基于AQP8和线粒体细胞凋亡途径相关性探讨马兰草乙醇提取物对活化的大鼠肝星细胞HSC-T6体外增殖和凋亡的影响  

Effect of Ethanol Extract of Malancao (Herba Kalimeridis) on Proliferation and Apoptosis of Activated HSC-T6 of Rats in Vitro Based on Correlation between AQP8 and Mitochondrial Cell Apoptosis Pathway

作　　者：李嘉 高玲 陈晓兰 罗乐 田源红 张雄 LJ Jia;GAO Ling;CHEN Xiaolan;LUO Le;TIAN Yuanhong;ZHANG Xiong(Guizhou University of Traditional Chinese Medicine,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州中医药大学,贵州贵阳550025

出　　处：《中华中医药学刊》2021年第4期145-150,I0054,共7页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金(81860887,81560771);贵州省教育厅青年科技人才成长项目(黔教和KY[2016]184);贵阳市科技计划(筑科合同[2017-4]20);贵州省中医药、民族医药科学技术专项课题项目(QZYY-2018-013);贵州中医药大学院内科研项目(200);贵州中医药大学黔苗医药项目(K字[2017]010);贵州省高层次创新型人才项目(黔人领发[2018]5)。

摘　　要：目的研究马兰草乙醇提取物在体外对转化生长因子-β1(TGF-β1)活化的大鼠肝星形细胞(HSC-T6)增殖、凋亡及基于AQP8蛋白靶点调控活化的HSC-T6细胞线粒体凋亡途径的影响,探究该药抗肝硬化腹水的作用机制。方法体外培养HSC-T6,用TGF-β1刺激24 h后,用不同浓度的马兰草乙醇提取物体外干预12、24、36、48 h,用MTT法检测马兰草乙醇提取物对活化HSC-T6存活率的影响,流式细胞术检测HSC-T6细胞凋亡和细胞线粒体膜电位的影响,实时荧光定量PCR(qRT-PCR)法检测AQP8mRNA的表达水平,Western Blot检测AQP8、Cty-C的蛋白表达。结果阴性对照组相比,各组OD值和细胞存活率均有下降,基质对照组的凋亡率下降(P<0.05),其余各组的凋亡率均有所上升(P<0.05),基质对照组和马兰提取物组(20、40μg/mL)的AQP8mRNA的表达水平明显上升(P<0.05);与基质组相比,大黄素各剂量组和马兰乙醇提取物各剂量组的细胞存活率均呈时间/剂量依赖性下降,大黄素组的早期凋亡有所上升(P<0.05),马兰提取物各剂量组各时期的凋亡增加的同时呈剂量依赖性升高(P<0.05),且线粒体细胞膜电位呈剂量依赖性显著下降(P<0.05)。Western Blot检测结果显示,Cty-C和AQP8的表达趋势一致,大黄素组的表达较较阴性对照组和基质对照组均有明显增多(P<0.05),马兰提取物组(40μg/mL)的表达量最多(P<0.05)。结论马兰草乙醇提取物在体外可抑制活化的HSC-T6增殖,促进细胞凋亡,其机制可能与激活线粒体细胞凋亡途径和上调AQP8表达有关。Objective To study the effect of ethanol extract of Malancao (Herba Kalimeridis) on the proliferation and apoptosis of transforming growth factor-beta-1 (TGF-beta1)-activated hepatic stellate cells (HSC-T6) of rats in vitro and the regulation of mitochondrial apoptosis pathway of HSC-T6 cells activated by AQP8 protein target and explore the mechanism of the anti-cirrhosis effect. Methods In vitro cultivation of HSC-T6, after TGF-beta1 stimulation for 24 h, different concentrations of ethanol extracts of Malancao (Herba Kalimeridis) was used to intervene for 12, 24, 36 and 48 h in vitro. MTT method was used to detect the influence of ethanol extract of Malancao (Herba Kalimeridis) on the survival rate of activated HSC-T6 and the effect on HSC-T6 cell apoptosis and mitochondrial membrane potential was detected by flow cytometry. The real-time fluorescent quantitative PCR (qRT-PCR) method was used to detect AQP8 mRNA expression. The protein expressions of AQP8 and Cty-C were detected by Western Blot. Results Compared with the negative control group, OD value and cell survival rate of each group decreased, the apoptosis rate of matrix control group decreased (P<0.05), and the apoptosis rate of the other groups increased (P<0.05). The expressions of AQP8 mRNA in the matrix control group and Malancao (Herba Kalimeridis) extract groups (20,40 μg/mL) were obviously increased (P<0.05). Compared with the matrix control group, the substrate emodin groups and ethanol extract of Malancao (Herba Kalimeridis) groups were in a time/dose-dependent manner. The cell survival rate of emodin group increased (P<0.05). The early apoptosis of Malancao (Herba Kalimeridis) extract different dose groups in different periods was increased in a dose-dependent manner (P<0.05), and mitochondrial membrane potential was a significant reduction in a dose-dependent manner (P<0.05). Western Blot results showed that the expression trends of Cty-C and AQP8 were consistent. The expression in the emodin group was significantly increased compared with

关 键 词：马兰草乙醇提取物 HSC-T6大鼠肝星形细胞 细胞凋亡 AQP8 线粒体途径 

分 类 号：R285.5[医药卫生—中药学]