基于STAT3/TGF-β1/Smads信号通路调控EMT探讨大黄灵仙颗粒干预肝内胆管结石形成机制研究

Choleithiasis is a commom clinical multiple disease , Some prophase research had proved that gallstones were closely related to cholangitis, The clinical research had proved it was definitly curative effect many times that Dahuanglingxian granules could prevent and control the gallstones effectively . Biliary tract inflammation is the base pathology feature of calculus of bile duct , biliary tract fibrosis is the necessary premise of calculus of bile duct , EMT is the definitely way of biliary tract fibrosis , EMT is the mainly way of TGF-β1/Smadssignal transduction pathways .We conjecture that biliary tract inflammation activated the TGF-β1 /Smadssignal transduction pathways and this urge the bile duct cells to participate in the formation of gallstones.This project strctured inflammation animal models, injected the TGF-β1 receptor blocking pharmacon, determined the pritein and mRNA expressions of groups liver tissue that S100A4、α-SMA、TGF-β1、smad3、IL-6、EGR-1 with the way of electron microscope technology , Western Blotand RT-PCR . Exporated the process of how the biliary tract inflammation ILurged the STAT3/TGF-β1/Smads signal transduction pathways to induce the EMT , what the intervene was and where was the target spot was.Revealed the mechanism of how Dahuanglingxian granules set-back the EMT process and blocking-up to coming into hepatolith , which with great theory meaning and practical value .

胆石病是临床常见多发病，前期研究发现胆石形成与胆管炎症紧密相关，中药复方大黄灵仙颗粒能有效防治结石，临床已多次验证其疗效确切。胆道炎症是胆管结石的基本病理特征，胆道纤维化是结石形成的必要前提，上皮-间叶样表型转化（EMT）是胆道纤维化的必经途径，TGF-β1 /Smads信号通路是致EMT的主要路径，我们推测胆道炎症激活信号通路促进胆管细胞EMT进程参与结石形成。项目通过炎症动物模型构建，注射TGF-β1受体阻断剂，采用电镜技术、Western Blot、RT-PCR测定各组肝组织样本S100A4、α-SMA、TGF-β1、smad3、IL-6、EGR-1蛋白和mRNA表达量，探究胆道炎症IL激活STAT3/TGF-β1/Smads信号通路诱导EMT过程及中药干预作用和靶点，揭示大黄灵仙颗粒逆转EMT进程阻断肝内胆管结石形成机理，对胆石病的防治具有重要的理论意义与实际价值。

肝内胆管结石是指发生在双侧汇管区以上的结石，其病因复杂，与胆管炎症密不可分。临床治疗主要以手术治疗方式为主，但因肝内胆管解剖复杂，往往需要多次手术，故亟需明白肝内胆管结石病因及术后辅助治疗。本课题以LPS构建急性胆管炎症模型大鼠，在胆管炎症模型大鼠的基础上，对大黄灵仙方进行药效评价。前后取120只雄性SD大鼠随机分为空白组、脂多糖模型组、熊去氧胆酸胶囊对照组、大黄灵仙方实验组、熊去氧胆酸+紫杉醇对照组、大黄灵仙+紫杉醇实验组，每组各20只。正常组予常规饲料饲养；模型组、对照组和实验组在胆总管一次性注射1.25mg/kg脂多糖，同时熊去氧胆酸对照组予熊去氧胆酸灌胃干预，大黄灵仙实验组予大黄灵仙方灌胃干预，熊去氧胆酸+紫杉醇对照组、大黄灵仙方+紫杉醇实验组分别在前两组的基础上腹腔注射紫杉醇，共7天。取血液进行ELISA检测，采取肝脏组织进行HE染色、WB检测、免疫组化检测及PCR检测。结果模型组大鼠的L-6、EGR-1、TGF-β1、Smad3、β-GD蛋白表达量相对较高，大黄灵仙治疗组大鼠的蛋白表达趋于正常水平；结论 LPS可以构建胆管炎模型；大黄灵仙颗粒调控TGF-β1/Smads信号通路调控EMT使之趋于正常生理水平，真正起到“胆病从肝论治”的作用。