氧化苦参碱调控RhoA/ROCK信号通路介导溃疡性结肠炎E-cadherin及TGF-β的影响
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摘要
目的:通过检测小鼠结肠组织中Rho激酶(ROCK),E-钙黏蛋白(E-cadherin)及转化生长因子-β(TGF-β)相关指标变化,探讨氧化苦参碱通过RhoA/ROCK信号通路介导上皮-间充质转化,防治溃疡性结肠炎(UC)及其相关癌变的机制。方法:48只SPF级Balb/c雄性小鼠随机分为正常组,模型组,氧化苦参碱低、中、高剂量组(25,50,100 mg·kg~(-1)),Rho激酶抑制剂(Y-27632)组(10 mg·kg~(-1))。采用3%葡聚糖硫酸钠(DSS)自由饮用1周法制备UC模型,腹腔注射的方式给药,正常组和模型组腹腔注射等体积的磷酸盐缓冲液(PBS)。自造模起,每天记录小鼠体质量、粪便的性状、隐血及肉眼血便情况,连续给药1周,第8天处死全部小鼠,评估UC小鼠疾病活动指数(DAI);对小鼠结肠进行病理学评分;采用透射电镜观察各组小鼠结肠组织超微结构变化;酶联免疫吸附法测定法(ELISA)检测结肠组织TGF-β含量;采用蛋白免疫印迹法(Western blot)及实时荧光定量PCR法(Real-time PCR)检测小鼠结肠组织Rho激酶-1(ROCK-1),Rho激酶-2(ROCK-2),E-cadherin,TGF-β蛋白及mRNA的表达。结果:与正常组比较,模型组光镜下见黏膜及黏膜下层大量炎性细胞浸润、腺体排列紊乱、伴有不同程度肠黏膜缺损、甚有溃疡形成,电镜下见肠上皮细胞表面微绒毛稀疏,细胞连接间隙增宽,杯状细胞减少,细胞器肿胀,DAI评分显著升高(P <0. 01),结肠组织ROCK-1,ROCK-2蛋白和mRNA含量均显著升高(P <0. 01),结肠长度显著降低(P <0. 01),结肠组织E-cadherin,TGF-β蛋白和mRNA表达显著减少(P <0. 01);与模型组比较,各治疗组小鼠光镜及电镜下病理表现均有不同程度的改善,DAI评分显著降低(P <0. 01),结肠长度显著增加(P <0. 01),结肠组织ROCK-1,ROCK-2蛋白和mRNA表达水平显著下降(P <0. 01),E-cadherin,TGF-β蛋白和mRNA表达显著上升(P <0. 01);与氧化苦参碱中剂量组比较,氧化苦参碱低、高剂量组ROCK-1,ROCK-2蛋白和mRNA含量均明显升高(P <0. 05,P <0. 01),TGF-β和E-cadherin蛋白和mRNA含量显著降低(P <0. 01)。结论:氧化苦参碱可通过下调Rho激酶表达,上调E-cadherin和TGF-β表达,诱导肠上皮细胞凋亡,介导上皮-间充质转化,从而达到缓解溃疡性结肠炎的功效。
Objective: To investigate the mechanism of Oxymatrine on epithelial-mesenchymal transition mediated by RhoA/Rho-associated kinase( ROCK) signaling pathway to prevent and treat ulcerative colitis( UC)and its related canceration by detecting the changes of ROCK,E-cadherin and transforming growth factor-β( TGF-β) in colon tissues of mice. Method: Totally 48 male Balb/c mice were randomly divided into normal control group,model group,low,medium and high-dose Oxymatrine groups( 25,50,100 mg·kg~(-1)) and Y-27632 group( 10 mg·kg~(-1)),with 8 mice in each group. Mice in control group received distilled water,while all the other mice were treated with 3% dextra sulfate sodium for 7 days to induce the ulcerative colitis model. Since the first day of modeling,Y-27632( 10 mg·kg~(-1)) and different doses of Oxymatrine( 25,50,100 mg·kg~(-1))were intraperitoneally injectedfor 7 days,and equal volume of PBS was intraperitoneally injected in normal group and model group. Body weight loss,stool consistency and fecal blood loss were observed on a daily basis. On the8 thday,mice were put to death,colon was collected and its length was measured; the scores of disease activity index( DAI) were evaluated; part of the colons were fixed and stained with hematoxylin and eosin( HE) for a histopathological analysis; the ultrastructural changes of mucosa tissue in ulcerative colitis were observed by transmission electron microscope. The expression levels of TGF-β in tissue mucosa were tested by enzyme-linked immunosorbentassay( ELISA). The expression levels of Rho-associated kinase-1,Rho-associated kinase-2,Ecadherin and TGF-β in colon were measured by Western blot and Real-time PCR. Result: Compared with normal group,model group showed the infiltration of a large number of inflammatory cells in mucosa and submucosa,disordered gland arrangement,varying degrees of intestinal mucosal defect and even ulcer formation. Under electron microscopy,microvilli were sparse on the surface of intestinal epithelial cells,the gap between cell junctions was widened,goblet cells were reduced and organelles were swollen. The disease activity index,and the expression levels of ROCK-1 and ROCK-2 proteins in the colonic mucosa of model group were increased( P <0. 01),while the colon length and the protein and mRNA contents of E-cadherin,TGF-β were decreased( P <0. 01). Compared with model group,there were different degrees of alleviations in pathological manifestationsunder the light and electron microscopy in each treatment group. DAI score and colon length reduction were significantly decreased in each treatment group( P < 0. 01). The proteins and mRNA expression levels of colonic mucosa ROCK-1 and ROCK-2 of the treatment group were decreased( P < 0. 01),while the protein and mRNA expression levels of E-caherin and TGF-β were increased( P < 0. 01),which was statistically significant compared with model group. Compared with middle-dose Oxymatrine group,the ROCK-1 and ROCK-2 protein and mRNA levels were significantly increased in low-dose and high-dose groups( P < 0. 05,P < 0. 01),and the TGF-β and E-cadherin protein and mRNA levels were significantly decreased( P < 0. 01). Conclusion: Oxymatrine may alleviate ulcerative colitis by down-regulating the expression of Rho kinase,up-regulating the expressions of E-cadherin and TGF-β,inducing the apoptosis of intestinal epithelial cells,and mediating epithelial-mesenchymal transition.
Objective: To investigate the mechanism of Oxymatrine on epithelial-mesenchymal transition mediated by RhoA/Rho-associated kinase( ROCK) signaling pathway to prevent and treat ulcerative colitis( UC)and its related canceration by detecting the changes of ROCK,E-cadherin and transforming growth factor-β( TGF-β) in colon tissues of mice. Method: Totally 48 male Balb/c mice were randomly divided into normal control group,model group,low,medium and high-dose Oxymatrine groups( 25,50,100 mg·kg~(-1)) and Y-27632 group( 10 mg·kg~(-1)),with 8 mice in each group. Mice in control group received distilled water,while all the other mice were treated with 3% dextra sulfate sodium for 7 days to induce the ulcerative colitis model. Since the first day of modeling,Y-27632( 10 mg·kg~(-1)) and different doses of Oxymatrine( 25,50,100 mg·kg~(-1))were intraperitoneally injectedfor 7 days,and equal volume of PBS was intraperitoneally injected in normal group and model group. Body weight loss,stool consistency and fecal blood loss were observed on a daily basis. On the8 thday,mice were put to death,colon was collected and its length was measured; the scores of disease activity index( DAI) were evaluated; part of the colons were fixed and stained with hematoxylin and eosin( HE) for a histopathological analysis; the ultrastructural changes of mucosa tissue in ulcerative colitis were observed by transmission electron microscope. The expression levels of TGF-β in tissue mucosa were tested by enzyme-linked immunosorbentassay( ELISA). The expression levels of Rho-associated kinase-1,Rho-associated kinase-2,Ecadherin and TGF-β in colon were measured by Western blot and Real-time PCR. Result: Compared with normal group,model group showed the infiltration of a large number of inflammatory cells in mucosa and submucosa,disordered gland arrangement,varying degrees of intestinal mucosal defect and even ulcer formation. Under electron microscopy,microvilli were sparse on the surface of intestinal epithelial cells,the gap between cell junctions was widened,goblet cells were reduced and organelles were swollen. The disease activity index,and the expression levels of ROCK-1 and ROCK-2 proteins in the colonic mucosa of model group were increased( P <0. 01),while the colon length and the protein and mRNA contents of E-cadherin,TGF-β were decreased( P <0. 01). Compared with model group,there were different degrees of alleviations in pathological manifestationsunder the light and electron microscopy in each treatment group. DAI score and colon length reduction were significantly decreased in each treatment group( P < 0. 01). The proteins and mRNA expression levels of colonic mucosa ROCK-1 and ROCK-2 of the treatment group were decreased( P < 0. 01),while the protein and mRNA expression levels of E-caherin and TGF-β were increased( P < 0. 01),which was statistically significant compared with model group. Compared with middle-dose Oxymatrine group,the ROCK-1 and ROCK-2 protein and mRNA levels were significantly increased in low-dose and high-dose groups( P < 0. 05,P < 0. 01),and the TGF-β and E-cadherin protein and mRNA levels were significantly decreased( P < 0. 01). Conclusion: Oxymatrine may alleviate ulcerative colitis by down-regulating the expression of Rho kinase,up-regulating the expressions of E-cadherin and TGF-β,inducing the apoptosis of intestinal epithelial cells,and mediating epithelial-mesenchymal transition.
引文
[1]孙盟朝,田晶晶,崔莉红.乌梅丸治疗溃疡性结肠炎的疗效分析及对T淋巴细胞的影响[J].中国中西医结合外科杂志,2018,24(5):545-549.
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[7]罗爽,罗霞,刘琦,等.大黄酸对DSS诱导溃疡性结肠炎小鼠的治疗作用及机制探讨[J].中国实验方剂学杂志,2017,23(11):109-113.
[8]付敏军,石荣珍,沈建君,等.新麦纤散对DSS诱导UC大鼠的治疗作用及机制分析[J].中国实验方剂学杂志,2018,24(5):126-130.
[9]杨佳,范恒.复方苦参汤对溃疡性结肠炎大鼠炎性反应和氧化应激的影响[J].中华中医药杂志,2017,32(8):3457-3461.
[10]CHEN Q,DUAN X,FAN H,et al.Oxymatrine protects against DSS-induced colitis via inhibiting the PI3K/AKT signaling pathway[J].Int Immunopharmacol,2017,53:149-157.
[11]ZOU Y,MA L,ZHAO Y,et al.Inhibition of Rho kinase protects against colitis in mice by attenuating intestinal epithelial barrier dysfunction via MLC and the NF-kappaB pathway[J].Int J Mol Med,2018,41(1):430-438.
[12]Sanchez-Fidalgo S,Cardeno A,Sanchez-Hidalgo M,et al.Dietary extra virgin olive oil polyphenols supplementation modulates DSS-induced chronic colitis in mice[J].J Nutr Biochem,2013,24(7):1401-1413.
[13]马艳,刘虹,张浩,等.TGF-β信号通路调控乳腺癌上皮-间质转化的研究进展[J].药学学报,2015,50(4):385-392.
[14]Demaio L,Buckley S T,Krishnaveni M S,et al.Ligand-independent transforming growth factor-beta type I receptor signalling mediates type I collagen-induced epithelial-mesenchymal transition[J].J Pathol,2012,226(4):633-644.
[15]Sanchez-Tillo E,LIU Y,de Barrios O,et al.EMT-activating transcription factors in cancer:beyond EMTand tumor invasiveness[J].Cell Mol Life Sci,2012,69(20):3429-3456.
[16]YANG J,Mani S A,Donaher J L,et al.Twist,a master regulator of morphogenesis,plays an essential role in tumor metastasis[J].Cell,2004,117(7):927-939.
[17]Ansieau S,Bastid J,Doreau A,et al.Induction of EMT by twist proteins as a collateral effect of tumorpromoting inactivation of premature senescence[J].Cancer Cell,2008,14(1):79-89.
[18]DU L,Kim J J,SHEN J,et al.Crosstalk between inflammation and ROCK/MLCK signaling pathways in gastrointestinal disorders with intestinal hyperpermeability[J].Gastroenterol Res Pract,2016,2016(5):7374197.
[19]Lopez-Posadas R,Becker C,Gunther C,et al.Rho-Aprenylation and signaling link epithelial homeostasis to intestinal inflammation[J].J Clin Invest,2016,126(2):611-626.
[20]ZHANG K,ZHANG H,XIANG H,et al.TGF-beta1induces the dissolution of tight junctions in human renal proximal tubular cells:role of the Rho A/ROCKsignaling pathway[J].Int J Mol Med,2013,32(2):464-468.
[21]WANG Q,YANG X,XU Y,et al.Rho A/Rho-kinase triggers epithelial-mesenchymal transition in mesothelial cells and contributes to the pathogenesis of dialysisrelated peritoneal fibrosis[J].Oncotarget,2018,9(18):14397-14412.
[22]Marafini I,Zorzi F,Codazza S,et al.TGF-beta signaling manipulation as potential therapy for IBD[J].Curr Drug Targets,2013,14(12):1400-1404.
[23]Skeen V R,Paterson I,Paraskeva C,et al.TGF-beta1signalling,connecting aberrant inflammation and colorectal tumorigenesis[J].Curr Pharm Des,2012,18(26):3874-3888.
[24]Monteleone G,Kumberova A,Croft N M,et al.Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease[J].J Clin Invest,2001,108(4):601-609.
[25]MA Y,GUAN Q,BAI A,et al.Targeting TGF-beta1by employing a vaccine ameliorates fibrosis in a mouse model of chronic colitis[J].Inflamm Bowel Dis,2010,16(6):1040-1050.
[26]LI G,REN J,HU Q,et al.Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-beta signaling in a murine colitis model[J].Biochem Pharmacol,2016,doi:10.1016/j.bcp.2016.08.002.
[2]DENG S,WANG H,FAN H,et al.Over-expressed miRNA-200b ameliorates ulcerative colitis-related colorectal cancer in mice through orchestrating epithelial-mesenchymal transition and inflammatory responses by channel of Akt2[J].Int Immunopharmacol,2018,61:346-354.
[3]SONG G L,JIN C C,ZHAO W,et al.Regulation of the Rho A/ROCK/AKT/beta-catenin pathway by arginine-specific ADP-ribosytransferases 1 promotes migration and epithelial-mesenchymal transition in colon carcinoma[J].Int J Oncol,2016,49(2):646-656.
[4]Korol A,Taiyab A,West-Mays J A.Rho A/ROCKsignaling regulates TGFbeta-induced epithelialmesenchymal transition of lens epithelial cells through MRTF-A[J].Mol Med,2016,22(1):713-723.
[5]LI M,WANG B,SUN X,et al.Upregulation of intestinal barrier function in mice with DSS-induced colitis by a defined bacterial consortium is associated with expansion of IL-17A producing gamma delta T cells[J].Front Immunol,doi:10.3389/fimmu.2017.00824.
[6]Richard M L,Liguori G,Lamas B,et al.Mucosaassociated microbiota dysbiosis in colitis associated cancer[J].Gut Microbes,2018,9(2):131-142.
[7]罗爽,罗霞,刘琦,等.大黄酸对DSS诱导溃疡性结肠炎小鼠的治疗作用及机制探讨[J].中国实验方剂学杂志,2017,23(11):109-113.
[8]付敏军,石荣珍,沈建君,等.新麦纤散对DSS诱导UC大鼠的治疗作用及机制分析[J].中国实验方剂学杂志,2018,24(5):126-130.
[9]杨佳,范恒.复方苦参汤对溃疡性结肠炎大鼠炎性反应和氧化应激的影响[J].中华中医药杂志,2017,32(8):3457-3461.
[10]CHEN Q,DUAN X,FAN H,et al.Oxymatrine protects against DSS-induced colitis via inhibiting the PI3K/AKT signaling pathway[J].Int Immunopharmacol,2017,53:149-157.
[11]ZOU Y,MA L,ZHAO Y,et al.Inhibition of Rho kinase protects against colitis in mice by attenuating intestinal epithelial barrier dysfunction via MLC and the NF-kappaB pathway[J].Int J Mol Med,2018,41(1):430-438.
[12]Sanchez-Fidalgo S,Cardeno A,Sanchez-Hidalgo M,et al.Dietary extra virgin olive oil polyphenols supplementation modulates DSS-induced chronic colitis in mice[J].J Nutr Biochem,2013,24(7):1401-1413.
[13]马艳,刘虹,张浩,等.TGF-β信号通路调控乳腺癌上皮-间质转化的研究进展[J].药学学报,2015,50(4):385-392.
[14]Demaio L,Buckley S T,Krishnaveni M S,et al.Ligand-independent transforming growth factor-beta type I receptor signalling mediates type I collagen-induced epithelial-mesenchymal transition[J].J Pathol,2012,226(4):633-644.
[15]Sanchez-Tillo E,LIU Y,de Barrios O,et al.EMT-activating transcription factors in cancer:beyond EMTand tumor invasiveness[J].Cell Mol Life Sci,2012,69(20):3429-3456.
[16]YANG J,Mani S A,Donaher J L,et al.Twist,a master regulator of morphogenesis,plays an essential role in tumor metastasis[J].Cell,2004,117(7):927-939.
[17]Ansieau S,Bastid J,Doreau A,et al.Induction of EMT by twist proteins as a collateral effect of tumorpromoting inactivation of premature senescence[J].Cancer Cell,2008,14(1):79-89.
[18]DU L,Kim J J,SHEN J,et al.Crosstalk between inflammation and ROCK/MLCK signaling pathways in gastrointestinal disorders with intestinal hyperpermeability[J].Gastroenterol Res Pract,2016,2016(5):7374197.
[19]Lopez-Posadas R,Becker C,Gunther C,et al.Rho-Aprenylation and signaling link epithelial homeostasis to intestinal inflammation[J].J Clin Invest,2016,126(2):611-626.
[20]ZHANG K,ZHANG H,XIANG H,et al.TGF-beta1induces the dissolution of tight junctions in human renal proximal tubular cells:role of the Rho A/ROCKsignaling pathway[J].Int J Mol Med,2013,32(2):464-468.
[21]WANG Q,YANG X,XU Y,et al.Rho A/Rho-kinase triggers epithelial-mesenchymal transition in mesothelial cells and contributes to the pathogenesis of dialysisrelated peritoneal fibrosis[J].Oncotarget,2018,9(18):14397-14412.
[22]Marafini I,Zorzi F,Codazza S,et al.TGF-beta signaling manipulation as potential therapy for IBD[J].Curr Drug Targets,2013,14(12):1400-1404.
[23]Skeen V R,Paterson I,Paraskeva C,et al.TGF-beta1signalling,connecting aberrant inflammation and colorectal tumorigenesis[J].Curr Pharm Des,2012,18(26):3874-3888.
[24]Monteleone G,Kumberova A,Croft N M,et al.Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease[J].J Clin Invest,2001,108(4):601-609.
[25]MA Y,GUAN Q,BAI A,et al.Targeting TGF-beta1by employing a vaccine ameliorates fibrosis in a mouse model of chronic colitis[J].Inflamm Bowel Dis,2010,16(6):1040-1050.
[26]LI G,REN J,HU Q,et al.Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-beta signaling in a murine colitis model[J].Biochem Pharmacol,2016,doi:10.1016/j.bcp.2016.08.002.