to drive M2 macrophage polarization. Furthermore, the persistence of its anti-inflammatory effects after CGRP inhibition strongly suggests the involvement of additional, non-CGRP-dependent pathways in modulating the immune response.

Electroacupuncture (EA) at the Neiguan acupoint (PC6) has shown significant cardioprotective effects. Sympathetic nerves play an important role in maintaining cardiac function after myocardial infarction (MI). Previous studies have found that EA treatment may improve cardiac function by modulating sympathetic remodeling after MI. However, the mechanism in how EA affects sympathetic remodeling and improves cardiac function remains unclear. The aim of this study is to investigate the cardioprotective mechanism of EA after myocardial ischemic injury by improving sympathetic remodeling and promoting macrophage M2 polarization. We established a mouse model of MI by occluding coronary arteries in male C57/BL6 mice. EA treatment was performed at the PC6 with current intensity (1 mA) and frequency (2/15 Hz). Cardiac function was evaluated using echocardiography. Heart rate variability in mice was assessed via standard electrocardiography. Myocardial fibrosis was evaluated by Sirius red staining. Levels of inflammatory factors were assessed using RT‐qPCR. Sympathetic nerve remodeling was assessed through ELISA, western blotting, immunohistochemistry, and immunofluorescence staining. Macrophage polarization was evaluated using flow cytometry. Our results indicated that cardiac systolic function improved significantly after EA treatment, with an increase in fractional shortening and ejection fraction. Myocardial fibrosis was significantly mitigated in the EA group. The sympathetic nerve marker tyrosine hydroxylase and the nerve sprouting marker growth‐associated Protein 43 were significantly reduced in the EA group, indicating that sympathetic remodeling was significantly reduced. EA treatment also promoted macrophage M2 polarization, reduced levels of inflammatory factors TNF‐α, IL‐1β, and IL‐6, and decreased macrophage‐associated nerve growth factor in myocardial tissue. To sum up, our results suggest that EA at PC6 attenuates sympathetic remodeling after MI to promote macrophage M2 polarization and improve cardiac function.

Objective To observe the effect of electroacupuncture(EA)at"Neiguan"(PC6)on pain response in mice injected with complete Freund's adjuvant(CFA)in the hind paw,so as to investigate the mechanism of orexin 1 receptor(OX1R)-endogenous cannabinoid 1 receptor(CB1R)pathway in acupuncture analgesia.Methods A total of 48 male C57BL/6 mice were used in the present study.In the first part of this study,18 mice were randomized into control,model and EA groups,with 6 mice in each group.In the second part of this study,30 mice were randomized into control,model,EA,EA+Naloxone,EA+OX1R antagonist(SB33486)groups,with 6 mice in each group.Inflammatory pain model was established by subcutaneous injection of 20 μL CFA solution in the left hind paw.EA(2 Hz,2 mA)was applied to bilateral PC6 for 20 min,once a day for 5 consecutive days.The mice in the EA+ Naloxone and EA+SB33486 groups were intraperitoneally injected with naloxone(10 mg/kg)or SB33486(15 mg/kg)15 min before EA intervention on day 5,respectively.Tail-flick method and Von Frey method were used to detect the thermal pain threshold and mechanical pain threshold of mice.Quantitative real-time PCR was used to detect the expression level of β-endorphin mRNA in periaqueductal gray(PAG)of mice.The expression of OX1R positive cells in the lateral hypothalamic area(LH)and CB1R positive cells in the ventrolateral periaqueductal gray(vlPAG)were detected by immunofluorescence.Results Compared with the control group,the thermal pain threshold and mechanical pain threshold of the model group were decreased(P<0.001),the expression level of β-endorphin mRNA in PAG was decreased(P<0.001),and the numbers of OX1R positive cells in LH and CB1R positive cells in vlPAG were decreased(P<0.05,P<0.001).Compared with the model group,the thermal pain threshold and mechanical pain threshold of the EA group were significantly increased(P<0.001),and the numbers of OX1R positive cells in LH and CB1R positive cells in vlPAG were increased(P<0.01,P<0.001).Compared with the EA group,the mechanical pain threshold in the EA+SB33486 group was significantly decreased(P<0.01),but there was no significant difference in the mechanical pain threshold between the EA+Naloxone group and EA group,and the numbers of OX1R positive neurons in LH and CB1R positive neurons in vlPAG were decreased in the EA+SB33486 group(P<0.001).Conclusion EA at PC6 can achieve analgesic effect on CFA mice by activating the OX1R-CB1R pathway in the brain,and this effect is opioid-independent.

Objective To observe the effect of electroacupuncture (EA) at u201CNeiguanu201D (PC6) on pain response in mice injected with complete Freundu2019s adjuvant (CFA) in the hind paw, so as to investigate the mechanism of orexin 1 receptor (OX1R) -endogenous cannabinoid 1 receptor (CB1R) pathway in acupuncture analgesia. Methods A total of 48 male C57BL/6 mice were used in the present study. In the first part of this study, 18 mice were randomized into control, model and EA groups, with 6 mice in each group. In the second part of this study, 30 mice were randomized into control, model, EA, EA+Naloxone, EA+OX1R antagonist (SB33486) groups, with 6 mice in each group. Inflammatory pain model was established by subcutaneous injection of 20 u03BCL CFA solution in the left hind paw. EA (2 Hz, 2 mA) was applied to bilateral PC6 for 20 min, once a day for 5 consecutive days. The mice in the EA+ Naloxone and EA+SB33486 groups were intraperitoneally injected with naloxone (10 mg/kg) or SB33486 (15 mg/kg) 15 min before EA intervention on day 5, respectively. Tail-flick method and Von Frey method were used to detect the thermal pain threshold and mechanical pain threshold of mice. Quantitative real-time PCR was used to detect the expression level of u03B2-endorphin mRNA in periaqueductal gray (PAG) of mice. The expression of OX1R positive cells in the lateral hypothalamic area (LH) and CB1R positive cells in the ventrolateral periaqueductal gray (vlPAG) were detected by immunofluorescence. Results Compared with the control group, the thermal pain threshold and mechanical pain threshold of the model group were decreased (Pu0026lt;0.001), the expression level of u03B2-endorphin mRNA in PAG was decreased (Pu0026lt;0.001), and the numbers of OX1R positive cells in LH and CB1R positive cells in vlPAG were decreased (Pu0026lt;0.05, Pu0026lt;0.001). Compared with the model group, the thermal pain threshold and mechanical pain threshold of the EA group were significantly increased (Pu0026lt;0.001), and the numbers of OX1R positive cells in LH and CB1R positive cells in vlPAG were increased (Pu0026lt;0.01, Pu0026lt;0.001). Compared with the EA group, the mechanical pain threshold in the EA+SB33486 group was significantly decreased (Pu0026lt;0.01), but there was no significant difference in the mechanical pain threshold between the EA+Naloxone group and EA group, and the numbers of OX1R positive neurons in LH and CB1R positive neurons in vlPAG were decreased in the EA+SB33486 group (Pu0026lt;0.001). Conclusion EA at PC6 can achieve analgesic effect on CFA mice by activating the OX1R-CB1R pathway in the brain, and this effect is opioid-independent.

Objective To observe the effects of electroacupuncture(EA)on local inflammatory mediators and macrophage polarization,and immune cells in the spleen of mice with chronic inflammatory pain induced by complete Freund's adjuvant(CFA)in the hind paw,so as to investigate the immunoinflammatory regulatory mechanisms of EA in relieving pain and swelling in mice with chronic inflammatory pain.Methods Thirty C57BL/6 mice were randomly di-vided into control,model,and EA groups,with 10 mice in each group.Chronic inflammatory pain model were estab-lished by subcutaneous injection of 20 μL CFA solution in the left hind paw for 7 consecutive days.After modeling,mice in the EA group received EA at bilateral"Zusanli"(ST36)for 20 min(2 Hz/100 Hz,1 mA)once a day for 18 consecu-tive days.Mechanical pain threshold,heat pain thresholds,and paw thickness were measured before and after mode-ling,and after interventions.Western blot was used to detect the expression of tumor necrosis factor-alpha(TNF-α),interleukin(IL)-1β,and NOD-like receptor protein 3(NLRP3)in the paw tissue.Immunohistochemistry was used to de-tect the positive expression of M1-type macrophage marker inducible nitric oride synthase(iNOS)and M2-type marker CD206 in the paw,and flow cytometry was used to detect the proportion of F4/80+ CD11b+ macrophages,Ly6G+ CD11b+ neutrophils,and CD25+ Foxp3+ regulatory T cells(Treg)in the spleen.Results Compared with the control group,mechanical pain and heat pain thresholds were significantly reduced(P<0.0001),while paw thickness,expres-sions of IL-1β,TNF-α,and NLRP3 in the paw,and positive expression of M1 macrophage marker iNOS in the paw,the proportions of macrophages and neutrophils in the spleen were significantly increased(P<0.0001,P<0.001)in the model group.Compared with the model group,mechanical pain threshold and heat pain thresholds,CD206 positive expression in the paw,and Treg cell proportion in spleen were significantly increased(P<0.01),while paw thickness,the expressions of IL-1β,TNF-α and NLRP3 in the paw,as well as the positive expression of M1 macrophage marker iNOS in the paw,the proportions of macrophages and neutrophils in the spleen were significantly reduced(P<0.001,P<0.01,P<0.05)in mice of the EA group after intervention.Conclusion EA may alleviate pain and swelling in mice with chronic inflammatory pain by regulating the numbers of macrophages,neutrophils,and Treg cells,as well as promoting M2 polarization of local macrophages and inhibiting the release of pro-inflammatory cytokines.

OBJECTIVE: To explore whether the regulation of matrix metalloproteinase 9(MMP-9)/tissue inhibitors of MMPs(TIMPs) gene expression through histone acetylation is a possible mechanism by which electroacupuncture(EA)protects blood-brain barrier(BBB) integrity in a middle cerebral artery occlusion(MCAO) rat model.METHODS: Male Sprague-Dawley rats were divided into four groups: the sham group, the MCAO group, the MCAO + EA(MEA) group, and the MCAO + EA + HAT inhibitor(HATi) group. The MCAO model was generated by blocking the middle cerebral artery. EA was applied to Baihui(GV20). Samples were collected 1 or 3 d after reperfusion. Neurological function scores and Evans blue extravasation were employed to evaluate the poststroke injury. The effect of EA on MMP-9/TIMPs gene expression was assessed by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) and chromatin immunoprecipitation(ChIP). RESULTS: Our results showed that EA treatment prominently improved neurological function and ameliorated BBB disruption. The RT-qPCR assay showed that EA reduced the expression of MMP-9 and promoted TIMP-2 mRNA expression, but HATi reversed these effects of EA. In addition, ChIP results revealed that EA decreased the enrichment of H3K9ace/H3K27ace at MMP-9 promoters and notably stimulated the recruitment of H3K9ace/H3K27ace at TIMP-2 promoter. CONCLUSION: EA treatment at Baihui(GV20) regulates the transcription of MMP-9 and TIMP-2 through histone acetylation modification in the acute stage of stroke, which preserves the structural integrity of the BBB in MCAO rats. These findings suggested that the histone acetylation-mediated transcriptional activity of target genes may be a crucial mechanism of EA treatment in stroke.

目的 观察电针对心肌缺血损伤小鼠心功能及心脏组织中巨噬细胞极化、TLR4、MyD88等表达的影响,探讨电针促进心肌损伤保护的可能机制.方法 27只雄性C57BL/6J小鼠随机分为假手术组、模型组、电针组,每组9只.模型组、电针组通过结扎心脏冠状动脉左前降支建立心肌缺血模型.电针组于造模成功后,电针小鼠双侧"内关"穴治疗:2/15 Hz,疏密波,1 mA,20 min·次-1,每日1次,共治疗7 d.运用超声心动图评价小鼠心脏射血分数(EF)及短轴收缩率(FS),天狼星红染色观察小鼠心脏纤维化情况,流式细胞术检测心肌组织中中性粒细胞、巨噬细胞数量及各表型巨噬细胞占比,qPCR法检测心肌组织中TNF-α、IL-1β的mRNA表达量,Western blot法检测心肌组织中TLR4、MyD88、IL-1β、IL-17A的蛋白表达水平.结果 与假手术组相比,模型组小鼠EF、FS值下降(P<0.0001),胶原容积分数升高(P<0.001),心脏组织中中性粒细胞、巨噬细胞数增多(P<0.0001,P<0.001),同时M1型巨噬细胞占比增高(P<0.05),心肌组织中TNF-α、IL-1β的mRNA表达量上升(P<0.01,P<0.001),TLR4、MyD88、IL-1β、IL-17A蛋白表达水平上升(P<0.05,P<0.01);与模型组相比,电针组小鼠EF、FS值升高(P<0.01,P<0.001),胶原容积分数降低(P<0.001),心脏组织中中性粒细胞、巨噬细胞数减少(P<0.0001,P<0.01),其中M2型巨噬细胞占比增高(P<0.05),心肌组织中TNF-α、IL-1β的mRNA表达量下降(P<0.001),TLR4、MyD88、IL-1β、IL-17A蛋白表达水平降低(P<0.05,P<0.001).结论 电针可能通过降低TLR4、MyD88在心肌组织中的表达,促进心肌缺血损伤后心脏巨噬细胞向M2型极化,减轻局部炎症,抑制心肌纤维化,实现心肌保护效应.

Carfilzomib (CFZ), a proteasome inhibitor commonly used in the treatment of multiple myeloma (MM), exhibits limited clinical application due to its cardiotoxicity. In our study, electroacupuncture (EA) at Neiguan acupoint (PC6) effectively reversed CFZ-induced reduction in ejection fraction (EF) and fractional shortening (FS), demonstrating great potential effect for heart protection. Through comparative analysis of the transcriptome profile from heart samples of mice treated with DMSO control, CFZ injection, and EA stimulation, we identified a total of 770 differentially expressed genes (DEGs) in CFZ (vs. Control) group and 329 DEGs in EA (vs. CFZ) group. Specifically, CFZ (vs. Control) group exhibited 65 up-regulated DEGs and 705 down-regulated DEGs, while EA (vs. CFZ) group displayed 251 up-regulated DEGs and 78 down-regulated DEGs. Metascape analysis revealed that among these treatment groups, there were 137 co-expressed DEGs remarkably enriched in skeletal system development, cellular response to growth factor stimulus, negative regulation of Wnt signaling pathway, and muscle contraction. The expression patterns of miR-8114, Myl4, Col1a1, Tmem163, Myl7, Sln, and Fxyd3, which belong to the top 30 DEGs, were verified by quantitative real-time PCR (RT-qPCR). In summary, this study firstly discloses novel insights into the regulatory mechanisms underlying PC6-based EA therapy against CFZ-induced cardiotoxicity, potentially serving as a theoretical foundation for further clinical applications.

BACKGROUND:Accumulating evidence suggests that acupuncture may serve as a potent strategy to mitigate the deleterious effects of ischemic stroke on neural tissue. The present investigation delineated the neuroprotective potential of electroacupuncture (EA) administered pre-and post-stroke, with a focus on determining the commonalities and disparities between these two therapeutic approaches in ameliorating ischemic stroke-induced brain injury. The ultimate objective is to inform optimal timing for acupuncture intervention in the clinical management and prevention of stroke.METHODS:The extent of cerebral infarction was quantified with 2,3,5-triphenyltetrazolium chloride staining. The integrity of the blood-brain barrier was assessed by evaluating the extravasation of Evans blue (EB) dye, while neurological function was appraised using the Longa neurological scoring system. RNA sequencing was employed to examine the transcriptomic landscape of ischemic brain tissue, with subsequent bioinformatics annotation of the sequencing data facilitated by Metascape.RESULTS:(1) A notable decrease in the ischemic infarct volume was observed in both the EA-preconditioned plus middle cerebral artery occlusion (MCAO), EA-preconditioned plus middle cerebral artery occlusion (EAM) and MCAO plus EA-treated (MEA) groups, compared to the MCAO group. Furthermore, the decreased leakage of EB and reduction in neurological function impairment scores were evident in the EAM and MEA groups compared with the MCAO group. (2) Relative to the Sham group, the MCAO group exhibited a total of 4798 differentially expressed genes (DEGs), with 67.84% demonstrating an expression fold change (FC) greater than 1.5, and 34.16% exceeding a FC of 2. The EAM and MEA groups displayed 4020 and 1956 DEGs, respectively, compared to the MCAO group. In both groups, more than 55% of DEGs showed an expression FC surpassing 1.5, whereas only approximately 10% exhibited a change greater than 2-fold. Remarkably, EA preconditioning and EA treatment resulted in the reversal of 18.72% and 28.91% of DEGs, respectively, in the MCAO group. (3) The DEGs upregulated in response to ischemic stroke were predominantly implicated in immune inflammatory processes and cellular apoptosis, whereas the downregulated DEGs were associated with neurogenesis and neuronal signal transduction. The MEA-induced upregulated DEGs were primarily involved in neural transmission and metabolic processes, whereas the downregulated DEGs were linked to excessive inflammatory responses to physical and chemical stimuli, as well as cell matrix adhesion chemotaxis. In the context of EAM, the upregulated DEGs were chiefly related to protein biosynthesis, and energy and metabolic processes, whereas the downregulated genes were connected to gene transcriptional activity, synaptic function, and neuronal architecture.CONCLUSIONS:Both preconditioning and post-event treatment with acupuncture demonstrated efficacy in mitigating pathological damage to brain tissue in a rat model of ischemic stroke, albeit with some divergences in their gene targets. The integration of EA preconditioning and treatment may potentially confer enhanced neuroprotection in the clinical management of stroke patients.

Background: Electro-acupuncture (EA) is an effective and safe treatment for ischemic stroke. It is not only capable of reducing cerebral damage but also alleviating intestinal inflammation. However, its mechanism has not been fully elucidated. Methods: All rats were randomly divided into three experimental groups: the SHAM group, the MCAO group, and the MEA (MCAO+EA) group. Ischemic-reperfusion (I/R) injury was induced by MCAO surgery. Rats in the MEA group were treated with EA stimulation in the "Baihui" acupoint (1 mA, 2/15 Hz, 20 min for each time). The Real-time (RT)-qPCR was used to evaluate the mRNA expression of inflammation factors in the ischemic brain and the small intestine after I/R injury. In addition, our research evaluated the effects of EA on regulatory T cells (Tregs) and gamma delta T cells in the small intestine and brain via Flow cytometry analysis. Finally, we applied CM-Dil and CFSE injection and explored the potential connections of T cells between the ischemic hemisphere and the small intestine. Results: Our results suggested that EA treatment could significantly reduce the inflammation response in the ischemic brain and small intestine 3 days after I/R injury in rats. To be specific, EA increased the percentage of Tregs in the brain and the small intestine and decreased intestinal and cerebral gamma delta T cells. Concomitantly, after EA treatment, the percentage of cerebral CD3+TCR gamma delta+CFSE+ cells dropped from 12.06% to 6.52% compared with the MCAO group. Conclusions: These findings revealed that EA could regulate the Tregs and gamma delta T cells in the ischemic brain and the small intestine, which indicated its effect on inhibiting inflammation. And, EA could inhibit the mobilization of intestinal T cells, which may contribute to the protection of EA after ischemic stroke.

Electro-acupuncture (EA) has an anti-inflammatory role in ischemic stroke, but whether the protective effect of EA involves the regulation of the intestine barrier and Treg/ γδ T cells is unclear. Cerebral ischemia-reperfusion (I/R) injury was induced by middle cerebral artery occlusion(MCAO) for 2 h followed by reperfusion for 24 h. The rats have treated with EA at the "Baihui" acupoint(GV20). Triphenyl tetrazolium chloride (TTC) staining and Longa neurologic score were performed to evaluate the outcomes after ischemic stroke. Inflammatory factor expression levels in the serum, ischemic hemisphere brain, and small intestine were detected by ELISA or RT-qPCR. Additionally, the morphology change of the small intestine was evaluated by analyzing villus height and smooth muscle thickness. Meanwhile, the expression of tight-junction proteins, including Zonula Occludens-1 (ZO-1), Occludin, and Claudin-1, were detected to evaluate the impact of EA on mucosal permeability in the small intestine. The percentages of regulatory T cells (Tregs) (CD45+CD4+Foxp3+) and γδ T cells (CD45+CD4-γδ T+) were measured to assess the effect of EA on intestinal T cells. EA decreased the brain infarction volume and intestine barrier injury in ischemic stroke rats. At the same time, it effectively suppressed the post-stroke inflammation in the brain and small intestine. More importantly, EA treatment increased the percentage of Tregs in the small intestine while reducing the rate of γδ T cells, and ultimately increased the ratio of Treg/ γδ T cells. These results demonstrated that EA ameliorated intestinal inflammation damage by regulating the Treg/ γδ T cell polarity shift and improving the intestine barrier integrity in rats with I/R injury. This may be one of the mechanisms underlying the anti-ischemic injury effects of acupuncture on stroke.

Much evidence suggests that gut immunity homeostasis plays a vital role in regulating neuroinflammatory response under ischemic stroke. Acupuncture is an effective treatment for post-stroke rehabilitation. However, little is known about the effect of acupuncture on intestinal injury after stroke. Herein, we observed the intestinal inflammatory response and barrier's impairment of the post-stroke rats to explore the mechanism of electroacupuncture (EA) against cerebral ischemia injury from the brain-gut axis. Rats were randomized to the sham operation group (SHAM), the middle cerebral artery occlusion group (MCAO), and the MCAO plus EA treatment group (MEA). Triphenyl tetrazolium chloride (TTC) staining and Longa neurologic score were performed to evaluate the outcomes after ischemic stroke. Inflammatory factors expression levels in the serum, ischemic hemisphere brain, and small intestine were detected. Additionally, the morphology change of small intestine was evaluated via analyzing villus height and smooth muscle thickness. Meanwhile, the expression of tight-junction proteins, including Zonula Occludens-1(ZO-1), Occludin and Claudin-1, was detected to evaluate the impact of EA on mucosal permeability in the small intestine. The percentage of Treg cells (CD45 + CD4 + Foxp3 + ) and γδ T cells (CD45 + CD4 − γδ T + ) were measured to assess the effect of EA on T cells. Our data showed that EA could significantly decrease the brain infarction volume and intestine barrier injury of the ischemic stroke rats. At the same time, it effectively suppressed the post-stroke inflammation response of the ischemic brain, small intestine, and peripheral serum. More importantly, EA treatment increased the percentage of Treg cells in the small intestine while reducing the rate of γδ T cells, and ultimately increased the ratio of Treg/ γδ T cells. These results suggested that EA could ameliorate intestinal inflammation damage by regulating the Treg/ γδ T cell polarity shift and improving the intestine barrier integrity post-stroke. It may be one of the mechanisms underlying the anti-ischemic injury effects of acupuncture on stroke.