晚期自噬在非酒精性脂肪肝中的促凋亡作用

The effect of late autophagy on inducing apoptosis in nonalcoholic fatty liver

目的在非酒精性脂肪肝体外模型中研究油酸长期刺激对自噬的影响及其作用。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。用抗M30抗体对培养的细胞爬片做免疫组化来确定凋亡细胞。荧光定量PCR确定凋亡相关基因BCL2和BAX的表达。结果油酸刺激肝癌HepG2细胞12h时自噬水平很低,36h后自噬水平再次显著升高。抗M30抗体免疫组化结果显示12h时肝癌HepG2细胞开始凋亡,至36h时有52%细胞出现凋亡。LY294002处理可使12h和36h的自噬消失,并且也明显减弱了凋亡的发生。凋亡相关基因BCL2和BAX的基因转录水平在油酸处理的12h和36h无显著性差异,LY294002处理也未引起两者转录的显著改变。结论油酸长期刺激肝癌HepG2细胞可以引起自噬介导的凋亡,但该凋亡发生的机制可能和BCL2/BAX基因转录水平的调节无关。

Objective To study the function of autophagy in in vitro model of nonalcoholic fatty liver （NAFL）. Methods HepG2 cells were cultured in DMEM and 400 μmol/L oleate, which was used as an in vitro model of NAFL. Transfeetion of plasmids encoding GFP-LC3 into HepG2 cells and auotophagosomes formation/autophagy development was confirmed by observing accumulation of LC3 puncta. PI3K inhibitor LY294002 was employed to inhibit autophagy. M30 immunoreaetivity was used as a method for detecting apoptosis development. The levels of BCL2 and BAX expression were determined by Real-time PCR. Results HepG2 cells showed a very low level of autophagy after 12 hours oleate stimulus, but autophagy level reached a high level after 36 hours oleate stimulus. M30 immunoreactivity showed apoptosis could be induced after 12 hours oleate stimulus and reached a high level at which 52% cells experiencing apoptosis. LY294002 treatment caused depletion of autophagy in HepG2 cells at 12 and 36 hours oleate stimulus, which was accompanied by a reduced response of apoptosis. Both oleate and LY294002 treatment did not cause significant change in BCL2 and BAX transcription. Conclusion Long term of stimulus of oleate can induce autophagy mediated apoptosis in HepG2 cells, however, this apoptosis may not be associated with the regulation of transcription of BCL2 and BAX.