大黄素甲醚通过调控Keap1/Nrf2信号通路抗大鼠肝纤维化的作用

Effect of Physcion on Hepatic Fibrosis in Rats via Regulating Keap1/Nrf2 Signaling Pathway

目的:探讨大黄素甲醚通过调控Keap1/Nrf2信号通路对肝纤维化大鼠的保护作用。方法:将SD大鼠随机分为模型对照组、秋水仙碱0.2 mg/kg组、大黄素甲醚20、40 mg/kg组,腹腔注射40%四氯化碳橄榄油混悬液以建立大鼠肝纤维化模型,另设正常对照组,从造模第2 d起分别灌胃给予相应药物,1次/d,连续8 w,试剂盒检测大鼠丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、白蛋白(ALB)、总蛋白(TP)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、丙二醛(MDA)、超氧化物歧化酶(SOD)、层粘连蛋白(LN)、透明质酸(HA)、Ⅲ型前胶原(PC-Ⅲ)和Ⅳ型胶原(Ⅳ-C)含量;HE和Masson染色观察肝组织病理并进行Ishak评分;免疫组化分析肝组织中转化生长因子β1(TGF-β1)和α-平滑肌肌动蛋白(α-SMA)的阳性表达情况;Western blot法检测肝组织中Kelch样环氧氯丙烷相关蛋白-1(Keapl)、核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)和醌氧化还原酶1(NQO1)的蛋白表达。结果:与正常对照组相比,模型对照组大鼠血清中AST、ALT、ALP、HA、LN、PC-Ⅲ和Ⅳ-C活力或含量显著升高,ALB和TP含量显著降低(P<0.01),肝组织匀浆中MDA含量显著升高(P<0.01),SOD、GSH-Px和CAT活力或含量显著降低(P<0.01),肝组织炎症和纤维化评分均显著升高(P<0.01),肝组织中α-SMA、TGF-β1、Keap1蛋白表达明显上调(P<0.01),Nrf2、HO-1和NQO1蛋白表达显著下调(P<0.01);与模型对照组相比,大黄素甲醚20、40 mg/kg组大鼠血清中AST、ALT、ALP、HA、LN、PC-Ⅲ和Ⅳ-C活力或含量明显降低,ALB和TP含量明显增加(P<0.05或P<0.01),肝匀浆中MDA含量降低(P<0.05或P<0.01),SOD、GSH-Px和CAT活力或含量显著升高(P<0.01),肝组织炎症和纤维化评分明显降低(P<0.05或P<0.01),肝组织中α-SMA、TGF-β1、Keap1蛋白表达明显下调(P<0.05或P<0.01),Nrf2、HO-1和NQO1蛋白表达明显上调(P<0.05或P<0.01)。结论:大黄素甲醚可以改善肝纤维化大鼠的肝功能及氧化应激指标,减轻肝纤维化程度,其机制可能与调控Keap1/Nrf2信号通路有关。

Objective:To investigate the protective effect of physcion on hepatic fibrosis in rats by regulating the Keap1/Nrf2 signaling pathway.Methods:SD rats were randomly divided into model control group,colchicine 0.2 mg/kg group,and physcion 20 mg/kg and 40 mg/kg groups.Hepatic fibrosis model in rats was established by intraperitoneal injection of 40%carbon tetrachloride-olive oil suspension.A normal control group was also set up.Starting from the 2nd day of modeling,each group was orally administered with corresponding drugs once a day for 8 weeks.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),albumin(ALB),total protein(TP),glutathione peroxidase(GSH-Px),catalase(CAT),malondialdehyde(MDA),superoxide dismutase(SOD),laminin(LN),hyaluronic acid(HA),typeⅢprocollagen(PC-Ⅲ),and typeⅣcollagen(Ⅳ-C)were measured using reagent kits.Liver tissues were subjected to HE and Masson staining to observe pathological changes,and Ishak scoring was performed.Immunohistochemistry was used to detect the positive expression of transforming growth factorβ1(TGF-β1)andα-smooth muscle actin(α-SMA)in liver tissues.Western blot was conducted to detect the protein expression of Kelch-like ECH-associated protein 1(Keap1),nuclear factor E2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and NAD(P)H quinone oxidoreductase 1(NQO1)in liver tissues.Results:Compared with the normal control group,the model control group showed significantly increased serum levels of AST,ALT,ALP,HA,LN,PC-Ⅲ,andⅣ-C activities or content,significantly decreased ALB and TP content(P<0.01),significantly increased MDA content in liver homogenate(P<0.01),significantly decreased SOD,GSH-Px,and CAT activities or content(P<0.01),significantly increased inflammation and fibrosis scores in liver tissues(P<0.01),significantly upregulated expression ofα-SMA,TGF-β1,and Keap1 in liver tissues(P<0.01),and significantly downregulated expression of Nrf2,HO-1,and NQO1 proteins(P<0.01).Compared with the model control group,the physcion 20 mg/kg and 40 mg/kg groups showed significantly decreased serum levels of AST,ALT,ALP,HA,LN,PC-Ⅲ,andⅣ-C activities or content,significantly increased ALB and TP content(P<0.05 or P<0.01),decreased MDA content in liver homogenate(P<0.05 or P<0.01),significantly increased SOD,GSH-Px,and CAT activities or content(P<0.01),significantly decreased inflammation and fibrosis scores in liver tissues(P<0.05 or P<0.01),significantly downregulated expression ofα-SMA,TGF-β1,and Keap1 in liver tissues(P<0.05 or P<0.01),and significantly upregulated expression of Nrf2,HO-1,and NQO1 proteins(P<0.05 or P<0.01).Conclusion:Physcion can improve liver function and oxidative stress indicators in liver fibrosis rats,alleviate the degree of hepatic fibrosis,and its mechanism may be related to the regulation of the Keap1/Nrf2 signaling pathway.