Effects Of BMP9 Overexpression On Triglyceride Metabolism And Its Mechanism

PART 1 THE EXPRESSION OF BMP9 IN DIFFERENT PATHOLOGICAL MODELS OF MICE AND HUMAN LIVERObjective: To investigate the expression of BMP9 mRNA and protein in liver of NAFLD patients,high fat diet mice and db mice.Methods: 7-week-old C57BL/6J mice were fed on a normal chow diet and high-fat diet for 16 weeks(ND,n=10;HFD,n=10).Received liver tissues from normal and NAFLD patients,db mice and wild mice age-matched,then detect the expression of BMP9 mRNA and protein by Q-PCR and Western blot.Results: The expression of BMP9 mRNA and protein was significantly lower in HFD mice,NAFLD patients and db mice than in the control group(p <0.01).Conclusions: The expression of BMP9 gene may be related to lipid metabolism.PART 2 EFFECTS OF BMP9 OVEREXPRESSION ON TRIGLYCERIDE METABOLISM IN HEPG2 CELLS INDUCED BY SATURATED FATTY ACIDSObjective: To investigate the effect of BMP9 uptake on triglyceride aggregation in HepG2 cells induced by saturated fatty acids.Methods: In the human hepatoma cell line HepG2,the hepatocyte steatosis cell model was induced by saturated fatty acid FFAs(OA: PA = 2: 1),and the appropriate concentration of FFAs was determined according to the amount of intracellular lipid droplets and the cell status.At the same time,the expression of BMP9 was overexpressed by BMP9 overexpressing adenovirus(Ad-BMP9),and the effect of BMP9 gene on lipid aggregation induced by FFAs was detected by oil red O staining and intracellular triglyceride content determination.Results: In human HepG2 cells,hepatocellular steatosis models were successfully induced.According to the results of oil red O staining,the optimal concentration of FFAs was 0.25 m M.The expression of BMP9 was successfully overexpressed according to the expression of BMP9 mRNA and protein in the cells.The expression of BMP9 mRNA decreased with the increase of FFAs concentration The expression of BMP9 was significantly reduced(P <0.01)at 0.25 m M FFAs compared to 0m M.Compared with Ad-GFP group,the fat droplets in Ad-BMP9 group were significantly decreased(p <0.01),and the content of TG was significantly decreased(p <0.01).Conclusions: Up regulation of BMP9 gene expression can reduce the aggregation of triglycerides.PART 3 THE POSSIBLE MECHANISM OF BMP9 OVEREXPRESSION REGULATE TRIGLYCERIDE METABOLISMObjective: To investigate the molecular mechanism of BMP9 in the regulation of triglyceride metabolism in fatty acid-induced hepatocellular steatosis.Methods: The mRNA and protein expression of SREBP1 C,FAS,SCD-1,ACC-1 was detected by Q-PCR and Western blot after the overexpression of BMP9 gene in human HepG2 cells by adding 0.25 m M FFAs to hepatic steatosis;Analyze the change of signal path AMPK;And the parts of BMP9 expression in cells was detected by immunofluorescence assay.Then the effect of BMP9 gene on the promoter of SREBP1 C gene was detected by double luciferase reporter assay and the possible binding sites were found.Results: Compared with Ad-GFP group,the mRNA expression of fatty acid de novo synthesis-related genes(SREBP1C,FAS,SCD-1,ACC-1)in Ad-BMP9 group was significantly decreased(P <0.01).The expression of FAS,SCD-1 and SREBP1 C protein was significantly decreased(p <0.01).The phosphorylation levels of ACC and AMPK protein were significantly increased(p <0.01),while the total protein level of AMPK was significantly decreased(p <0.01).Cell immunofluorescence assay showed that BMP9 was expressed in the nucleus and cytoplasm,but the expression of BMP9 in the cytoplasm was stronger.The results of double luciferase reporter assay showed that the BMP9 gene could significantly down-regulate the activity of SREBP1 C gene promoter(P <0.05).After further truncation of the promoter,it was found that the binding region of BMP9 gene regulated SREBP1 C gene promoter may be located between SREBP1 C gene promoter-212 to-381 and the binding site is LXRE1.Conclusions: BMP9 overexpression can activate AMPK signaling pathway,reduce the accumulation of triglycerides in hepatocytes by reducing the de novo lipogenesis;and BMP9 gene can inhibit the promoter activity of SREBP1 C gene through LXRE1.