油酸通过TLR4/NF-κB信号通路诱导体外肝细胞脂肪变性的研究  

Research of oleic acid induces hepatocyte steatosis in vitro through TLR4/NF-κB signaling pathway

作　　者：刘洋[1] 赖铭裕[1] 陈晖[1] 崔萍 LIU Yang;LAI Mingyu;CHEN Hui;CUI Ping(Elderly Gastroenterology Department,the First Affiliated Hospital of Guangxi Medical University,Nanning 530021;Life Sciences Institute,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学第一附属医院老年消化内科,广西南宁530021 [2]广西医科大学生命科学研究院,广西南宁530021

出　　处：《临床医学研究与实践》2025年第4期15-19,共5页Clinical Research and Practice

基　　金：国家自然科学基金项目(No.8226030252)。

摘　　要：目的研究油酸体外诱导WRL68人正常肝细胞脂肪变性的最适浓度,探究肝细胞脂肪变性的可能信号传导通路及评价体外细胞实验的应用前景。方法采用WRL68人正常肝细胞系作为实验材料,根据完全DMEM培养基中油酸浓度的不同共分为7个组,即DMEM对照组、0.4%DMSO组、0.1 mmol/L油酸组、0.3 mmol/L油酸组、0.5 mmol/L油酸组、0.7 mmol/L油酸组和0.9 mmol/L油酸组,各组细胞均体外培养24 h。采用油红Ο染色实验观察细胞脂滴聚集情况;采用CCK-8法检测肝细胞活力;采用酶联免疫吸附法(ELISA)检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、甘油三酯(TG)及核因子κB(NF-κB)含量,确定最适诱导浓度,建立肝细胞脂肪变性的离体细胞模型。确定模型构建条件后,采用RT-PCR检测模型肝细胞中Toll样受体4(TLR4)、NF-κB、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)基因的相对表达量。结果0.7 mmol/L油酸组、0.9 mmol/L油酸组的肝细胞活力低于DMEM对照组(P<0.05)。0.5 mmol/L油酸组的TG含量高于DMEM对照组(P<0.05)。0.3 mmol/L油酸组、0.5 mmol/L油酸组的AST含量高于DMEM对照组(P<0.05);0.5 mmol/L油酸组的NF-κB含量高于DMEM对照组(P<0.05)。0.5 mmol/L油酸组的TLR4、NF-κB、TNF-α及IL-6基因的相对表达量高于DMEM对照组(P<0.05)。结论在WRL68人正常肝细胞中添加诱导浓度为0.5 mmol/L的油酸可建立离体肝细胞脂肪变性模型,其作用机制是通过调节TLR4/NF-κB信号通路增加肝细胞脂肪变性,这可以为从细胞水平研究非酒精性脂肪性肝病(NAFLD)的发病机制及药物治疗提供实际应用价值。Objective To study the optimal concentration of oleic acid in inducing steatosis of WRL68 human normal hepatocytes in vitro,explore the possible signal transduction pathway of hepatocyte steatosis and evaluate the application prospect of in vitro cell experiments.Methods WRL68 human normal hepatocyte line was used as experimental material,according to the concentration of oleic acid in complete DMEM medium,the cells were divided into seven groups:DMEM control group,0.4%DMSO group,0.1 mmol/L oleic acid group,0.3 mmol/L oleic acid group,0.5 mmol/L oleic acid group,0.7 mmol/L oleic acid group and 0.9 mmol/L oleic acid group,the cells in each group were cultured in vitro for 24 h.Oil red O staining experiment was used to observe the aggregation of lipid droplets in cells;the viability of hepatocytes was detected by CCK-8 method;the contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG)and nuclear factor-κB(NF-κB)were detected by enzyme-linked immunosorbent assay(ELISA)to determine the optimal induction concentration and establish an in vitro model of hepatocyte steatosis.After the model construction conditions were determined,the relative expression levels of Toll-like receptor 4(TLR4),NF-κB,tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)genes in model hepatocytes were detected by RT-PCR.Results The viability of hepatocytes in the 0.7 mmol/L oleic acid group and 0.9 mmol/L oleic acid group was lower than that in the DMEM control group,respectively(P<0.05).The content of TG in the 0.5 mmol/L oleic acid group was higher than that in the DMEM control group(P<0.05).The content of AST in the 0.3 mmol/L oleic acid group and 0.5 mmol/L oleic acid group was higher than that in the DMEM control group,respectively(P<0.05);the content of NF-κB in the 0.5 mmol/L oleic acid group was higher than that in the DMEM control group(P<0.05).The relative expression levels of TLR4,NF-κB,TNF-αand IL-6 genes in the 0.5 mmol/L oleic acid group were higher than those in the DMEM control group(

关 键 词：非酒精性脂肪性肝病 TLR4/NF-κB信号通路 油酸 

分 类 号：R575.5[医药卫生—消化系统]