The Effect And Mechanism Of Down-regulated Fsp27 Gene Combined With Myricetin On Lipolysis Of 3T3-L1 Adipocytes

Objective:To investigate the effect and regulatory mechanism on lipid metabolism of 3T3-L1 adipocytes by down regulating Fsp27 gene expression,to provide a new theoretical foundation and drug target for the prevention and treatment of obesity.Methods:1.Using RNAi technology to build three positive and a negative shRNA recombinant vectors targeting mouse Fsp27 gene,identified by DNA sequencing.2.3T3-L1 preadipocytes were routinely cultured and induced differentiation by the hormonal cocktail.The shRNA recombinant vectors were transfected into the differentiated 3T3-L1 adipocytes with lipofectamine.In addition,adipocytes were treated with 100?mol/L Myric for 72 hours when Myric intervented adipocytes.3.After combined intervention,the oil red O staining photographed the morphology and sizes of lipid droplets.The content level of adipocytes cellular TG and glycerol were tested through enzymatic method to evaluate the lipolysis.4.Western blot measured the expression levels of Fsp27,the adipose triglyceride lipase(ATGL),hormone sensitive lipase(HSL),p38 MAPK,p-p38 MAPK and PPAR?.Results:1.As the differentiation of the 3T3-L1 adipocytes,amount and size of lipid droplets incresaed.After the oil red O staining,intracellular lipid droplets were distributed around the nucleus like finger ring.2.After lipofectin transfection,the three positive shRNA recombinant vectors could significantly inhibit the protein expression of Fsp27(P<0.05).3.After combined intervention,compared to the control group,content of adipocytes cellular TG decreased significantly with the increased glycerol in sh-RNA group and Myric group(P<0.05),but with no difference between the two groups(P>0.05).Meanwhile,compared to the other three group,the TG content in Myric+sh-RNA group was lower,and the glycerol content increased(P<0.05).4.After combined intervention,compared to the control group,the expression levels of Fsp27 in the other three group decreased significantly,and the expression levels of ATGL and PPAR?increased significantly(P<0.05).In addition,the expression levels of HSL and p-p38MAPK/p38 MAPK in Myric+sh-RNA group and Myric group was higher than that in sh-RNA group and control group,the difference was statistically significant(P<0.05).Conclusions:1.The vectors was successfully constructed.The three positive shRNA recombinant vectors could down-regulate the expression of Fsp27.2.The combination of Fsp27 gene silencing and Myric could more obviously improve the efficiency of lipolysis than individual.3.The regulating effect of Myric on lipolysis of 3T3-L1 adipocytes might be exerted through the MAPK pathway by up-regulating the level of ATGL and HSL.sh-Fsp27 vectors could promote lipolysis by blocking Fsp27 gene and increasing the expression level of ATGL.