摘要
目的探讨氧化苦参碱(OM)对转化生长因子-β1(TGF-β1)诱导的大鼠胰腺星状细胞LTC-14中细胞外基质(ECM)分泌的影响及其分子机制。方法取生长良好的LTC-14细胞株,分为对照组(正常培养的LTC-14细胞),TGF-β1组(10μg/L TGF-β1刺激12 h),TGF-β1+OM组(1 g/L OM预处理30 min,再加入10μg/L TGF-β1刺激12 h),TGF-β1+SiZNF850组(LTC-14细胞中瞬时转染ShRNA-ZNF580质粒,24 h后加入10μg/L TGF-β1刺激12h)。实时定量PCR、Western blot检测细胞内Smad2、Smad3、ZNF580、α-肌动蛋白(α-SMA)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶组织抑制剂1(TIMP1)的mRNA和蛋白表达情况,酶联免疫吸附试验(ELISA)检测ECM中胶原蛋白-Ⅰ(Col-Ⅰ)、Col-Ⅲ、纤维连接蛋白(FN)、肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)的分泌情况。结果 TGF-β1诱导的LTC-14细胞中Smad2、Smad3、ZNF580和α-SMA的mRNA和蛋白表达水平均升高(P<的分泌水平升高(P<0.05);而用OM预处理或沉默ZNF580基因后,LTC-14细胞中TGF-β1/Smads通路Smad2、Smad3、α-SMA和ZNF580的mRNA和蛋白表达水平均下调(P<0.05),MMP-2/TIMP1表达比值升高(P<0.05),细胞外基质Col-Ⅰ、Col-Ⅲ、FN、TNF-α和IL-1β分泌水平均降低(P<0.05)。结论 OM通过抑制胰腺星状细胞LTC-14中TGF-β1/Smads通路激活,下调ZNF580,阻滞胰腺星状细胞的活化,使ECM分泌减少、降解增多,减轻胰腺纤维化。ZNF580可能是OM作用的分子靶点。
Objective To probe the molecular mechanism and effect of oxymatrine(OM)on the secretion of extracellular matrix(ECM)in rat pancreatic stellate cell line LTC-14 induced by TGF-β1.Methods LTC-14 cells were divided into control group(LTC-14 cells in normal culture),TGF-β1 group(LTC-14 cells stimulated by 10μg/L TGF-β1 for 12 h),TGF-β1+OM group(LTC-14 cells pretreated with 1 g/L OM for 30 min before TGF-β1 stimulation)and TGF-β1+SiZNF850 group(LTC-14 cells were transiently transfected with ShRNA-ZNF580 plasmids for 24 h before TGF-β1 stimulation).After cultivation,cell supernatant was collected and total RNA and total protein were extracted.The expressions of Smad2,Smad3,ZNF580,α-SMA,MMP2 and TIMP1 were detected by Real-time PCR and Western blot assay.The secretion of ECM components,Col-I,Col-III,FN,TNF-αand IL-1βwere detected by ELISA.Results The mRNA and protein expressions of Smad2,Smad3,ZNF580 andα-SMA were increased(P<0.05),the expression ratio of MMP2/TIMP1 was down-regulated(P<0.05),and the secretion of ECM components was increased in LTC-14 cells induced by TGF-β1(P<0.05).However,after OM intervention or ZNF580 gene knockdown,the mRNA and protein expressions of Smad2 and Smad3 were suppressed(P<0.05),the mRNA and protein expressions of ZNF580 decreased,the expression ratio of MMP-2/TIMP1 was up-regulated(P<0.05),and the secretion of ECM components was reduced(P<0.05).Conclusion Oxymatrine can reduce ECM secretion,increase ECM degradation and alleviate pancreatic fibrosis by inhibiting TGF-β1/Smads/ZNF580 signaling pathway and blocking the activation of pancreatic stellate cells.ZNF580 may be a molecular target of OM in reducing pancreatic fibrosis.
作者
陈凯
张青
刘百庆
陈伟
周芳
张兰
夏时海
许威
CHEN Kai;ZHANG Qing;LIU Bai-qing;CHEN Wei;ZHOU Fang;ZHANG Lan;XIA Shi-hai;XU Wei(Tianjin Key Laboratory of Hepatopancreatic Fibrosis and Molecular Diagnosis & Treatment,Characteristic Medical Center of PAP,Tianjin 300162,China;Tianjin Xiqing Hospital)
出处
《天津医药》
CAS
北大核心
2019年第4期376-381,共6页
Tianjin Medical Journal
基金
天津市自然科学基金(17JCYBJC26700)
天津市救援医学临床医学研究中心(15ZXLCSY00040)
天津市西青医院科研基金(xqyylx201603)
