附子理中汤对非酒精性脂肪肝细胞模型脂肪沉积的影响与相关机制研究

Effects of Fuzi Lizhong Decoction on fat deposition in nonalcoholic fatty liver cell model and related mechanism

目的:探究附子理中汤对非酒精性脂肪肝(NAFLD)细胞模型脂肪沉积的影响及相关机制。方法:采用1 ml/l的脂肪乳剂培养肝细胞建立NAFLD细胞模型,并采用高(1.5 g/ml)、中(1 g/ml)、低(0.5 g/ml)附子理中汤及易善复(5μmol/l)干预48 h,MTT检测细胞活力,油红O染色观察脂肪沉积,酶联免疫吸附法检测丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、甘油三酯(TG)、干扰素-α(INF-α)、干扰素-β(INF-β)和白介素-10(IL-10)水平,Annexin V-FITC/PI双染检测细胞凋亡水平,实时荧光定量PCR及免疫印迹检测细胞中Toll样受体4(TLR4)、TRIF相关的接头分子(TRAM)、肿瘤坏死因子受体相关因子3(TRAF3)及干扰素调节因子3(IRF3)的表达水平。结果:与模型组比较,各治疗组细胞活力与INF-α、INF-β、IL-10、TRAM、TRAF3和IRF3水平显著增加,脂肪沉积能力与ALT、AST、TG及TLR4水平显著降低。结论:附子理中汤可降低NAFLD模型细胞的脂肪沉积能力,这可能与附子理中汤对TLR4/TRAM/TRAF3信号通路的有效调节密切相关。

Objective:To investigate the effect and related mechanism of Fuzi Lizhong decoction on the adipose deposition of non-alcoholic fatty liver disease(NAFLD)cells model.Methods:NAFLD cell model was established by culturing hepatocytes with 1ml/l fat emulsion.High(1.5 g/ml),medium(1.0 g/ml)and low(0.5 g/ml)Fuzilizhong decoction and Yishanfu(5μmol/l)were used for 48 h.Cell viability was detected by MTT method and the fat deposition in cells was observed by oil red O staining.Cell culture medium of each group was collected to detect the contents of ALT,AST and TG by ELISA method,as well as the secretion levels of inflammatory cytokines INF-α,INF-βand IL-10.Annexin V-FITC/PI double staining was used to detect the level of apoptosis in each group.The expression levels of TLR4,TRAM,TRAF3 and IRF3 were detected by qRT-PCR and Western bolt.Results:Compared with the model group,the cell viability and the levels of INF-α,INF-β,IL-10,TRAM,TRAF3 and IRF3 in each treatment group were significantly increased,while the fat deposition ability and the levels of ALT,AST,TG and TLR4 were all significantly decreased.Conclusion:Fuzilizhong decoction can reduce the adipose deposition ability of NAFLD model cells,which may be closely related to effective regulation of TLR4/TRAM/TRAF3 signaling pathway.