基于miR-135a/FOXO1/PINK1通路探讨柔肝化纤颗粒调控线粒体自噬抑制肝星状细胞活化的机制  

Exploring Mechanism of Regulating Mitochondrial Autophagy and Inhibiting Hepatic Stellate Cell Activation by Ruangan Huaxian Granules(柔肝化纤颗粒)Based on miR-135a/FOXO1/PINK1 Pathway

作　　者：张文富[1] 吴姗姗 戴铭 吕建林[1,5] 黄晶晶 李晓龙[6] 王振常 ZHANG Wenfu;WU Shanshan;DAI Ming;LYU Jianlin;HUANG Jingjing;LI Xiaolong;WANG Zhenchang(The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530023,Guangxi,China;Guangxi International Zhuang Medical Hospital,Nanning 530201,Guangxi,China;Guangxi University of Chinese Medicine,Nanning 530299,Guangxi,China;Guangxi Key Laboratory of Basic Research of Traditional Chinese Medicine,Nanning 530299,Guangxi,China;Guangxi Key Laboratory of Integrated Traditional Chinese and Western Medicine and Transformational Medicine for High Incidence Infectious Diseases,Nanning 530299,Guangxi,China;Guangxi Medical University,Nanning 530021,Guangxi,China)

机构地区：[1]广西中医药大学第一附属医院,广西南宁530023 [2]广西国际壮医医院,广西南宁530201 [3]广西中医药大学基础医学院,广西南宁530299 [4]广西中医基础研究重点实验室,广西南宁530299 [5]广西高发传染病中西医结合转化医学重点实验室,广西南宁530299 [6]广西医科大学基础医学院,广西南宁530021

出　　处：《中华中医药学刊》2024年第4期30-34,I0011-I0014,共9页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81960910,81860839);广西自然科学基金项目(2020GXNSFDA297021,2020GXNSFAA238020);戴铭广西名中医工作室建设项目(2023017-05-09)。

摘　　要：目的基于miR-135a/FOXO1/PINK1通路探讨柔肝化纤颗粒通过调控线粒体自噬来抑制肝星状细胞活化与增殖的影响及其机制。方法HSC-T6分为空白组、H_(2)O_(2)组、miR-135a-NC组、miR-135a-mimic组、miR-135a-in-hibitor-NC+H 2 O2组、miR-135a-inhibitor+H_(2)O_(2)组、柔肝化纤颗粒含药血清对照组、H_(2)O_(2)+含药血清对照组、H_(2)O_(2)+含药血清组、H_(2)O_(2)+含药血清+miR-135a-NC组及H_(2)O_(2)+含药血清+miR-135a-mimic组。分别采用Real-time PCR、Western Blot、ELISA及流式细胞术检测miR-135a、FOXO1、PINK1、Parkin、LC3Ⅱ、Smad2、p-Smad2、转化生长因子-β1(transforming growth factor beta 1,TGF-β1)、NF-κB p65、p-NF-κB p65、α-SMA、Ⅰ型胶原、Ⅲ型胶原、TNF-α表达及线粒体膜电位、活性氧(reactive oxygen species,ROS)生成。结果给予H_(2)O_(2)及过表达miR-135a可显著上调HSC-T6 miR-135a、α-SMA、Ⅰ型胶原、Ⅲ型胶原、p-Smad2、TGF-β1、p-NF-κB p65、TNF-α表达及ROS生成(P<0.01);下调FOXO1、PINK1、Parkin、LC3Ⅱ表达与线粒体膜电位(P<0.01)。给予柔肝化纤颗粒及抑制miR-135a表达可显著下调HSC-T6 miR-135a、α-SMA、Ⅰ型胶原、Ⅲ型胶原、p-Smad2、TGF-β1、p-NF-κB p65、TNF-α表达及ROS生成(P<0.01);上调FOXO1、PINK1、Parkin、LC3Ⅱ表达与线粒体膜电位(P<0.01)。给予柔肝化纤颗粒同时过表达miR-135a可抑制柔肝化纤颗粒对HSC-T6的影响(P<0.01)。结论线粒体自噬可抑制HSC-T6活化,其机制与线粒体自噬抑制ROS生成,进而抑制TGF-β1/Smad2通路及炎症反应有关;柔肝化纤颗粒亦可抑制HSC-T6活化,其机制与柔肝化纤颗粒抑制miR-135a表达,活化FOXO1/PINK1通路,从而促进线粒体自噬有关。Objective Based on miR-135a/FOXO1/PINK1 pathway,the effect and mechanism of Ruangan Huaxian Granules(柔肝化纤颗粒)on inhibiting activation and proliferation of hepatic stellate cells by regulating mitochondrial autophagy.Methods HSC-T6 was divided into blank group,H_(2)O_(2) group,miR-135a-NC group,miR-135a-mimic group,miR-135a inhibitor NC+H_(2)O_(2) group,miR-135a-inhibitor+H_(2)O_(2) group,drug-containing serum(Ruangan Huaxian Granules)control group,H_(2)O_(2)+drug-containing serum control group,H_(2)O_(2)+drug containing serum group,H_(2)O_(2)+drug-containing serum+miR-135a-NC group and H_(2)O_(2)+drug-containing serum+miR-135a-mimic group.The expressions of MiR-135a,recombinant forkhead box protein O1(FOXO1),PTEN induced putative kinase 1(PINK1),Parkin,human microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ),Smad2,p-Smad2 and transforming growth factor beta 1(TGF-β1),nuclear factor kappa-B(NF-κB)p65,p-NF-κB p65,α-smooth muscle actin(α-SMA),type I collagen,typeⅢ collagen and tumor necrosis factor-α(TNF-α),mitochondrial membrane potential and reactive oxygen species(ROS)production were detected by Real time PCR,Western Blot,ELISA and flow cytometry,respectively.Results Administration of H_(2)O_(2) and over-expression of miR-135a significantly up-regulated the expressions of HSC-T6 miR-135a,α-SMA,type I collagen,typeⅢ collagen,p-Smad2,TGF-β1,p-NF-κB p65,TNF-αand ROS production(P<0.01),down-regulate the expressions of FOXO1,PINK1,Parkin and LC3Ⅱand mitochondrial membrane potential(P<0.01).Administration of Ruogan Huaxian Granules and inhibition of miR-135a expression can significantly down-regulate the expressions of HSC-T6 miR-135a,α-SMA,type I collagen,typeⅢ collagen,p-Smad2,TGF-β1,p-NFκB p65 and TNF-α and ROS production(P<0.01),up-regulate the expressions of FOXO1,PINK1,Parkin and LC3Ⅱand mitochondrial membrane potential(P<0.01).Simultaneous over-expression of miR-135a with Ruogan Huaxian Granules can inhibit the effect of Ruogan Huaxian Granules on HSC-T6(P<0.01).Conclusio

关 键 词：线粒体自噬 HSC-T6 柔肝化纤颗粒 miR-135a FOXO1 

分 类 号：R289.5[医药卫生—方剂学]