隔药灸对肿瘤坏死因子-α介导克罗恩病肠上皮细胞凋亡途径的影响  

Herbal cake-partitioned moxibustion improves intestinal epithelial barrier dysfunction by suppressing TNF-α-mediated apoptosis pathway of intestinal epithelium in rats with Crohn's disease

作　　者：孙怡 周竞 郭娅静 陈柳[1] 李涛 高艳玲 王雨宁 赵继梦[1,2] 吴焕淦[1,2] 施茵[1,2] SUN Yi;ZHOU Jing;GUO Ya-jing;CHEN Liu;LI Tao;GAO Yan-ling;WANG Yu-ning;ZHAO Ji-meng;WU Huan-gan;SHI Yin(Yueyang Clinical Medical School,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shanghai Research Institute of Acupuncture-moxibustion and Meridian,Shanghai 200030)

机构地区：[1]上海中医药大学岳阳临床医学院,上海201203 [2]上海市针灸经络研究所,上海200030

出　　处：《针刺研究》2019年第1期1-7,共7页Acupuncture Research

基　　金：国家自然科学基金项目(No.81273844;81473757);国家重点基础研究发展计划"973"计划项目(No.2015CB554500)

摘　　要：目的:观察隔药灸对肿瘤坏死因子-α(TNF-α)-肿瘤坏死因子受体相关死亡结构域(TRADD)-Fas相关的死亡结构域(FADD)途径介导克罗恩病(Crohn's disease,CD)肠上皮细胞凋亡的影响,探讨其作用机制。方法:将48只SD雄性大鼠随机分成正常组、模型组、隔药灸组和西药组,每组12只。采用2,4,6-三硝基苯磺酸(TNBS)制备CD模型,造模成功后隔药灸组采用隔药饼灸"天枢""气海"穴,每日1次,每次每穴隔药灸2壮,共灸10d。西药组采用美沙拉嗪灌胃,每日2次,每次2mL,共灌胃10d。治疗结束后取各组大鼠结肠上皮组织,分离、纯化和培养结肠上皮细胞建立体外肠上皮细胞屏障模型,将100ng/mL TNF-α分别与各组肠上皮细胞孵育培养24h,应用荧光黄透过率检测各组肠上皮细胞屏障通透性,Western blot法检测各组肠上皮细胞凋亡途径相关蛋白肿瘤坏死因子受体1(TNFR1)、TRADD、受体作用蛋白1(RIP1)、FADD、锌指蛋白A20(A20)表达以及流式细胞术观察各组肠上皮细胞凋亡情况。结果:与正常组比较,模型组的荧光黄透过率显著升高(P<0.001),结肠上皮细胞TNFR1、TRADD、RIP1表达量明显升高(P<0.01),A20表达量明显降低(P<0.01),细胞凋亡率显著增高(P<0.001)。与模型组比较,隔药灸组和西药组的荧光黄透过率显著降低(P<0.001),结肠上皮细胞TRADD、RIP1、FADD表达量明显降低(P<0.01),A20表达量明显升高(P<0.01),结肠上皮细胞凋亡率显著降低(P<0.001)。结论:隔药灸可能是通过调控TNF-α介导CD肠上皮细胞凋亡途径的异常,达到保护或修复CD肠上皮屏障损伤之效应。Objective To observe the effect of herbal cake-partitioned moxibustion(Moxi)on tumor necrosis factor(TNF)-α/TNF receptor 1(TNFR1)-associated death domain(TRADD)/Fas-associated death domain(FADD)pathway-mediated apoptosis of intestinal epithelial cells in Crohn’s disease(CD)rats,so as to explore its underlying mechanisms in the treatment of CD.Methods Forty-eight SD male rats were randomly divided into normal,model,Moxi and medication groups(n=12 rats in each).The CD model was established by intra-annual perfusion of 2,4,6-trinitrobenzene sulfonic acid(TNBS)solution(TNBS∶50% alcohol=2∶1,3 mL/kg),once every 7 days,4 times altogether.For rats of the Moxi group,moxibustion was given to"Tianshu"(ST25)and"Qihai"(CV6),two moxa-cones every time,once daily for 10 days.For rats of the medication group,intragastric perfusion of mesalazine solution was given twice daily for 10 days.After the treatment,the colonic epithelium tissue was sampled.The epithelial cells were purified and cultured to establish an in vitro intestinal epithelial barrier,and added with TNF-α(apro-inflammatory factor,100 ng/mL)in the culture medium for 24 hfor making an increased epithelial permeability model.The permeability of intestinal epithelial cell barrier was evaluated by detecting the fluorescence yellow transmittance of the TNF-α-incubated cell medium.Western blot was used to detect the expression levels of TNFR1,TRADD,receptor-interacting protein 1(RIP1),FADD and zinc finger protein A20(A20,a ubiquitination enzyme for inhibiting activation of TRADD and RIP1)of the cultured intestinal epithelium cells.The apoptosis of the TNF-α-incubated intestinal epithelial cells was detected by flow cytometry.Results After modeling and compared with the normal group,the fluorescence yellow transmittance of intestinal epithelia cells,apoptosis rate,and expression levels of TNFR1,TRADD,and RIP1 proteins were significantly increased(P<0.001,P<0.01),and the expression of A20 was significantly decreased(P<0.01)in the model group.In comparison with the

关 键 词：隔药灸 克罗恩病 肿瘤坏死因子-Α 细胞凋亡途径 

分 类 号：R245.82[医药卫生—针灸推拿学]