黄连碱促进肝细胞自噬及胆固醇外流改善脂肪肝的脂质蓄积

目的用油酸和棕榈酸处理HepG2细胞构建脂肪肝细胞模型，观察黄连碱（COP）对脂肪肝细胞中脂质蓄积的影响及其作用机制。方法采用100 μmol/L油酸和50 μmol/L棕榈酸诱导HepG2细胞脂肪变性形成细胞内脂质蓄积，构建脂肪肝细胞模型；通过CCK8检测不同浓度的黄连碱对HepG2细胞增殖活性的影响，设置4组细胞模型:对照组、脂肪肝细胞模型组（FFA组）、COP+HepG2组以及COP+FFA组；通过RT-PCR和Western blotting检测3组细胞的自噬基因LC3Ⅰ/Ⅱ、ATG5及胆固醇外流介导因子ABCA1 mRNA及蛋白水平的表达变化；采用油红O染色观察对照组、FFA组和COP+FFA组3组细胞的脂滴变化。结果油红O染色结果显示相较于对照组，HepG2细胞经过油酸和棕榈酸共同处理后的FFA组细胞内脂质明显增多；而COP+FFA组中细胞的脂滴明显减少。CCK8结果显示0~25 μmol/L的黄连碱对HepG2细胞活性无明显影响（P>0.05）。Western blotting与RT-PCR结果显示:相对于对照组，FFA组细胞自噬以及胆固醇外流ABCA1水平明显降低，而COP+HepG2组自噬基因LC3Ⅰ/Ⅱ和ATG5以及ABCA1表达水平明显升高；相对于FFA组，COP+FFA组细胞中脂质蓄积明显减少，差异均有统计学意义（P < 0.05~P < 0.01），且自噬与胆固醇外流障碍也被改善。结论黄连碱可通过促进HepG2细胞自噬及胆固醇外流，减少油酸与棕榈酸诱导形成的脂肪肝细胞中的脂质蓄积。

Coptisine promots hepatocyte autophagy and cholesterol efflux to improve lipid accumulation in fatty liver

ObjectiveTo construct a model of fatty liver cell of HepG2 cells treated with oleic acid and palmitic acid, and observe the effects of coptisine(COP) on the accumulation of lipid droplets in fatty liver cells and its mechanism of action.MethodsThe fatty liver cell model of HepG2 cells was constructed by induction of 100 μmol/L oleic acid and 50 μmol/L palmitic acid.The CCK8 was used to detect the effects of COP at different concentrations on cell proliferation.The cells were divided into the control group, the fatty liver cell model group(FFA group), COP+HepG2 group and COP+FFA group.The RT-PCR and Western blotting were used to detect the mRNA and protein levels of autophagy genes LC3Ⅰ/Ⅱ, ATG5 and cholesterol outflow-mediated factor ABCA1 in four groups.The changes of lipid droplets in contral, FFA and COP+FFA groups were observed using oil red O staining.ResultsThe results of oil red O staining showed that compared with contral group, the lipid droplets in HepG2 cells treated with palmitic acid and oleic acid increased significantly in FFA group, and the lipid droplets decreased significantly in COP+FFA group.The results of CCK8 showed that the activity of HepG2 cells was not affected by 0-25 μmol/L COP(P>0.05).The results of Western blotting and RT-PCR showed that compared with the control group, the levels of autophagy and cholesterol outflow-mediated factor ABCA1 in the FFA group significantly reduced, while the expression levels of autophagy genes LC3Ⅰ/Ⅱ, ATG5 and ABCA1 in the COP+HepG2 group significantly increased(P < 0.05 to P < 0.01).Compared with the FFA group, the lipid accumulation in COP+FFA group significantly reduced, and the difference of which between FFA group and COP+FFA group was statistically significnat(P < 0.05 to P < 0.01).The autophagy and cholesterol efflux disorders were also improved in the COP+FFA group.ConclusionsCOP can promote the autophagy and cholesterol efflux of HepG2 cells, and alleviate the accumulation of lipids in fatty liver cells induced by oleic acid and palmitic acid.