利拉鲁肽减轻脂毒性肝细胞损伤并促进自噬改善非酒精性脂肪肝

Liraglutide alleviates lipotoxic liver cell damage and promotes autophagy to improve non-alcoholic fatty liver

目的研究胰高血糖样肽-1受体激动剂(GLP-1RAs)对游离脂肪酸(FFA)诱导的肝细胞脂肪变性的影响,并探讨细胞自噬在该过程中的作用。方法体外人肝细胞培养诱导出非酒精性脂肪性肝病(NAFLD)细胞模型,加入浓度梯度的利拉鲁肽(LRG)观察其对细胞存活率和肝细胞脂肪变性的影响;用氯喹抑制细胞自噬,雷帕霉素(RAPA)激活自噬,探究利拉鲁肽改善肝细胞脂肪变性与细胞自噬的关系。实验分组:对照组,DMEM完全培养基培养的细胞中加入一定浓度BSA;FFA模型组,用1 mmol/L的FFA(OA∶PA=2∶1)诱导肝细胞脂肪变性;LRG组,细胞中同时加入FFA(1 mmol/L)和LRG(100 nmol/L);自噬抑制组,细胞中同时加入FFA(1 mmol/L)、LRG(100 nmol/L)和氯喹(20μmol/L);自噬激活组,细胞中同时加入FFA(1 mmol/L)和RAPA(1μmol/L)。用油红O染色、全自动生化分析仪观察肝细胞内脂质沉积情况,蛋白质免疫印迹法检测自噬相关蛋白(Beclin1、P62、LC3B)水平,观察细胞自噬水平。多组间均数比较用单因素方差分析。结果在一定浓度范围内,随着LRG浓度的增加,肝细胞存活率不断上升,胞内脂质沉积程度不断减轻,LRG浓度达到100 nmol/L时效果最佳,且与FFA组相比差异有统计学意义(P<0.01)。在肝细胞脂肪变性程度与细胞自噬关系的探究中,LRG组肝细胞内甘油三酯含量显著低于FFA组(P<0.01),Beclin1、LC3B-II/LC3B-I水平较FFA组升高,P62水平较FFA组下降,细胞自噬增强;自噬抑制组与LRG组相比,胞内甘油三酯含量有所增高(P<0.01),Beclin1、LC3B-II/LC3B-I水平下降,P62水平增高;自噬激活组中RAPA显著减轻了FFA诱导的肝细胞内甘油三酯沉积,自噬相关蛋白水平变化与LRG作用效果一致。结论GLP-1RAs可以改善FFA诱导的脂毒性肝细胞损伤,并通过促进细胞自噬来改善NAFLD中肝细胞脂肪变性。

Objective To study the effect of glucagon-like peptide-1 receptor agonists(GLP-1RAs)on free fatty acid(FFA)-induced hepatocyte steatosis,and to explore its autophagic role in this process.Methods Human hepatocytes were cultured in vitro to induce NAFLD cell model.Liraglutide(LRG)concentration gradient was added to observe the effect on cell survival rate and fatty degeneration of liver cells.The relationship between liraglutide and autophagy was investigated with chloroquine inhibition and rapamycin(RAPA)activation in hepatocyte steatosis.Experimental group:control group:a certain concentration of BSA was added to cells cultured in DMEM complete medium;FFA model group:fatty degeneration of hepatocytes was induced by 1mmol/L FFA(OA:PA=2:1);LRG group:FFA(1 mmol/L)and LRG(100 nmol/L)were added to the cells at the same time;autophagy inhibition group:FFA(1 mmol/L),LRG(100 nmol/L),and chloroquine(20μmol/L)were added to the cells at the same time;autophagy activated group:FFA(1 mmol/L)and RAPA(1μmol/L)were added to the cells at the same time.Oil red O staining and fully automated biochemistry analyzer were used to observe the intracellular lipidosis condition.Western blotting was used to detect the levels of autophagy-related proteins(Beclin1,P62,and LC3B).One-way analysis of variance was used to compare the means between multiple groups.Results Within a certain concentration range,with the increase of LRG concentration,the hepatocytes survival rate was increased and the degree of intracellular lipidosis had continued to decrease.The best effect was achieved when LRG concentration reached 100nmol/L,and the difference was statistically significant when compared with the FFA group(P<0.01).During the exploration of the relationship between the degree of hepatic steatosis and autophagy,LRG group intracellular triglyceride content was significantly lower than FFA group(P<0.01),and the levels of Beclin1,LC3B-II/LC3B-I were higher than FFA group.Additionally,FFA group had reduced P62 level,and enhanced autophagy.Compared with the LRG group,autophagy inhibition group intracellular triglyceride content was increased(P<0.01),while the levels of Beclin1,LC3B-II/LC3B-I was decreased,and P62 level was increased.Autophagy activated group RAPA had significantly reduced FFA-induced intrahepatic triglyceride deposition,and the changes in autophagy-related protein levels were consistent with the effect of LRG.Conclusion GLP-1RAs can alleviates FFA-induced lipotoxic liver cell damage,and promote autophagy to improve fatty degeneration of hepatocytes in NAFLD.