电针刺激上巨虚、天枢对溃疡性结肠炎模型大鼠Th17相关特异性因子及Treg的影响

Effects of electroacupuncture at'Shangjuxu'(ST37)and'Tianshu'(ST25)on Th17-related specificity factors and Treg cells in rats with ulcerative colitis

目的观察电针刺激上巨虚、天枢对溃疡性结肠炎(ulcerative colitis,UC)模型大鼠辅助性T细胞17(T helper cell 17,Th17)相关特异性因子及调节性T细胞(T regulatory cell,Treg)的影响,探讨合募配穴理论指导下电针刺激大肠腑下合穴、募穴治疗UC的作用机制,为特定穴的临床应用提供实验依据。方法50只SD大鼠随机分出10只作为空白组,剩余40只UC模型造模成功的大鼠随机分为模型组、合募配穴组、天枢组和上巨虚组,每组10只。2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzene sulfonic acid,TNBS)/乙醇法制备UC大鼠模型。合募配穴组予以电针刺激双侧天枢、上巨虚,天枢组予以电针刺激双侧天枢,上巨虚组予以电针刺激双侧上巨虚,每次20 min,每天1次,共10次。观察大鼠一般情况,计算大鼠疾病活动指数(disease active index,DAI);肉眼观察大鼠结肠黏膜损伤情况,评估结肠黏膜损伤指数(colonic mucosa damage index,CMDI),HE染色观察结肠形态学变化;流式细胞术检测大鼠脾脏CD4+T细胞中Treg细胞百分比,荧光定量PCR检测大鼠结肠组织中转化生长因子-β(transforming growth factor-β,TGF-β)、维甲酸相关孤儿受体γt(retinoid acid related orphan receptor gamma t,RORγt)、白细胞介素-17A(interleukin-17A,IL-17A)mRNA相对表达量。结果(1)与空白组比较,模型组一般情况不佳,肠黏膜坏死,有明显溃疡面,炎性浸润严重,DAI及CMDI评分显著升高(P<0.05),Treg细胞百分比及TGF-βmRNA相对表达量显著降低(P<0.05),RORγt、IL-17A mRNA相对表达量显著升高(P<0.05);(2)与模型组比较,合募配穴组、天枢组及上巨虚组一般情况明显好转,炎性浸润和结肠黏膜坏死情况明显减轻,DAI及CDMI评分显著降低(P<0.05),Treg细胞百分比及TGF-βmRNA相对表达量显著升高(P<0.05),RORγt、IL-17A mRNA相对表达量显著降低(P<0.05);(3)与天枢组和上巨虚组比较,合募配穴组Treg细胞百分比显著升高(P<0.05),RORγt mRNA相对表达量显著降低(P<0.05)。结论电针刺激大肠合募穴上巨虚、天枢对UC有良好的治疗作用,合募配穴疗效优于单穴疗效,其作用机制可能是通过调节Th17/Treg平衡,降低炎性细胞因子表达,以调控肠黏膜免疫失衡状态,恢复肠黏膜免疫稳态,减轻炎症反应。

Objective To observe the effects of electroacupuncture(EA)at'Shangjuxu'(ST37)and'Tianshu'(ST25),the lowe He-sea point and front-Mu point of the large intestine respectively,on T helper cell 17(Th17)-related specificity factors and T regulatory(Treg)cells in rats with ulcerative colitis(UC),and to explore the mechanism of EA at these two points in treating UC under the guidance of the theory of"He and Mu point combination",so as to provide experimental evidence for the clinical application of the specific points.Methods A total of 10 out of 50 SD rats were randomly selected as blank group,and the remaining 40,after being successfully used to establish UC models,were randomly subdivided into model group,"He and Mu point combination"group,'Tianshu'(ST25)group and'Shangjuxu'(ST37)group,with 10 rats in each group.The UC rat model was established by enema of 2-4-6 trinitrobenzene sulfonic acid(100 mg/kg)+50%ethanol(0.25 mL).EA(10 Hz/50 Hz)was respectively performed at bilateral'Shangjuxu'(ST37)and'Tianshu'(ST25)of the rats in"He and Mu point combination"group,bilateral'Shangjuxu'(ST37)in'Shangjuxu'(ST37)group and bilateral'Tianshu'(ST25)in'Tianshu'(ST25)group for 20 min,once a day,for a total of 10 times.The general condition of the rats was recorded to calculate the score of disease activity index(DAI,0-4 points).Colonic tissue was sampled for colonic mucosal damage index(CMDI)scoring by naked eyes,and histopathological changes were observed by HE staining.Flow cytometry was used to determine the percentage of Treg cells in rat spleen CD4+T cells.The relative quantity of transforming growth factor-β(TGF-β),retinoid acid related orphan receptor gammat(RORγt)and interleukin-17A(IL-17A)mRNA in colonic tissue was examined by quantitative real-time PCR.Results Compared with blank group,with poor general condition,intestinal mucosal necrosis,obvious ulcers and severe inflammatory infiltration,the scores of DAI and CMDI in model group significantly increased(P<0.05),so did the relative quantity of RORγt and IL-17A mRNA(P<0.05),while the percentage of Treg cells,and the relative quantity of TGF-βmRNA significantly decreased(P<0.05);compared with model group,with the better general condition and obviously alleviated inflammatory infiltration and intestinal mucosal necrosis,the scores of DAI and CDMI in"He and Mu point combination"group,'Tianshu'(ST25)group and'Shangjuxu'(ST37)group significantly dropped(P<0.05),so did the relative quantity of RORγt and IL-17A mRNA(P<0.05),while the percentage of Treg cells and the relative quantity of TGF-βmRNA significantly elevated(P<0.05);in contrast with'Tianshu'(ST25)group and'Shangjuxu'(ST37)group,the percentage of Treg cells in"He and Mu point combination"group significantly went up(P<0.05),while the relative quantity of RORγt mRNA significantly went down(P<0.05).Conclusion EA at'Shangjuxu'(ST37)and'Tianshu'(ST25)under the guidance of"He and Mu point combination"theory has good therapeutic effects on UC rats.The efficacy of He and Mu point combination is better than that of single acupoint selection,and its mechanism of action may be regulating Th17/Treg balance and reducing the expression of inflammatory cytokines,so as to regulate intestinal mucosal immune imbalance,restore intestinal mucosal immune homeostasis and relieve inflammatory response.