摘要
目的 探讨柔肝化纤颗粒对肝纤维化的影响及机制.方法 SD大鼠分为正常组、模型组、生理盐水对照组、柔肝化纤颗粒低、中、高剂量组、柔肝化纤颗粒中剂量+3-MA组.腹腔注射CCl4制备肝纤维化模型,分别灌胃柔肝化纤颗粒低、中、高剂量(2.5 g·kg-1、5 g·kg-1、10 g·kg-1)和3-MA(15 mg·kg-1).采用Real-time PCR、Western Blot、及ELISA检测大鼠肝组织miR-135a、FOXO1、PINK1、Parkin、LC3 Ⅱ、Smad2、p-Smad2、TGF-β1、NF-κB p65、p-NF-κB p65、α-SMA、Collagen Ⅰ、Collagen Ⅲ、TNF-α表达及ROS含量.结果 模型组miR-135a、α-SMA、Collagen Ⅰ、Collagen Ⅲ、p-Smad2、TGF-β1、p-NF-κB p65、TNF-α表达及ROS含量显著上调(P<0.05);FOXO1、PINK1、Parkin、LC3 Ⅱ表达显著下调(P<0.05).柔肝化纤颗粒低剂量组可明显抑制miR-135a、α-SMA、Collagen Ⅰ、Collagen Ⅲ、p-Smad2、TGF-β1、p-NF-κB p65、TNF-α表达及ROS生成;上调FOXO1、PINK1、Parkin、LC3 Ⅱ表达(P<0.05).柔肝化纤颗粒中、高剂量组可显著抑制miR-135a、α-SMA、Collagen Ⅰ、Collagen Ⅲ、p-Smad2、TGF-β1、p-NF-κB p65、TNF-α表达及ROS生成;上调FOXO1、PINK1、Parkin、LC3Ⅱ表达(P<0.05).柔肝化纤颗粒中剂量组药效明显强于低剂量组(P<0.05),中、高剂量组药效无明显差异.线粒体自噬抑制剂(3-MA)可明显抑制柔肝化纤颗粒药效(P<0.05).结论 柔肝化纤颗粒可抑制肝纤维化,其机制与柔肝化纤颗粒抑制miR-135a表达,激活FOXO1/PINK1通路,进而促进线粒体自噬,抑制氧化应激反应,抑制TGF-β1/Smad2活化有关.
Abstract
Objective To investigate the effect and mechanism of Rougan Huaxian particle on hepatic fibrosis.Methods SD rats were divided into normal group,model group,normal saline control group,Rougan Huaxian particle low,middle,high dose group,Rougan Huaxian particle middle dose+3-MA group.Liver fibrosis model was induced by intraperitoneal injection of CCl4.Rougan Huaxian particle was administered with low,middle and high dose(2.5 mg·kg-1,5 mg·kg-1,10 mg·kg-1)and 3-MA(15 mg·kg-1)respectively.The expression of miR-135a,FOXO1,PINK1,Parkin,LC3 II,Smad2,p-Smad2,TGF-β1,NF-κB p65,p-NF-κB p65,α-SMA,Collagen I,Collagen III,TNF-α and the content of ROS were detected by Real-time PCR,Western Blot and ELISA.Results The expression of miR-135a,α-SMA,Collagen I,Collagen III,p-Smad2,TGF-β1,p-NF-κB p65,TNF-α and ROS in model group were significantly up-regulated(P<0.05),and the expression of FOXO1,PINK1,Parkin,LC3 II were significantly down-regulated(P<0.05).The middle and high dose of Rougan Huaxian particle could significantly inhibit the expression of miR-135a,α-SMA,Collagen I,Collagen III,p-Smad2,TGF-β1,p-NF-κb p65,TNF-α and ROS,and up-regulate the expression of FOXO1,PINK1,Parkin,LC3 II(P<0.05).The expression of miR-135a,α-SMA,Collagen Ⅰ,Collagen Ⅲ,p-Smad2,TGF-β1,p-NF-κB p65 and TNF-α and the production of ROS were significantly inhibited in the medium and high dose groups.Up-regulated expression of FOXO1,PINK1,Parkin and LC3Ⅱ(P<0.05).The effect of Rougan Huaxian Particle in middle dose group was stronger than that in low dose group(P<0.05),and there was no significant difference between middle and high dose groups.Mitophagy inhibitor(3-MA)could significantly inhibit the efficacy of rougan Huaxian particle(P<0.05).Conclusion Ruugan Huaxian granules can inhibit liver fibrosis,and its mechanism is related to the inhibition of miR-135a expression,activation of FOXO1/PINK1 pathway,promotion of mitochondrial autophagy,inhibition of oxidative stress response,and inhibition of TGF-β1/Smad2 activation.
基金项目
广西壮族自治区科学技术厅重点项目(2020GXNSFDA297021)
国家自然科学基金委员会地区基金项目(81960910)
国家自然科学基金委员会地区基金项目(81660745)
NETL
NSTL
万方数据