隔药灸对克罗恩病大鼠结肠黏膜巨噬细胞M1/M2型极化的影响

Herbal cake-partitioned moxibustion regulates M1and M2macrophage polarization of colonic mucosa in rats with Crohn’s disease

目的:观察隔药灸对克罗恩病(CD)大鼠结肠黏膜巨噬细胞M1/M2型极化及炎性因子表达的影响,探讨其作用机制。方法:将40只雄性SD大鼠随机分为正常组、模型组、西药组、隔药灸组,每组10只。采用2,4,6-三硝基苯磺酸灌肠法制备CD模型。造模成功后隔药灸组大鼠给予隔药饼灸“天枢”“气海”,每次每穴灸2壮,每日1次,共10 d。西药组大鼠给予美沙拉嗪灌胃,每日2次,共10 d。治疗结束后取各组大鼠结肠上皮组织,分离、纯化和培养结肠黏膜固有层巨噬细胞。HE染色法观察结肠组织病理变化;透射电镜观察结肠组织超微结构;Western blot法检测结肠黏膜巨噬细胞α7烟碱型乙酰胆碱受体(α7nAChR)、核因子κB(NF-κB) p65、肿瘤坏死因子(TNF)-α的表达水平;流式细胞术检测结肠黏膜组织M1、M2型巨噬细胞的数量;免疫荧光法检测结肠黏膜组织M1、M2型巨噬细胞的表达情况。结果:与正常组比较,模型组大鼠结肠组织呈现巨大溃疡及炎性表现,结肠上皮细胞连接疏松,细胞器结构破坏;结肠黏膜巨噬细胞α7nAChR表达水平降低(P<0.01),NF-κB p65和TNF-α表达水平升高(P<0.01);结肠黏膜M1、M2型巨噬细胞数量和表达增加(P<0.01,P<0.05)。与模型组比较,隔药灸组和西药组大鼠结肠形态结构改善,肠道炎性反应减轻,结肠上皮细胞连接较紧密,细胞器形态较正常;结肠黏膜巨噬细胞α7nAChR表达水平升高(P<0.01),NF-κB p65及TNF-α表达水平降低(P<0.01),结肠黏膜M1型巨噬细胞数量和表达减少(P<0.05),M2型巨噬细胞数量和表达增加(P<0.05)。结论:隔药灸可能是通过上调CD大鼠结肠黏膜巨噬细胞α7nAChR的表达来抑制NF-κB活化,并促使巨噬细胞由M1向M2型极化,减少M1巨噬细胞比例,抑制结肠黏膜TNF-α的表达,从而有效抑制肠道炎性反应。

Objective To observe the effect of herbal cake-partitioned moxibustion(Moxi)on the expressions of inflam-matory factors and M1/M2polarization in colonic mucosal macrophages in Crohn’s disease(CD)rats,so as to explore its under-lying mechanisms in the treatment of CD.Methods Forty male SD rats were randomly divided into normal,model,Moxi and medication groups(n=10).The CD model was established by enema of 2,4,6-trinitrobenzene sulfonic acid(TNBS)solution(5%TNBS∶50%alcohol=2∶1,3mL/kg),once every 7days,4times altogether.For rats of the Moxi group,cake-partitioned moxibustion was given to“Tianshu”(ST25)and“Qihai”(CV6),two moxa-cones for each acupoint every time,once daily for 10days.For rats of the medication group,intragastric perfusion of mesalazine solution was given twice daily for 10days.After the treatment,the colonic mucosa tissue was sampled,and the macrophages were isolated,purified and cultured.The pathologi-cal changes of colon tissues were observed by H.E.staining.The ultrastructure of colon tissue was observed by transmission electron microscopy.The expression levels ofα7nAChR,NF-κB p65and TNF-αin colon mucosal macrophages were detected by Western blot.The number of M1and M2macrophages in colon mucosa was detected by flow cytometry and immunofluorescence assay.Results Compared with the normal group,the colon tissue of rats presented huge ulceration and inflammatory manifesta-tions,the junction of colon epithelial cells was loose,the structure of organelles was damaged;the expression level ofα7nAChR in macrophages of colon mucosa was significantly decreased(P<0.01),while the expression levels of NF-κB p65and TNF-α,and the number of M1and M2macrophages were increased(P<0.01,P<0.05)in the model group.In comparison with the model group,the morphology and structure of colon mucosa tissues of rats in Moxi and medication groups were improved;the expression level ofα7nAChR,the number of M2macrophage in colon mucosa were significantly increased(P<0.01,P<0.05),while the expression levels of NF-κB p65and TNF-α,and the number of M1macrophage were significantly decreased(P<0.01,P<0.05)in both the Moxi and medication groups.Conclusion Herbal cake-partitioned moxibustion may inhibit NF-κB activation by up-regulating the expression level ofα7nAChR to promote the polarization of macrophages from M1to M2type,and reduce the proportion of M1macrophages,inhibit the expression of TNF-αin colonic mucosa of CD rats,so as to relieve the intestinal inflam-mation.