基于线粒体氧化应激损伤探究异钩藤碱对小鼠肝纤维化的保护作用

Study on protective effect of isorhychophylline on hepatic fibrosis in mice based on oxidative stress damage of mitochondria

目的 探讨异钩藤碱(IHY)对四氯化碳(CCl_(4))诱导的小鼠肝纤维化(HF)的影响及其潜在机制。方法 C57BL/6小鼠随机分对照(CON)组、CCl_(4)组、IHY 20 mg/kg组和IHY 40 mg/kg组,每组各10只。20%CCl_(4)橄榄油溶液(0.05 ml/10 g)灌胃8周制备肝纤维化模型,造模后第5周开始每天连续灌胃给予IHY治疗4周。白蛋白(ALB)、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平分别采用溴甲酚绿法、丙氨酸底物法及天门冬氨酸底物法检测。ELISA检测白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)及转化生长因子-β1(TGF-β1)的水平。HE和Masson染色观察肝组织病理变化及胶原沉积情况。透射电镜观察肺组织线粒体结构。免疫组化检测肝组织Ⅰ型胶原(COL1A1)和基质金属蛋白酶2(MMP2)的表达。比色法检测肝组织丙二醛(MDA)及4-羟基壬烯醛(4-HNE)含量。制备肝组织单细胞悬液,使用DCFH-DA探针检测细胞内活性氧(ROS)水平。Western blot法检测IL-6、TNF-α、TGF-β1、8-氧鸟嘌呤DNA糖基化酶1(OGG1)及沉默信号调节因子3(SIRT3)的蛋白水平。结果 与CON组相比,CCl_(4)组肝组织结构紊乱,肝细胞样结节再生,中央静脉周围可见炎症细胞浸润和大量蓝色胶原纤维沉积;COL1A1和MMP2蛋白表达明显升高;肝功能明显下降;炎症因子IL-6、TNF-α和TGF-β1水平明显升高;ROS、MDA及4-HNE水平明显增加,OGG1和SIRT3的表达明显降低,线粒体损伤明显加剧。与CCl_(4)组相比,IHY连续给药4周后,小鼠肝组织病理损伤明显减轻,胶原沉积明显减少,COL1A1和MMP2蛋白表达显著降低;肝功能明显改善;炎症因子IL-6、TNF-α和TGF-β1水平明显降低;ROS、MDA及4-HNE的水平也明显下降,OGG1和SIRT3的表达明显增加,线粒体损伤明显减轻。结论 IHY具有抗肝纤维化作用,其机制可能与其抗氧化,减轻线粒体氧化应激损伤,抑制炎症反应有关。

Objective To investigate the effects of Isorhychophylline(IHY) on carbon tetrachloride(CCl_(4)) induced hepatic fibrosis(HF) in mice and its potential mechanism.Methods C57BL/6 mice were randomly divided into the control(CON) group,the CCl_(4) group,the IHY 20 mg/kg group and the IHY 40 mg/kg group,10 mice in each group.The HF model was established by intragastric administration of 20%CCl_(4) olive oil solution(0.05 ml/10 g) for 8 weeks.Since the fifth week after modeling,IHY was given daily by intragastric administration for 4 weeks.The levels of albumin(ALB),alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected by bromocresol green method,alanine substrate method and aspartate substrate method,respectively.The levels of inflammatory cytokines interleukin-6(IL-6),tumor necrosis factor-α(TNF-α) and transforming growth factor-β1(TGF-β1) in plasma were detected by ELISA.Hepatic histopathology was analyzed by HE staining and collagen deposition was observed by Masson staining.Mitochondrial morphology of hepatic tissues was observed by transmission electron microscopy.The expression of collagen type I alpha 1(COL1A1) and matrix metallopeptidase 2(MMP2) was determined by immunohistochemistry.The contents of Malondialdehyde(MDA) and 4-hydroxynonenal(4-HNE) in hepatic tissues were determined by colorimetry.Single cell suspension of hepatic tissues was prepared and reactive oxygen species(ROS) levels in the cells was examined using a DCFH-DA fluorescent probe.The protein levels of IL-6,TNF-α,TGF-β1,8-oxoguanine DNA glycosylase 1(OGG1) and silent information regulator 3(SIRT3) were detected by Western blot analysis.Results Compared with the CON group,the hepatic structure was disordered,with regeneration of hepatocyte-like nodules,around the central veins,there were inflammatory cell infiltration and a large amount of blue collagen fiber deposition;the expression of COL1A1 and MMP2 was markedly up-regulated;the liver function decreased significantly;the content of inflammatory factors such as IL-6,TNF-α and TGF-β1 and the levels of ROS,MDA and 4-HNE were significantly increased;the expression of OGG1 and SIRT3 were obviously decreased;mitochondrial damage was aggravated in the CCl_(4)group.Compared with the CCl_(4)group,IHY robustly alleviated hepatic fibrosis and improved liver function.Meanwhile,IHY down-regulated the expression of and COL1A1,MMP2 and inflammatory cytokines(IL-6,TNF-α and TGF-β1).In addition,the levels of ROS,MDA and 4-HNE were significantly decreased,the expressions of OGG1 and SIRT3 were significantly increased,and the mitochondrial damage was significantly alleviated after4 weeks of IHY administration.Conclusions IHY can reverse hepatic fibrosis,and its mechanism may be related to its antioxidant,alleviating mitochondrial oxidative stress damage and inhibiting inflammatory response.