摘要
目的:研究肝豆灵(GDL)治疗Wilson病肝纤维化可能的作用机制及途径.方法:60只雄性SD大鼠随机分为6组:正常组、模型组、GDL低、中、高剂量组(0.24、0.48、0.96 g·kg-1)及青霉胺组(90 mg·kg-1),每组10只.除正常组以外,其余各组以300 mg·kg-1五水合硫酸铜灌胃造模,构建铜负荷Wilson病大鼠模型.采用苏木素-伊红(HE)和马松(Masson)染色观察肝脏病理形态改变;酶联免疫吸附测定法(ELISA)检测透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原肽(PC-Ⅲ)、Ⅳ型胶原(C-Ⅳ)水平;透射电镜观察肝组织超微结构;实时荧光定量聚合酶链式反应(Real-time PCR)检测肝组织肝脏和血清外泌体长链非编码核糖核酸H19(LncRNAH19)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)表达水平;蛋白免疫印迹法(Western blot)检测肝组织中PI3K/Akt/mTOR通路蛋白及其磷酸化蛋白和自噬效应蛋白1(Beclin1)、微管相关蛋白1轻链3B(LC3-Ⅱ/LC3-Ⅰ)蛋白表达水平;免疫荧光观察Beclin1、LC3-Ⅱ信号强度.结果:与正常组比较,模型组染色结果显示肝细胞出现炎性细胞浸润,细胞核边界不清伴有裂解、坏死,汇管区周围可见胶原纤维沉积;HA、LN、PC-Ⅲ、C-Ⅳ含量升高(P＜0.01);电镜下观察到自噬小体的数量增多,出现大多数包浆被降解后呈现单层膜结构的自噬溶酶体;Beclin1、LC3-Ⅱ/LC3-Ⅰ 表达升高(P＜0.05,P＜0.01);Beclin1、LC3-Ⅱ 荧光信号明显增强;PI3K、Akt、mTOR、p-PI3K、p-Akt、p-mTOR蛋白表达降低(P＜0.01);LncRNA H19 mRNA表达水平升高(P＜0.01),PI3K、Akt、mTOR mRNA表达水平降低(P＜0.01).经GDL治疗后,肝组织纤维化程度明显改善;HA、LN、PC-Ⅲ、C-Ⅳ水平降低;自噬小体数量明显减少;Beclin1、LC3-Ⅱ/LC3-Ⅰ蛋白表达水平降低(P＜0.05,P＜0.01);Beclin1、LC3-Ⅱ荧光信号随药物剂量呈不同程度的减弱;P13K、Akt、mTOR、p-PI3K、p-Akt、p-mTOR 蛋白表达升高(P＜0.01);LncRNA H19 mRNA 表达水平降低(P＜0.01),PI3K、Akt、mTOR mRNA 表达水平升高(P＜0.05,P＜0.01).结论:GDL可能通过LncRNAH19调控PI3K/Akt/mTOR自噬信号通路减轻肝纤维化程度,减少肝损伤.
Abstract
Objective:To investigate the potential mechanisms and pathways through which Gandouling(GDL)exerts its effects in the treatment of liver fibrosis in Wilson disease.Methods:Sixty male SD rats were randomly divided into six groups:the normal group,the model group,the GDL low-,medium-,and high-dose groups(0.24,0.48,0.96 g·kg-1),and the penicillamine group(90 mg·kg-1),with 10 rats in each group.A copper-loaded Wilson disease rat model was established by gavage administration of 300 mg·kg-1 copper sulfate pentahydrate to all groups except the normal group.Hematoxylin-eosin(HE)staining and Masson staining were used to observe the pathomorphological changes in the liver.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the levels of hyaluronic acid(HA),laminin(LN),procollagen type-Ⅲ peptide(PC-Ⅲ),and collagen type-Ⅳ(C-Ⅳ).Transmission electron microscopy was used to examine the ultrastructure of liver tissues.Real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the expression levels of liver tissues and serum exosomal long noncoding RNA H19(LncRNA H19),phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),and mammalian target of rapamycin(mTOR).Western blot analysis was performed to assess the expression levels of PI3K,Akt,mTOR,and their phosphorylated forms,as well as autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3B(LC3-Ⅱ/LC3-Ⅰ)in liver tissues.Beclin1 and LC3-Ⅱ fluorescence signal intensity was observed by immunofluorescence.Results:Compared with the normal group,the model group exhibited inflammatory cell infiltration in hepatocytes,unclear nuclear boundaries with cell cleavage and necrosis,and collagen fiber deposition around confluent areas.The levels of HA,LN,PC-Ⅲ,and C-Ⅳ were significantly elevated(P＜0.01).Transmission electron microscopy revealed an increased number of autophagic vesicles,with autophagic lysosomes exhibiting a single-layer membrane structure following degradation of most envelopes.Expression levels of Beclin1 and LC3-Ⅱ/LC3-Ⅰ were significantly increased(P＜0.01),and fluorescence signals of Beclin1 and LC3-Ⅱ were markedly enhanced.The protein expression levels of PI3K,Akt,mTOR,p-PI3K,p-Akt,and p-mTOR were reduced(P＜0.01),while LncRNA H19 expression was increased(P＜0.01),and mRNA expression levels of PI3K,Akt,and mTOR were decreased(P＜0.01).After treatment with GDL,the degree of liver fibrosis was significantly improved,with decreased levels of HA,LN,PC-Ⅲ,and C-Ⅳ.The number of autophagic vesicles was significantly reduced,and expression levels of Beclin1 and LC3-Ⅱ/LC3-Ⅰ proteins were lower(P＜0.01).The fluorescence signals of Beclinl and LC3-Ⅱ weakened dose-dependently.The protein levels of PI3K,Akt,mTOR,p-PI3K,p-Akt,and p-mTOR were elevated(P＜0.01),while the expression level of LncRNA H19 was reduced(P＜0.01).Furthermore,the mRNA expression levels of PI3K,Akt,and mTOR increased(P＜0.05,P＜0.01).Conclusion:GDL may alleviate liver fibrosis and reduce liver injury by regulating the PI3K/Akt/mTOR autophagy signaling pathway via LncRNA H19.
国家科技期刊平台
NSTL
维普
万方数据