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目的:研究硫化氢(H2S)供体GYY4137释放的外源性H2S对小鼠原代肝细胞内脂质自噬分解的影响。方法:采用2步原位灌流法分离C57BL/6小鼠原代肝细胞并分为4组:用正常培养基培养正常组细胞;模型组用含1.2 mmol/L油酸(溶于10%的BSA)的培养基培养48 h;H2S组和炔丙基甘氨酸(PAG)组则用1.2 mmol/L油酸(溶于10%的BSA)的培养液培养48 h,然后换无血清无酚红的RPMI-1640培养液(同时分别给予1 mmol/L GYY4137和200μmol/L PAG)处理6 h。收集各组细胞做LC3免疫荧光染色,拍摄荧光和相差图片;Western blot检测肝细胞中LC3-Ⅰ/Ⅱ的蛋白表达量;透射电镜观察肝细胞超微结构。结果:和模型组相比,H2S组肝细胞内LC3荧光颗粒和蛋白表达量增加,自噬溶酶体数量增加,细胞内空泡增多。结论:外源性H2S可促进脂肪变性肝细胞内脂质自噬分解。
AIM: To investigate the effect of exogenous H2S released from hydrogen donor (GHS) donor GYY4137 on lipid peroxidation in mouse primary hepatocytes. Methods: Primary hepatocytes of C57BL / 6 mice were isolated by 2-step in situ perfusion and divided into 4 groups: normal cells were cultured in normal medium; the model group was treated with 1.2 mmol / L oleic acid (dissolved in 10% BSA) for 48 h. The H2S and PGA groups were incubated with 1.2 mmol / L oleic acid (10% BSA) for 48 h, then replaced with serum-free phenol red Of RPMI-1640 medium (with 1 mmol / L GYY4137 and 200 μmol / L PAG respectively) for 6 h. The cells in each group were collected for LC3 immunofluorescence staining, fluorescence and phase difference images were taken; the protein expression of LC3-Ⅰ / Ⅱ in hepatocytes was detected by Western blot; the ultrastructure of hepatocytes was observed by transmission electron microscope. Results: Compared with the model group, the expression of LC3 fluorescent particles and protein in hepatocytes of H2S group increased, the number of autophagy lysosomes increased, and the number of intracellular vacuoles increased. Conclusion: Exogenous H2S can promote the lipid autophagy in fatty degeneration hepatocytes.