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、H2O2组、miR-135a-NC组、miR-135a-mimic组、miR-135a-in-hibitor-NC+H2O2组、miR-135a-inhibitor+H2O2组、柔肝化纤颗粒含药血清对照组、H2O2+含药血清对照组、H2O2+含药血清组、H2O2+含药血清+miR-135a-NC组及H2O2+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、LC3 Ⅱ、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H2O2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、LC3 Ⅱ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、LC3 Ⅱ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.
Abstract
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.Meth-ods HSC-T6 was divided into blank group,H2O2 group,miR-135a-NC group,miR-135a-mimic group,miR-135a inhibi-tor NC+H2O2 group,miR-135a-inhibitor+H2O2 group,drug-containing serum(Ruangan Huaxian Granules)control group,H2O2+drug-containing serum control group,H2O2+drug containing serum group,H2O2+drug-containing serum+miR-135a-NC group and H2O2+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 3 Ⅱ(LC3 Ⅱ),Smad2,p-Smad2 and transforming growth factor beta 1(TGF-β1),nuclear factor kappa-B(NF-KB)p65,p-NF-κB p65,α-smooth muscle actin(α-SMA),type Ⅰ collagen,type Ⅲ 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 H2O2 and over-expression of miR-135a sig-nificantly up-regulated the expressions of HSC-T6 miR-135a,α-SMA,type Ⅰ collagen,type Ⅲ collagen,p-Smad2,TGF-β1,p-NF-κB p65,TNF-α and ROS production(P＜0.01),down-regulate the expressions of FOXO1,PINK1,Parkin and LC3 Ⅱ and 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 1 collagen,type Ⅲcollagen,p-Smad2,TGF-β1,p-NF κB p65 and TNF-α and ROS production(P＜0.01),up-regulate the expressions of FOXO1,PINK1,Parkin and LC3 Ⅱ and 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).Conclusions-Mitochondrial autophagy can inhibit HSC-T6 activation,and its mechanism is related to the inhibition of ROS production by mi-tochondrial autophagy,which in turn inhibits TGF-β1/Smad2 pathway and inflammatory response.Rougan Huaxian Granules can also inhibit the activation of HSC-T6,and its mechanism is related to the inhibition of miR-135a expression by Ruogan Huaxian Granules,activation of the FOXO1/PINK1 pathway and promotion of mitochondrial autophagy.
基金项目
国家自然科学基金项目(81960910)
国家自然科学基金项目(81860839)
广西自然科学基金项目(2020GXNSFDA297021)
广西自然科学基金项目(2020GXNSFAA238020)
戴铭广西名中医工作室建设项目(2023017-05-09)
NETL
NSTL
万方数据