柔肝降酶方含药血清抑制人肝星状细胞的活化及自噬  

Rougan Jiangmei Formula medicated serum inhibits activation and autophagy of human hepatic stellate cells

作　　者：陶柏楠 王咏兰 江露 张宗星 刘道忠 万星 黄德斌 袁林 Tao Bonan;Wang Yonglan;Jiang Lu;Zhang Zongxing;Liu Daozhong;Wan Xing;Huang Debin;Yuan Lin(Hubei Provincial Key Laboratory of Occurrence and Intervention of Rheumatic Diseases,Hubei Minzu University,Enshi 445000,Hubei Province,China;West China School of Preclinical Medicine and Forensic Medicine,Sichuan University,Chengdu 610064,Sichuan Province,China)

机构地区：[1]湖北民族大学,风湿性疾病发生与干预湖北省重点实验室,湖北省恩施州445000 [2]四川大学,华西基础医学与法医学院,四川省成都市610064

出　　处：《中国组织工程研究》2023年第33期5334-5341,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(82060745),项目负责人:黄德斌;湖北省卫健委面上项目(ZY2019M076),项目负责人:袁林;恩施州科技局科技计划性项目(E20190001),项目负责人:袁林。

摘　　要：背景:自噬在肝纤维化中发挥重要作用,可以通过诱导肝星状细胞活化促进肝纤维化的进程。前期研究表明,柔肝降酶方具有保护肝功能、抗炎、抗氧化应激反应等作用,其是否通过自噬抗肝纤维化的作用机制尚未阐明。目的:探讨柔肝降酶方含药血清对脂多糖诱导的人肝星状细胞活化及自噬的影响及其潜在的作用机制。方法:用脂多糖建立人肝星状细胞活化模型,分成不同的处理组:(1)空白组:人肝星状细胞+空白血清;(2)模型组:人肝星状细胞+空白血清+脂多糖;(3)柔肝降酶方低、中、高剂量含药血清组:人肝星状细胞+低、中、高剂量柔肝降酶方含药血清+脂多糖;(4)柔肝降酶方高剂量含药血清联合雷帕霉素组:高剂量组基础上+雷帕霉素;(5)柔肝降酶方高剂量含药血清联合PI3K抑制剂组:高剂量组基础上+LY294002。CCK-8法观察细胞增殖情况;采用单丹磺酰尸胺染色法观察细胞自噬情况;WesternBlot检测细胞通路蛋白(PI3K、P-PI3K、AKT、P-AKT、mTOR、P-mTOR)、自噬相关蛋白(P62、Beclin-1、LC3-Ⅰ、LC3-Ⅱ)、人肝星状细胞活化标志蛋白(α-平滑肌肌动蛋白、Ⅰ型胶原纤维)表达水平的影响。结果与结论:(1)柔肝降酶方含药血清能够降低人肝星状细胞的增殖活性,下调Ⅰ型胶原纤维、α-平滑肌肌动蛋白的蛋白表达,上调自噬P62蛋白的表达,降低LC3-Ⅱ、Beclin-1蛋白的表达,且呈现剂量依赖性;(2)MDC荧光染色结果显示,柔肝降酶方含药血清可使细胞绿色斑点荧光强度降低;(3)加入自噬激动剂雷帕霉素后,P62蛋白表达降低,LC3-Ⅱ、Beclin-1蛋白表达升高;(4)经过柔肝降酶方含药血清处理后,PI3K、Akt、mTOR蛋白磷酸化水平升高,呈剂量依赖性,这种作用能被PI3K抑制剂LY294002减弱;(5)提示柔肝降酶方含药血清可能通过PI3K/Akt/mTOR信号通路抑制自噬,减少脂多糖诱导的人肝星状细胞活化。BACKGROUND:Autophagy can play an important role in liver fibrosis by inducing the progression of hepatic stellate cell activation.Previous studies have shown that Rougan Jiangmei Formula could protect liver function,anti-inflammation,and anti-oxidative stress response.The mechanism of fighting liver fibrosis through autophagy has not been elucidated.OBJECTIVE:To investigate the effect of Rougan Jiangmei Formula medicated serum on activation and autophagy of human hepatic stellate cells induced by lipopolysaccharide and its underlying mechanism.METHODS:Human hepatic stellate cell activation models were established using lipopolysaccharide and divided into various treatment groups:(1)Blank group:human hepatic stellate cells+blank serum;(2)model group:human hepatic stellate cells+blank serum+lipopolysaccharide;(3)low-,medium-and high-dose Rougan Jiangmei Formula medicated serum groups:human hepatic stellate cells+low-,medium-and high-dose Rougan Jiangmei Formula medicated serum+lipopolysaccharide;(4)high-dose Rougan Jiangmei Formula medicated serum combined with rapamycin group:based on high-dose group+rapamycin;(5)high-dose Rougan Jiangmei Formula medicated serum combined with PI3K inhibitor group:based on high-dose group+LY294002.Cell proliferation was observed by CCK-8 assay.The autophagy was observed by monodansylcadaverine staining.The effects of Rougan Jiangmei Formula medicated serum on the expression levels of cell pathway-related proteins(PI3K,P-PI3K,AKT,P-AKT,mTOR,P-mTOR),autophagy-related proteins(P62,Beclin-1,LC3-I,LC3-Ⅱ),the activation marker protein of hepatic stellate cells(α-smooth-muscle actin and collagen I)were determined by western blot assay.RESULTS AND CONCLUSION:(1)Rougan Jiangmei Formula medicated serum significantly reduced the proliferation of activated human hepatic stellate cells,down-regulated the protein expression ofα-smooth-muscle actin and collagen I,up-regulated the expression of autophagy protein P62,and decreased the expression of Beclin-1 and LC3-Ⅱin a dose-dependent manner

关 键 词：柔肝降酶方 细胞自噬 Akt mTOR LC3-Ⅱ 人肝星状细胞 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R256.42