微小RNA-377-3p调控自噬改善脂多糖/D-半乳糖胺诱导的急性肝衰竭的机制研究  

Mechanism of microRNA-377-3p regulating autophagy to ameliorate lipopolysaccharide/D-galactosamine-induced acute liver failure

作　　者：陈玲[1] 李楠[2] 杨建乐 Ling Chen;Nan Li;Jianle Yang(Department of Infectious Diseases,Zhejiang Hospital,Hangzhou 310013,China;Department of Gastroenterology,Zhejiang Hospital,Hangzhou 310013,China)

机构地区：[1]浙江医院感染疾病科,杭州310013 [2]浙江医院消化内科,杭州310013

出　　处：《中华危重症医学杂志（电子版）》2023年第2期89-97,共9页Chinese Journal of Critical Care Medicine:Electronic Edition

基　　金：浙江省医药卫生科技计划项目(2022KY002、2021KY422)。

摘　　要：目的本研究旨在研究微小RNA-377-3p(miR-377-3p)通过成纤维细胞生长因子受体1(FGFR1)对急性肝衰竭(ALF)自噬和凋亡的影响。方法分别通过D-半乳糖胺(D-GalN)/脂多糖(LPS)和D-GalN/肿瘤坏死因子α(TNF-α)诱导ALF动物模型和细胞凋亡模型。将12只C57BL/6J小鼠分为对照组和模型组,每组6只。将细胞分为对照组、模型组(经D-GalN、TNF-α诱导)、NC mimic组(D-GalN、TNF-α诱导并转染无意义miRNA对照序列)、miR-377-3p mimic组(D-GalN、TNF-α诱导并转染miR-377-3p模拟物)和miR-377-3p mimic+3-MA组(D-GalN、TNF-α诱导并转染miR-377-3p模拟物的同时应用自噬抑制剂3-MA干预)。检测小鼠肝脏组织病理情况、血清中生化参数水平、凋亡相关蛋白、自噬相关蛋白以及各组细胞中miR-377-3p RNA、自噬相关蛋白、FGFR1蛋白表达水平。通过双荧光素酶报告基因实验检测miR-377-3p和FGFR1靶向结合关系,并采用流式细胞术检测细胞凋亡情况。结果苏木素-伊红(HE)染色结果显示,对照组小鼠中央静脉周围肝细胞呈放射状排列,无炎症细胞浸润;模型组小鼠中肝小叶结构破坏,大量肝细胞坏死,周围炎症细胞浸润。5组细胞的凋亡率和自噬蛋白Beclin-1、LC3Ⅱ/Ⅰ、p62的表达比较,差异均有统计学意义(F=88.520、42.760、95.870、62.930,P均<0.001),且miR-377-3p mimic组细胞凋亡率和p62蛋白水平均较模型组显著降低,Beclin-1、LC3Ⅱ/Ⅰ水平则均显著升高(P均<0.05)。对照组、模型组、NC mimic组、miR-377-3p mimic组FGFR1蛋白表达水平比较,差异具有统计学意义(F=84.670,P<0.001),且miR-377-3p mimic组FGFR1表达水平较模型组显著降低(P<0.05)。对照组、模型组、miR-377-3p mimic组、miR-377-3p mimic+NC组(D-GalN、TNF-α诱导并转染miR-377-3p模拟物和空白载体)和miR-377-3p mimic+FGFR1组(D-GalN、TNF-α诱导并转染miR-377-3p模拟物和FGFR1目的序列载体)自噬相关蛋白Beclin-1、LC3Ⅱ/Ⅰ和p62的表达水平比较,差异均有�Objective To explore the effect of microRNA-377-3p(miR-377-3p)on autophagy and apoptosis in acute liver failure(ALF)through fibroblast growth factor receptor 1(FGFR1).Methods D-galactosamine(D-GalN)/lipopolysaccharide and D-GalN/tumor necrosis factor-alpha(TNF-α)were respectively used to induce ALF animal models and cell apoptosis models.Twelve C57BL/6J mice were divided into a control group and a model group,with six mice in each group.Cells were divided into a control group,a model group(induced by D-GalN and TNF-α),a NC mimic group(induced by D-GalN and TNF-αand transfected with nonsense miRNA control sequence),a miR-377-3p mimic group(induced by D-GalN and TNF-αand transfected with miR-377-3p mimics)and a miR-377-3p mimic+3-MA group(induced by D-GalN and TNF-αand transfected with miR-377-3p mimics while applying autophagy inhibitor 3-MA intervention).The pathology of liver tissue,the serum levels of biochemical parameters,apoptosis-related proteins,autophagy-related proteins and the cellular expression levels of miR-377-3p RNA,autophagy-related proteins,FGFR1 protein were examined in each group.The targeted binding relationship between miR-377-3p and FGFR1 was measured by dual-luciferase reporter gene experiment and apoptosis was determined by flow cytometry.Results Hematoxylin-eosin staining showed that the hepatocytes around the central vein of the control group were arranged radially,and there was no inflammatory cell infiltration.In the model group,the structure of hepatic lobule was destroyed,a large number of liver cells were necrotic,and surrounding inflammatory cells were infiltrated.There were significant differences in the apoptosis rate and expressions of autophagy proteins Beclin-1,LC3Ⅱ/Ⅰand p62 in the five groups(F=88.520,42.760,95.870,62.930;all P<0.001).Compared with the model group,the apoptosis rate and p62 protein level of the miR-377-3p mimic group were significantly decreased,while the levels of Beclin-1 and LC3Ⅱ/Ⅰwere significantly increased(all P<0.05).The FGFR1 protein expr

关 键 词：微小RNA-377-3p 成纤维细胞生长因子受体 自噬 急性肝衰竭 

分 类 号：R575.3[医药卫生—消化系统]