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
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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 model group,the fluorescence yellow transmittance of intestinal epithelial cells,the apoptosis rate and expression levels of TRADD,RIP1 and FADD were remarkably down-regulated(P<0.001,P<0.01),and the expression of A20 was significantly up-regulated(P<0.01)in both the Moxi and medication groups.Conclusion Herbal cake-partitioned moxibustion may down-regulate the permeability of intestinal epithelial barrier and the apoptosis of intestinal epithelial cells by way of suppressing TNF-α-mediated cellular apoptosis pathway of intestinal epithelium in CD rats.
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 model group,the fluorescence yellow transmittance of intestinal epithelial cells,the apoptosis rate and expression levels of TRADD,RIP1 and FADD were remarkably down-regulated(P<0.001,P<0.01),and the expression of A20 was significantly up-regulated(P<0.01)in both the Moxi and medication groups.Conclusion Herbal cake-partitioned moxibustion may down-regulate the permeability of intestinal epithelial barrier and the apoptosis of intestinal epithelial cells by way of suppressing TNF-α-mediated cellular apoptosis pathway of intestinal epithelium in CD rats.
引文
[1]NIKOLAUS S,SCHREIBER S.Diagnostics of inflammatory bowel disease[J].Gastroenterology,2007,133(5):1670-1689.
[2]BAUMGART D C,SANDBORN W J.Crohn's disease[J].Lancet,2012,380(9853):1590-1605.
[3]严瑾,欧阳钦,刘卫平,等.肿瘤坏死因子-α在溃疡性结肠炎中的表达及其作用探讨[J].胃肠病学,2005,10(5):269-272.
[4]GUBERNATOROVA E O,TUMANOV A V.Tumor necrosis factor and lymphotoxin in regulation of intestinal inflammation[J].Biochemistry(Mosc),2016,81(11):1309-1325.
[5]KOLODZIEJ L E,LODOLCE J P,CHANG J E,et al.TN-FAIP3maintains intestinal barrier function and supports epithelial cell tight junctions[J].PLoS One,2011,6(10):e26352.
[6]BAO C H,ZHANG J Z,WU L Y,et al.Effect of electroacupuncture and herbal cake-partitioned moxibustion on anxiety and depression in patients with Crohns disease in remission[J].JAcupunct Tuina Sci,2016,14(2):87-92.
[7]包春辉,吴璐一,吴焕淦,等.针灸治疗活动期克罗恩病:随机对照研究[J].中国针灸,2016,36(7):683-688.
[8]SHANG H X,WANG A Q,BAO C H,et al.Moxibustion combined with acupuncture increases tight junction protein expression in Crohns disease patients[J].World J Gastroenterol,2015,21(16):4986-4996.
[9]施茵,包春辉,吴焕淦,等.隔药灸结合针刺对克罗恩病患者肠黏膜TNF-α、TNFR1、TNFR2表达及肠上皮细胞凋亡的影响[J].上海中医药杂志,2011,45(1):46-50.
[10]BAO C H,WU L Y,WU H G,et al.Moxibustion inhibits apoptosis and tumor necrosis factor-alpha/tumor necrosis factor receptor 1in the colonic epithelium of Crohns disease model rats[J].Dig Dis Sci,2012,57(9):2286-2295.
[11]BAO C H,WU L Y,SHI Y,et al.Moxibustion down-regulates colonic epithelial cell apoptosis and repairs tight junctions in rats with Crohn's disease[J].World J Gastroenterol,2011,17(45):4960-4970.
[12]施茵,包春辉,吴焕淦,等.艾灸对克罗恩病大鼠结肠肿瘤坏死因子-α含量及艾灸鼠结肠上清液对培养的结肠上皮细胞内紧密连接蛋白及其基因表达的影响[J].针刺研究,2011,36(4):235-241.
[13]MORRIS G P,BECK P L,HERRIDGE M S,et al.Hapteninduced model of chronic inflammation and ulceration in the rat colon[J].Gastroenterology,1989,96(3):795-803.
[14]李忠仁.《实验针灸学》[M].2版,北京:中国中医药出版社,2007.
[15]徐淑云,卞如濓,陈修.《药理实验方法学》[M].3版,北京:人民卫生出版社,2002.
[16]TURNER J R.Molecular basis of epithelial barrier regulation:from basic mechanisms to clinical application[J].Am JPathol,2006,169(6):1901-1909.
[17]VAN DULLEMEN H M,VAN DEVENTER S J,HOMMES DW,et al.Treatment of Crohns disease with anti-tumor necrosis factor chimeric monoclonal antibody(cA2)[J].Gastroenterology,1995,109(1):129-135.
[18]KINDLER V,SAPPINO A P,GRAU G E,et al.The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BGG infection[J].Cell,1989,56(5):731-740.
[19]SHI Y,ZHOU E H,WU H G,et al.Moxibustion treatment restoring the intestinal epithelium barrier in rats with Crohns disease by down-regulating tumor necrosis factor alpha,tumor necrosis factor receptor 1,and tumor necrosis factor receptor 2[J].Chin J Integr Med,2011,17(3):212-217.
[20]MICHEAU O,TSCHOPP J.Induction of TNF receptor 1-mediated apoptosis via two sequential signaling complexes[J].Cell,2003,114(2):181-190.
[21]SPRICK M R,WEIGAND M A,RIESER E,et al.FADD/MORT1and caspase-8are recruited to TRAIL receptors 1and 2and are essential for apoptosis mediated by TRAIL receptor 2[J].Immunity,2000,12(6):599-609.
[22]FLUSBERG D A,SORGER P K.Surviving apoptosis:lifedeath signaling in single cells[J].Trends Cell Biol,2015,25(8):446-458.
[23]HARPER N,HUGHES M,MACFARLANE M,et al.Fasassociated death domain protein and caspase-8are not recruited to the tumor necrosis factor receptor 1signaling complex during tumor necrosis factor-induced apoptosis[J].J Biol Chem,2003,278(28):25534-25541.
[24]STANGER B Z,LEDER P,LEE T H,et al.RIP:a novel protein containing a death domain that interacts with Fas/APO-1(CD95)in yeast and causes cell death[J].Cell,1995,81(4):513-523.
[25]SCHNEIDER-BRACHERT W,TCHIKOV V,NEUMEY-ER J,et al.Compartmentalization of TNF receptor 1signaling:internalized TNF receptosomes as death signaling vesicles[J].Immunity,2004,21(3):415-428.
[26]VEREECKE L,BEYAERT R,VAN LOO G.The ubiquitinediting enzyme A20(TNFAIP3)is a central regulator of immunopathology[J].Trends Immunol,2009,30(8):383-391.
[27]HE K L,TING A T.A20inhibits tumor necrosis factor(TNF)alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1complex in Jurkat Tcells[J].Mol Cell Biol,2002,22(17):6034-6045.
[28]KOLODZIEJ L E,LODOLCE J P,CHANG J E,et al.TN-FAIP3maintains intestinal barrier function and supports epithelial cell tight junctions[J].PLoS One,2011,6(10):e26352.
[29]JIN Z,LI Y,PITTI R,et al.Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8mediate extrinsic apoptosis signaling[J].Cell,2009,137(4):721-735.
[2]BAUMGART D C,SANDBORN W J.Crohn's disease[J].Lancet,2012,380(9853):1590-1605.
[3]严瑾,欧阳钦,刘卫平,等.肿瘤坏死因子-α在溃疡性结肠炎中的表达及其作用探讨[J].胃肠病学,2005,10(5):269-272.
[4]GUBERNATOROVA E O,TUMANOV A V.Tumor necrosis factor and lymphotoxin in regulation of intestinal inflammation[J].Biochemistry(Mosc),2016,81(11):1309-1325.
[5]KOLODZIEJ L E,LODOLCE J P,CHANG J E,et al.TN-FAIP3maintains intestinal barrier function and supports epithelial cell tight junctions[J].PLoS One,2011,6(10):e26352.
[6]BAO C H,ZHANG J Z,WU L Y,et al.Effect of electroacupuncture and herbal cake-partitioned moxibustion on anxiety and depression in patients with Crohns disease in remission[J].JAcupunct Tuina Sci,2016,14(2):87-92.
[7]包春辉,吴璐一,吴焕淦,等.针灸治疗活动期克罗恩病:随机对照研究[J].中国针灸,2016,36(7):683-688.
[8]SHANG H X,WANG A Q,BAO C H,et al.Moxibustion combined with acupuncture increases tight junction protein expression in Crohns disease patients[J].World J Gastroenterol,2015,21(16):4986-4996.
[9]施茵,包春辉,吴焕淦,等.隔药灸结合针刺对克罗恩病患者肠黏膜TNF-α、TNFR1、TNFR2表达及肠上皮细胞凋亡的影响[J].上海中医药杂志,2011,45(1):46-50.
[10]BAO C H,WU L Y,WU H G,et al.Moxibustion inhibits apoptosis and tumor necrosis factor-alpha/tumor necrosis factor receptor 1in the colonic epithelium of Crohns disease model rats[J].Dig Dis Sci,2012,57(9):2286-2295.
[11]BAO C H,WU L Y,SHI Y,et al.Moxibustion down-regulates colonic epithelial cell apoptosis and repairs tight junctions in rats with Crohn's disease[J].World J Gastroenterol,2011,17(45):4960-4970.
[12]施茵,包春辉,吴焕淦,等.艾灸对克罗恩病大鼠结肠肿瘤坏死因子-α含量及艾灸鼠结肠上清液对培养的结肠上皮细胞内紧密连接蛋白及其基因表达的影响[J].针刺研究,2011,36(4):235-241.
[13]MORRIS G P,BECK P L,HERRIDGE M S,et al.Hapteninduced model of chronic inflammation and ulceration in the rat colon[J].Gastroenterology,1989,96(3):795-803.
[14]李忠仁.《实验针灸学》[M].2版,北京:中国中医药出版社,2007.
[15]徐淑云,卞如濓,陈修.《药理实验方法学》[M].3版,北京:人民卫生出版社,2002.
[16]TURNER J R.Molecular basis of epithelial barrier regulation:from basic mechanisms to clinical application[J].Am JPathol,2006,169(6):1901-1909.
[17]VAN DULLEMEN H M,VAN DEVENTER S J,HOMMES DW,et al.Treatment of Crohns disease with anti-tumor necrosis factor chimeric monoclonal antibody(cA2)[J].Gastroenterology,1995,109(1):129-135.
[18]KINDLER V,SAPPINO A P,GRAU G E,et al.The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BGG infection[J].Cell,1989,56(5):731-740.
[19]SHI Y,ZHOU E H,WU H G,et al.Moxibustion treatment restoring the intestinal epithelium barrier in rats with Crohns disease by down-regulating tumor necrosis factor alpha,tumor necrosis factor receptor 1,and tumor necrosis factor receptor 2[J].Chin J Integr Med,2011,17(3):212-217.
[20]MICHEAU O,TSCHOPP J.Induction of TNF receptor 1-mediated apoptosis via two sequential signaling complexes[J].Cell,2003,114(2):181-190.
[21]SPRICK M R,WEIGAND M A,RIESER E,et al.FADD/MORT1and caspase-8are recruited to TRAIL receptors 1and 2and are essential for apoptosis mediated by TRAIL receptor 2[J].Immunity,2000,12(6):599-609.
[22]FLUSBERG D A,SORGER P K.Surviving apoptosis:lifedeath signaling in single cells[J].Trends Cell Biol,2015,25(8):446-458.
[23]HARPER N,HUGHES M,MACFARLANE M,et al.Fasassociated death domain protein and caspase-8are not recruited to the tumor necrosis factor receptor 1signaling complex during tumor necrosis factor-induced apoptosis[J].J Biol Chem,2003,278(28):25534-25541.
[24]STANGER B Z,LEDER P,LEE T H,et al.RIP:a novel protein containing a death domain that interacts with Fas/APO-1(CD95)in yeast and causes cell death[J].Cell,1995,81(4):513-523.
[25]SCHNEIDER-BRACHERT W,TCHIKOV V,NEUMEY-ER J,et al.Compartmentalization of TNF receptor 1signaling:internalized TNF receptosomes as death signaling vesicles[J].Immunity,2004,21(3):415-428.
[26]VEREECKE L,BEYAERT R,VAN LOO G.The ubiquitinediting enzyme A20(TNFAIP3)is a central regulator of immunopathology[J].Trends Immunol,2009,30(8):383-391.
[27]HE K L,TING A T.A20inhibits tumor necrosis factor(TNF)alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1complex in Jurkat Tcells[J].Mol Cell Biol,2002,22(17):6034-6045.
[28]KOLODZIEJ L E,LODOLCE J P,CHANG J E,et al.TN-FAIP3maintains intestinal barrier function and supports epithelial cell tight junctions[J].PLoS One,2011,6(10):e26352.
[29]JIN Z,LI Y,PITTI R,et al.Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8mediate extrinsic apoptosis signaling[J].Cell,2009,137(4):721-735.
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