| Objective:In recent years,the incidence of nonalcoholic fatty liver disease(NAFLD)has increased rapidly,and it has became the most serious chronic liver disease in China.Daily exposure of endocrine disrupting chemicals may be the risk factor of NAFLD.Benzo [a] pyrene(BaP)is a common environmental pollutant,which can interfere with lipid metabolism of cells.However,it is unknown whether low-dose BaP exposure can induce lipid deposition in hepatocytes,thereby promoting the occurrence of NAFLD.The aim of the present study was to establish an in vitro model of low-dose BaP exposure to mouse primary hepatocytes to investigate the effect and the mechanism of action of BaP exposure on Hepatocyte lipid accumulation,This study can also provide clues for understanding the facilitation and mechanism of environmental chemical pollutants to the occurrence of NAFLD,and valuable scientific basis for the prevention and treatment of NAFLD.Methods:1.Isolation and identification of mouse primary hepatocytesPrimary hepatocytes were isolated by using a two-step perfusion method and purified by differential centrifugation.Primary hepatocytes were identified by morphological observation and PAS glycogen staining.2.Detection of lipid deposition in primary hepatocytesThe deposition of lipid droplets in the cells was visualized by Oil red O staining and the area of red lipid droplets was analyzed using Image J.The intracellular TG contents after BaP exposure were detected by TG quantitative detection kit detection.3.Detection of key genes for fatty acid synthesis and decomposition in primary hepatocytesThe m RNA expressions of key genes for de novo synthesis of fatty acids(LXR-α and FANS),key genes for lipid uptake(FABP1 and FATP2),key genes for fatty acid β-oxidation(CPT1A and CPT2)and key gene for lipoprotein synthesis(DGAT1)were detected by real-time quantitative PCR.4.Detection of mTOR pathway and macrolipophagy in primary hepatocytesThe expression of macrolipophagy marker SIRT1 m RNA was examined by Real-time fluorescence quantitative PCR.The expression of mTOR,p-mTOR and the maker proteins(Atg5、p62、LC3-Ⅱ/LC3-Ⅰ)of autophagy were analyzed by Western blot analyses.5.Detection of methylation status of PP2 Ac and regulation of its methylation-related enzymes in primary hepatocytesThe expression of the total PP2 Ac,methylated PP2 Ac,demethylated PP2 Ac,the enzyme of regulating PP2 Ac methylation(LCMT1)and the enzyme of regulating PP2 Ac demethylation(PME)in primary hepatocytes were analyzed by Western blot analyses.Results:1.Isolation and identification of primary mouse hepatocytesThe primary hepatocytes obtained from mouse had adhered well.The cell membrane is clear and the cells did not shrink.It can be seen that the small dots of blue-purple glycogen were scattered in the cytoplasm of primary hepatocytes uniformly distributed after adherent.The purity of primary hepatocytes was >90 %.2.The Activity determination of primary mouse hepatocytes in mouse treated with BaPThe primary mouse hepatocytes were exposed to BaP(0,0.001,0.01,0.1and 1 nmol / L).After 72 hours,the primary mouse hepatocytes treated with different concentrations of BaP had adhered well,The cell membrane is clear and the cells did not shrink.Except the obvious fatty vacuoles in the cells after BaP treatment,the morphology of the cells in each group did not change significantly.Hepatocyte viability of each dose group,was typically > 97%,determined by trypan blue staining.There for,we selected this dose range and treatment time for follow-up research.3.Detection of BaP exposure promoting lipid accumulation in primary mouse hepatocytesThe primary hepatocytes were exposed to BaP(0,0.001,0.01,0.1 and 1nmol / L)for 72 h.In contrast to the control group,with the increase of BaP concentration,more and more lipid droplets stained by Oil Red O were formed in primary mouse hepatocytes,The content of TG in cells increases with the increase of BaP concentrations,the difference was statistically significant(P<0.05).4.Effects of low-dose BaP exposure on the expression of key genes in primary mouse hepatocyte lipid metabolismThe primary mouse hepatocytes were exposed to BaP(0,0.001,0.01,0.1and 1 nmol / L)for 72 h.In contrast to the control group,there were no significant alterations in the expression of the key genes of de novo synthesis of fatty acids(LXR-α and FASN),lipid uptake(FABP1 and FATP2)and lipoprotein synthesis(DGAT1),whereas the expression of the key genes of fatty acidβ-oxidation(CPT1a and CPT2)had increased,the difference was statistically significant(P<0.05).5.Inhibition of macrolipophagy in primary mouse hepatocytes induced by low-dose BaP exposureAfter the primary mouse hepatocytes were treated with different concentrations of BaP(0,0.001,0.01,0.1,and 1 nmol / L),the expression of m RNA in SIRT1 decreased,the protein expression level of Atg5 decreased,and the protein expression level of p62 and LC3-II / LC3 I increased,the differences were statistically significant(P<0.05).6.Low-dose BaP inhibits macrolipophagy to promote lipid accumulation through mTOR pathwayThe intervention of rapamycin(mTOR inhibitor)increased macrolipophagy and alleviated lipid accumulation in primary mouse hepatocytes caused by exposure to Bap:(1)After co-incubations with rapamycin and different concentrations of BaP(0,0.001,0.01,0.1,and 1 nmol / L),the protein expression level of mTOR and p-mTOR decreased,the protein expression level of m RNA in SIRT1 increased,the protein expression level of Atg5 increased,and the protein expression level of p62 and LC3-II / LC3 I decreased,the differences were statistically significant(P<0.05).(2)After co-incubations with rapamycin and different concentrations of BaP(0,0.001,0.01,0.1,and 1 nmol /L),the numbers of lipid droplets,area of red lipid droplets stained by Oil Red O,and intracellular TG content decreased,the differences were statistically significant(P<0.05).7.Low-dose BaP regulates PP2 Ac and its methylation levelThe results show that low-dose BaP exposure can promoting lipid accumulation in primary mouse hepatocytes.At the same time,low-dose BaP exposure can effect PP2 Ac and its methylation status.After the primary mouse hepatocytes were treated with different concentrations of BaP(0,0.001,0.01,0.1,and 1 nmol / L),the protein expression level of total PP2 Ac,methylated PP2 Ac and LCMT1 increased,and the protein expression level of demethylated PP2 Ac and PME decreased,the differences were statistically significant(P<0.05).8.Low-dose BaP activates mTOR pathway through PP2 Ac to inhibit macrolipophagy to promote lipid accumulationThe intervention of OA(PP2Ac inhibitor)increased macrolipophagy and alleviated lipid accumulation in primary hepatocytes caused by Bap exposure :(1)After co-incubations with OA and different concentrations of BaP(0,0.001,0.01,0.1,and 1 nmol / L),the protein expression level of total PP2 Ac,methylated PP2 Ac and LCMT1 decreased,and the protein expression level of demethylated PP2 Ac and PME increased,the differences were statistically significant(P<0.05).(2)After co-incubations with OA and different concentrations of BaP(0,0.001,0.01,0.1,and 1 nmol / L),the protein expression level of mTOR and p-mTOR decreased,the expression of m RNA of SIRT1 increased,the expression of Atg5 increased,and the protein expression level of p62 and LC3-II / LC3-I decreased,the differences were statistically significant(P<0.05).(3)After co-incubations with OA and different concentrations of BaP(0,0.001,0.01,0.1,and 1 nmol / L),the numbers of lipid droplets,area of red lipid droplets stained by Oil Red O,and intracellular TG content decreased,the differences were statistically significant(P<0.05).Conciusion:1.Low-dose BaP exposure promotes lipid deposition in mouse primary hepatocytes.2.Low-dose BaP regulates PP2 Ac to activate mTOR to inhibit macrolipophagy to promote lipid deposition,which is the mechanism of low dose BAP promoting lipid deposition in primary mouse hepatocytes. |
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