目的:研究硫化氢(H2 S)供体GYY4137释放的外源性H2 S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;H2 S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,H2 S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H2 S可促进脂肪变性肝细胞内脂质自噬分解.
AIM:To evaluate the effect of exogenous hydrogen sulfide ( H2 S) from GYY4137 on lipophagy in mouse primary hepatocytes .METHODS:The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups:the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h;the cells in H2S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which con-tained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h.The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope , phase-contrast microscope or transmission electron microscope . The protein expression of LC 3-Ⅰ/Ⅱin the hepatocytes was determined by Western blot .RESULTS:In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H 2 S group increased .CONCLUSION: In steatosis hepatocytes , exogenous H 2 S promotes the lipophagy .