The Regulation Of Lipophagy On Hepatic Lipid Accumulation Induced By Bisphenola

Objective:One of environmental endocrine disruptors BPA (Bisphenol A) can promote liver lipid deposition, but its mechanism is unclear. The loss of lipophagy promote hepatic lipid deposition, it is not clear that whether lopophagy is mediated lipid deposition caused by BPA. To in vestigate the effects of autophagy on hepatic lipid deposition caused by BPA in vivo and in intro.Methods:In vivtro, HepG2 cells were cultured and divided into control group, different concentrations of BPA groups (0.001,1,4, 10μmol/L), and positive control group (a mixture of free fatty acids FFAs:oleate 200μM plus palmitate 400μM), treated for 24 h. The content of intracellular triglyceride (TG) was tested using enzymic assay kit, oil red 0 staining and bodipy 493/503 fluorescence staining were used to determine the intracellular lipid droplet formation visually. Western blot and real-time PCR as well as immunofluorescence were used to detect the expression of LC3 and P62 respectively. Immunofluorescence was used to detect the expression of Atg5. Electron microscope was used to detect the number of autophagy some. ① The initiation phase of autophagy:detected the the expression of p-mTOR、 p-ULK1、Atg5.②The fusion of the autophagosome and lysosome: immunofluorescence was used to detect the merge of LC3 and LAMP1. ③ The stage of lysosomal degradation:AO staining was used to observe the lysosomal acidification, Western blot and real-time PCR was used to detected the the expression of calthepsin L. Further, primary hepatocytes were cultured with 10μM BPA, and the above results for validation. In vivo, CD1 mice was treated with different concentrations of BPA groups (0,5,50, 500μg/kg/day) for 42 days, different metholds were used to detected the above indicators.Results:In vitro, the level of TG was significantly higher in 10μM BPA group than in control group. Oil red O and bodipy 493/503 staining also showed that 10μM BPA promoted lipid accumulation. The expression of LC3-Ⅱ and P62 were increased in 10μM BPA compared with control group. The autophagosome was increased in 10μM BPA by electron microscope. The expression of p-ULK1、p-mTOR were increased and Atg5 was decreased in 10μM BPA compared with control group. The autophagosome-lysosome fusion was decreased as indicated with the merge of autophagosome and lysosome in10μM BPA. Compared with control group, the cytoplasmic AO-red dots dramatically decreased in 10μM BPA group, indicating a reduced number of acidified compartments. The expression of cathepesin L was decreased. In vivo, liver triglycerides were significantly increased in mice exposed to 50 and 500 μg BPA/kg/day, compared with control mice. The expression of LC3-Ⅱ and P62 were increased, Atg5and cathepesin L was decreased in 500μg BPA/kg/day group compared with control group.Conclusion:In vivo and in vitro, we found that BPA can cause liver lipid deposition, at the same time, autophagosome-lysosome fusion and lysosomal degradative capacity, lysosomal acidification as well as the initiation phase of autophagy was impaired in 10μM BPA treatment. These results suggest that autophagy could be involved in hepatic lipid deposition induced by BPA.