早期自噬在非酒精性脂肪肝中的保护作用

The protective role of early autophagy in nonalcoholic fatty liver

目的探讨非酒精性脂肪肝细胞模型中早期自噬的发生及作用。方法用含浓度为400μmol/L油酸的DMEM高糖培养基培养肝癌HepG2细胞,制作体外非酒精性脂肪肝细胞模型。向肝癌HepG2细胞转染GFP-LC3质粒,通过观察LC3Ⅱ绿色荧光斑点确定自噬的发生。采用PI3k抑制剂LY294002抑制自噬。CalceinAM和碘化丙啶(PI)对培养的细胞进行染色来确定活细胞和晚期凋亡细胞。荧光定量PCR确定促凋亡基因BAX的表达。结果油酸刺激的肝癌HepG2细胞在第8小时可以出现强烈的自噬反应,但未刺激细胞没有出现LC3Ⅱ绿色斑点。CalceinAM/PI染色未观察到第8小时出现晚期凋亡细胞。但通过LY294002抑制自噬后第8小时自噬水平明显下调,并且有7.8%的细胞出现晚期凋亡。荧光定量结果显示LY294002抑制自噬的第8小时BAX出现高表达。结论油酸刺激肝癌HepG2细胞的早期,自噬的发生可能对细胞有一定的保护作用,其机制可能与减少促凋亡基因的表达有关。

Objective To study the role of autophagy in nonalcoholic fatty liver （NAFL）. Methods The in vitro model of NAFL of HepG2 cells was made by culturing cells in DMEM which contained 400μmol/L oleate. Transient transfection of GFP-LC3 plasmid was performed with Fugenes and autophagy was detected by whether LC3 puncta had formed. The PI3K inhibitor LY294002 was used to inhibit autophagy. Caleein AM and propidium iodide （PI） were used to detect whether apoptosis had developed. Real-time PCR was employed to detect the level of BAX mRNA. Results A bold autophagy could be detected in HepG2 cells cultured with oleate at 8 hours and there were little positive responses of LC3 puncta in HepG2 cells without oleate stimulus. Apoptosis development was not found by using Calcein AM/PI in HepG2 cells cultured with oleate at 8 hours. However, LY294002 treatment could decrease the level of autophagy and induce apoptosis development among 7.8% HepG2 cells. Real-time PCR revealed that LY294002 inhibited autophagy could induce a high expression of BAX at 8 hours in HepG2 ceils cultured with oleate. Conclusion At early stage of culturing with oleate, autophagy development could protect HepG2 ceils from apoptosis and the mechanism might be associated to decrease the expression of pro-apoptotic genes.