利拉鲁肽调控miR-20b及TXNIP对非酒精性脂肪性肝病的影响机制研究
Mechanism effect of liraglutide on non-alcoholic fatty liver disease via regulating miR-20b and TXNIP
徐理茂 1吴成凤 1董和英 1黄晨曦 1王菲1
作者信息
- 1. 成都市郫都区人民医院,四川 成都 611730
- 折叠
摘要
目的 探讨利拉鲁肽(LG)对非酒精性脂肪性肝病(NAFLD)的影响机制,以及其对微小RNA20b(miR-20b)和硫氧还蛋白相互作用蛋白(TXNIP)的调控机制.方法 人肝细胞L02购自通派(上海)生物科技有限公司.50只雄性成年SD大鼠,鼠龄9～10周,体质量275～300 g.细胞实验部分:采用棕榈酸(PA)诱导人肝细胞L02形成脂肪变性细胞;由油红O、Rh123染色依次检测各组细胞中脂质沉积和线粒体膜电位的变化;经miRNA生物芯片分析及聚合酶链式反应(PCR)实验确定LG的作用靶点.动物实验部分:大鼠被随机分为正常对照组(Normal组)、NAFLD组(Model组)、NAFLD+LG组(LG组)、NAFLD+LG+miR-20b-antagomiR 组(LG+antagomiR组)、NAFLD+LG+miR-20b-antagomiR+siR-NA-TXNIP组(LG+antagomiR+si组).高脂饲料连续喂养12周构建大鼠NAFLD模型;生化分析仪检测大鼠血清中总胆固醇(TC)、甘油三酯(TG)的含量;实时荧光定量聚合酶链反应(RT-qPCR)检测大鼠肝组织中miR-20b和TXNIP的表达;JC-1染色检测大鼠肝组织细胞的线粒体膜电位的变化;油红O检测大鼠肝组织中的脂质沉积;Western blot检测各组大鼠肝组织中TXNIP、硫氧还蛋白还原酶(TRXR)、线粒体融合蛋白2(MFN2)、视神经萎缩蛋白1(OPA1)和脂滴包被蛋白2(PLIN2)的表达.结果 LG能明显降低PA诱导的L02细胞中脂滴沉积,升高其线粒体的膜电位;经miRNA生物芯片分析及PCR实验确定LG的作用靶点为miR-20b,双荧光素酶实验确定miR-20b靶向调控TXNIP的表达.LG能明显降低NAFLD大鼠血清中TC、TG的含量,升高肝组织中miR-20b、TRXR、MFN2、OPA1的表达及肝组织中的线粒体膜电位,降低肝组织中TXNIP和PLIN2表达及脂滴沉积.LG+antagomiR能部分逆转LG对肝组织的保护作用,LG+an-tagomiR+si能部分恢复药物这些作用,差异均具有统计学意义(均P＜0.05).结论 利拉鲁肽可以调控miR-20b的表达,靶向TXNIP的表达,改善NAFLD肝组织中线粒体的功能障碍,下调脂滴沉积.
Abstract
Objective To investigate the influencing mechanism of liraglutide(LG)on non-alcoholic fatty liver disease(NAFLD)and its regulatory mechanism on microRNA20b(miR-20b)and thioredoxin interacting protein(TXNIP).Methods A total of 55 male adult SD rats were sacrificed,with age of 9-10 weeks and body mass of 275-300 g.For cell experi-ment:Palmitic acid(PA)was used to induce human liver cell L02 to form steatotic cells,the lipid deposition and mitochondri-al membrane potential changes in each group were detected by oil red O and Rh123 staining.The target of LG was deter-mined by miRNA biochip analysis and polymerase chain reaction(PCR).For animal experiment:Rats were randomly divided into normal control guoup(Normal group),NAFLD group(Model group),NAFID+LG group(LG group),NAFLD+LG+miR-20b-antagomiR group(LG+antagomiR group),NAFLD+LG+miR-20b-antagomiR+siRNA-TXNIP group(LG+an-tagomiR+si group).The NAFLD rat model was established by feeding high-fat diet for 12-week.The contents of total cholesterol(TC)and triglyceride(TG)in serum of rats were detected by biochemical analyzer.The expression of miR-20b and TXNIP in rat liver tissue was detected by real-time fluorescence quantitative PCR(RT-qPCR).JC-1 staining was used to detect changes of mitochondrial membrane potential in rat liver tissue cells.Lipid deposition in rat liver tissue was detected by oil red O,and Western blot was used to detect expression of TXNIP,thioredoxin reductase(TRXR),mitochondrial fusion protein 2(MFN2),optic atrophy protein 1(OPA1)and lipid droplet-coated protein 2(PLIN2)in liver tissues of rats.Re-sults LG significantly reduced PA-induced lipid droplet deposition in L02 cells and increased mitochondrial membrane po-tential.The miRNA biochip analysis and PCR experiments determined that the target of LG was miR-20b,and dual luciferase assay determined that miR-20b targeted the expression of TXNIP.LG significantly reduced the content of TC and TG in serum of NAFLD rats,increased the expression of miR-20b,TRXR,MFN2,OPA1 and mitochondrial membrane potential in liver tis-sue,and decreased the expression of TXNIP and PLIN2 in liver tissue and lipid droplet deposition.LG+antagomiR partially reversed the protective effect of LG on liver tissue,and LG+antagomiR+si partially restored the drug effect,and the differ ences were statistically significant(P＜0.05).Conclusion It is demonstrated that LG can regulate miR-20b expression,target TXNIP expression,improve mitochondrial dysfunction and down-regulate lipid droplet deposition in NAFLD liver tissue.
关键词
利拉鲁肽/非酒精性脂肪性肝病/微小RNA20b/硫氧还蛋白相互作用蛋白Key words
liraglutide/non-alcoholic fatty liver disease/miR-20b/thioredoxin interacting protein引用本文复制引用
基金项目
成都市医学科研项目(2021)(2021373)
出版年
2024
NETL
NSTL
万方数据