摘要目的:探究针灸足三里穴(Stomach-36，ST36)通过调控外周免疫细胞及细胞因子对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis，EAE)疾病的影响。方法:对实验鼠随机分为两组，EAE组和针灸组，并且进行体重监测以及临床症状评估，苏木精伊红染色(hematoxylin-eosin staining，HE)检测脊髓炎性细胞浸润以及免疫荧光检测脱髓鞘情况，流式细胞术检测T细胞亚群的比例，超敏多因子电化学发光技术(Meso scale discovery，MSD)检测外周血清中相关细胞因子的浓度。结果:成功构建EAE小鼠模型并给予针灸治疗。临床评分显示，EAE组在第22天达到高峰期，发病分数为2.5分，针灸组在第24天达到高峰期，发病分数为2分。疾病起病时间统计结果显示，EAE组在第12天开始起病，针灸组在第17天开始起病，针灸组比EAE组的起病时间明显较晚。HE及免疫荧光检测结果显示，针灸组的脊髓炎性细胞浸润较少，髓鞘结构完整，边缘较光滑。流式细胞术实验结果显示，针灸组CD4 +IFN-γ +T、CD4 +IL-17 +T细胞比例较EAE组显著下降，差异具有统计学意义[(0.49±0.10)%比(0.92±0.25)%，(0.21±0.05)%比(0.34±0.04)%， t值分别为2.91和3.70， P值均<0.05]；针灸组CD4 +IL-4 +T、CD4 +Foxp3 +、CD8 +Foxp3 +细胞比例较EAE组明显上调，差异具有统计学意义[(0.78±0.22)%比(0.5±0.07)%，(1.38±0.23)%比(0.9±0.08)%，(0.38±0.12)%比(0.14±0.09)%， t值分别为2.55、3.35和4.16， P值均<0.05)]。MSD检测外周血清细胞因子结果显示，针灸组抑炎性细胞因子白细胞介素(interleukin，IL)-5和IL-16的浓度较EAE组明显上升，促炎性细胞因子干扰素-γ(interferon-γ，IFN-γ)的浓度较EAE组明显下降，差异均具有统计学意义[(4.53±0.95)pg/mL比(2.78±1.25)pg/mL，(2298.77±298.67)pg/mL比(1402.28±216.30)pg/mL，(6.6±0.548)pg/mL比(8.84±0.98)pg/mL， t值分别为9.00、4.84和3.57， P值均<0.05)]。本研究实验结果显示，针灸治疗后抑制辅助性T细胞( helper T cell，Th)1/Th17并促进Th2的比例，同时上调调节性T细胞(regulatory T cell，Treg)的比例。针灸治疗后，促进抑炎因子IL-5，IL-16的表达，抑制促炎因子IFN-γ的表达。 结论:针灸ST36通过纠正T细胞亚群的失衡，调节细胞因子的表达，治疗缓解EAE疾病。

abstractsObjective:To explore the effect of acupuncture at Zusanli (Stomach-36, ST36) on alleviating experimental autoimmune encephalomyelitis (EAE) by regulating immune cells and cytokines of peripheral immune system.Methods:Experimental mice were randomly divided into EAE group and acupuncture group. The body weight and clinical symptom of the experimental mice were monitored. The infiltration of inflammatory cells in spinal cord was detected by hematoxylin-eosin staining (HE) staining, and demyelination was detected by immunofluorescence. The proportion of T cell subtypes was detected by flow cytometry, and the concentration of related cytokines in peripheral serum was detected by Meso scale discovery (MSD).Results:EAE mouse model was successfully constructed and treated with acupuncture. The clinical score showed that EAE group reached the peak on the 22nd day with the score of 2.5 points. Acupuncture group reached the peak on the 24th day with the score of 2 points. The onset time of EAE group began on the 12th day, and that of acupuncture group began on the 17th day. The onset time of acupuncture group was later than that of EAE group. The results of HE and immunofluorescence showed that the spinal cord inflammatory cell infiltration was less, the myelin sheath structure was complete and the edge was smooth in the acupuncture group. The results of flow cytometry showed that the proportion of CD4 + IFN-γ + T cells and CD4 + IL-17 + T cells in acupuncture group was decreased significantly compared with EAE group [(0.49±0.10)% vs (0.92±0.25)%, (0.21±0.05)% vs (0.34±0.04)%, t values were 2.91 and 3.70 respectively, both P values<0.05]. The proportion of CD4 + IL-4 + T, CD4 + Foxp3 + and CD8 + Foxp3 + cells in acupuncture group was significantly higher than that in EAE group [(0.78±0.22)% vs (0.5±0.07)%, (1.38±0.23)% vs (0.9±0.08)%, (0.38±0.12)% vs (0.14± 0.09)%, t values were 2.55, 3.35 and 4.16 respectively, all P values <0.05]. The results of peripheral serum cytokines detected by MSD showed that the concentrations of anti-inflammatory cytokines interleukin (IL)-5 and IL-16 in acupuncture group were significantly higher than those in EAE group, and the concentration of pro-inflammatory cytokines IFN-γ was significantly lower than that in EAE group [(4.53±0.95) pg/mL vs (2.78±1.25) pg/mL, (2298.77±298.67) pg/mL vs (1402.28± 216.30) pg/mL, (6.6±0.548) pg/mL vs (8.84±0.98) pg/mL, t values were 9.00, 4.84 and 3.57 respectively, all P values <0.05)]. The results of this study showed that acupuncture treatment may inhibit the proportion of helper T cell (th) 1/Th17, promot the percentage of Th2, and increase the proportion of regulatory T cell (Treg). In addition, acupuncture treatment also can promote the expression of anti-inflammatory factors IL-5 and IL-16, and inhibit the expression of pro-inflammatory factor interferon-γ(IFN-γ). Conclusion:Acupuncture ST36 could alleviate EAE by correcting the imbalance of T cell subtypes and regulating the expression of cytokines.