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

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).Conclusions Mitochondrial autophagy can inhibit HSC-T6 activation,and its mechanism is related to the inhibition of ROS production by mitochondrial 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.