自噬相关蛋白Atg5在BPA所致肝脏脂质沉积中的作用研究

Regulation of Atg5 on hepatic lipid deposition induced by bisphenol A

摘要:

目的：观察自噬相关蛋白Atg5是否介导双酚A（bisphenol A，BPA）所致的肝脏脂质沉积。方法：将HepG2肝细胞分为对照组、BPA不同浓度（1、10 μmol/L）干预组、阳性对照组即200 μmol/L油酸（oleic acid，OA）联合400 μmol/L软脂酸（Palmitic acid，PA），分别给予上述不同干预24 h。采用化学酶促-比色法定量检测细胞内甘油三酯（triglyceride，TG）含量，油红O染色观察细胞内脂滴沉积情况。Real-time PCR及Western blot、免疫荧光方法检测在不同干预情况下HepG2细胞内Atg5的表达水平。结果：①不同浓度BPA干预后，HepG2细胞内TG含量均有不同程度的升高，其中10 μmol/L BPA组升高具有统计学意义（0.197± 0.033；0.094±0.014，P=0.031）；②油红O染色结果与TG定量一致。③Real-time PCR结果表明，与对照组相比10 μmol/L BPA组Atg5 mRNA表达并无明显变化（0.93±0.088；1.017 5±0.062 4，t=0.811，P=0.428）；Western blot结果显示与对照组相比10 μmol/L BPA组其蛋白表达明显减少（0.519±0.059；1，t=0.8101，P=0.000）；免疫荧光进一步检测发现，与对照组相比10 μmol/L BPA组Atg5荧光小点数量明显减少。结论：自噬相关蛋白Atg5可能介导BPA所致的肝脏脂质沉积的发生。

Abstract:

Objective：To investigate the effects of autophagy related protein Atg5 on hepatic lipid deposition caused by bisphenol A （BPA） in HepG2 cells. Methods：HepG2 cells were cultured and divided into control group，different concentrations of BPA groups（1，10 μmol/L），and positive control group（a mixture of free fatty acids（FFAs）：oleate 200 μmol/L plus palmitate 400 μmol/L），treated for 24 h. The content of intracellular triglyceride（TG） was measured by enzymic assay kit，and oil red O staining was used to determine the intracellular lipid droplet formation visually. Western blot and real-time PCR were used to detect the protein and mRNA expression of Atg5，respectively. Immunofluorescence was used to detect the expression of Atg5. Results：①The level of TG was significantly higher in 10 μmol/L BPA group than in control group（0.197±0.033，0.094±0.014，P=0.031）. ②Oil red O staining also showed that 10 μmol/L BPA promoted lipid accumulation in HepG2 cells. ③The mRNA expression levels of Atg5 in 10μM BPA group did not change compared with that in control group（0.93±0.088；1.017 5±0.062 4，t=0.811，P=0.428），however，the protein expression levels of Atg5 was lower in 10 μmol/L BPA group than that in control group（0.519±0.059；1.00，t=0.8101，P=0.000）. Besides，immunofluo－rescence indicated that the fluorescent dots were obviously reduced in 10 μmol/L BPA group than in control group. Conclusion：Atg5 could be involved in hepatic lipid deposition induced by BPA.