Effect Of DHEA On Lipid Deposition In Primary Chicken Hepatocytes And Its Cell Biological Mechanism

Dehydroepiandrosterone(DHEA),as the most important intermediatet in the cholesterol metabolism,could directly or indirectly exert its biological actions in the target organ.Many studies had certified that DHEA reduces lipid deposition,but its mechianms is not full clear.Lipid deposition depends on the increasing in the number of adipocyte and adipocyte hypertrophy.Previoius study mainly the changes of factors related to lipolysis to illustrate the mechanism of DHEA on lipid metabolism,but its effect on cell biological characteristics are neglected.Therefore,the aim of the current study was to explore the effects of DHEA on lipid metabolism,cell proliferation,cell cycle and mitochondrial function in primary chicken hepatocytes,which resuts will clarify the mechanism of DHEA regulating body fat deposition from the perspective of cell biology characteristics in primary chicken hepatocytes.These results provide an important theoretical basis for revealing the biological function and mechanism of DHEA.Meanwhile,it would provide a guiding significance for improving the quality of livestock and poultry products and preventing nutrition and metabolic diseases.1.Effect of DHEA on lipid deposition in primary chicken hepatocytesThis study investigate to the effect of DHEA on lipid deposition and the factors expression of realte to lipid metabolism,which wouldclarify the mechanism of DHEA on lipid deposition in primary chiken hepatocytes.The cell was treated with the final concentration of 0 μmol·L-1,0.1 μmol·L-1,1μmol·L-1,10 μmol·L-1,50 μmol· L-1 and 100μmol L-1 DHEAfor 1 h,3 h,6 h,12 h,24 h and 48 h.After treated,the cell viability was detected using MTT assay,the content of triglyceride(TG)was measured by the commercial kit,and the lipid deposition was observated by oil red staining.In addition,the factors expression of relate to lipid metabolism,including the ACC,FAS,CPT-1,PPARa,SREBP-1c and AMPK,were detected by real-time quantitative PCR.Results showed that 0.1-50 μmol·L-1 DHEA treatment for 1-48 h significantly increased hepatocytes cells viability(P<0.05)The addition of 0.1～100 μmol·L-1 DHEA treated for 1～48 h significantly reduced TG content in primary chicken hepatocytes(P<0.05).Oil red staining anlysis showed that 0.1～100 μmol·L-1 DHEA treatment significantly reduced the number of lipid droplets and total area of lipid droplets in primary chicken hepatocytes(P<0.05).0.1～100 μmol·L-1 DHEA treatment significantly reduced the FAS,ACC and SREBP-1c mRNA level,whilesignificantly increased CPT-I mRNA expression level than that in the control group(P<0.05).Compared with the control group,50 μmol·L-1 and 100 μmol·L-1 DHEA treatment significantly increased PPARa mRNA expression level(P<0.05).No significant differences were observed on the AMPKβ1,AMPKβ2,AMPKyl,AMPKy2and AMPKγ3mRNA level,while 0.1～100 μmol·L-1 DHEA treatment significantly increased AMPKα1and AMPKa2 mRNAexpression levelin primary chicken hepatocytes(P<0.01).These results indicated that DHEA treated inhibites lipid deposition by inhibiting lipid synthesis related genes expression and enhanced lipolysis genes expression,and these action maybe achieved by it enhanced AMPKa expression level in primary chicken hepatocytes.2.Effect of DHEA on cell proliferation characteristics in primary chicken hepatocytesThis study investigate the effect of DHEA on cell proliferation,cell cycle and the expression of cycle related factors in primay chicken hepatocytes,and whick would illustrate the mechanism of DHEA regulating lipid droplets accumaulation from the perspective of cell proliferation characteristics in primary chicken hepatocytes.Primary chicken hepatocytes were treated with 0 μmol·L-1,0.1 μmol·L-1,1 μmol-L-1,10 μmol·L-1,50 μmol·L-1 and 100 μmol·L-1 DHEA for 24 h.Cell proliferation was detected using a EdU Assay kit,cell cycle assessment performed using flow cytometry And the expression levels of CyclinA,CyclinB and CDK2 were detected by Real-time quantitative PCR.Results showed that 1～100 μmol·L-1 DHEA treatment obviously reduced the total number of cells and the number of newly proliferated cells in the primary chicken hepatocytes than that in the control group,and it demonstrate an obvious dose-effect relationship.Flow cytometry analysis showed that 1～100 μmol·L-1 DHEA treatment significatnly increased S cell population(P<0.01),and significatnly reduced G2/M cell population(P<0.01)when compared to the control group.Compared with the control group,1～100 μmol·L-1 DHEA treatment significantly reduced CyclinA mRNA level(P<0.05).In addition,1 μmol·L-1,50μmol(L-1 and 100 μmol·L-1 DHEA treatment significantly decreased CDK2 mRNA level(P<0.05),while no significant differences were observed on the CyclinB mRNA expression in primary chicken hepatocytes treated with different concentrations of DHEA(P>0.05).These results indicated that DHEAarrests cell in S phase and inhibits cell proliferation by decreasing CyclinA and CDK2 mRNA level,whick lead to the reducing of the number and total area of lipid droplets in primary chicken hepatocytes.3.Effect of DHEA on mitochondrial function in primary chicken hepatocytesThis study investagate to the effect of DHEA on the number of mitochondrial,membrane potential and succinate dehydrogenase activity,whick would clarify the effect of DHEA on lipid deposition from the perspective of regulating mitochondrial function in primary chicken hepatocytes.Primary chicken hepatocytes was treated with 0 μmol·L-1,0.1μmol·L-1,1 μmol·L-1,10 μmol·L-1,50 μmol L-1 and 100 pmol·L-1 DHEA for 24 h.The morphology and number of mitochondria were viewed in the transmission electron microscopy.Mitochondrial membrane potential was detected by flow cytometry,and succinate dehydrogenase(SDH)activity was determined by commercial assay kit.The results showed that no significant effect on the morphology and the number of mitochondria in primary chicken hepatocytes after DHEA treated(P>0.05).10 μmol· L-1 DHEA treatment significantly reduced the mitochondrial membrane potential(P<0.05),whereas 50 μmol ·L-1 and 100 μmol ·L-1 DHEA treatment substantially reduced the mitochondrial membrane potential(P<0.01).1 μmol·L-1 DHEA treatment could significantly increased SDH activity(P<0.05),10～100 μmol ·L-1 DHEA treatment significantly increased the activity of SDH activity(P<0.01)than that in the control group.These results indicates that DHEA treatment inhibited lipid droplets and promotes cell oxidation metabolism by increasing the permeability of the mitochondrial membrane and SDH activity in primary chicken hepatocytes.