Study On The Lipid Metabolism Regulation And Mechanism Of Flavonoid Derivative Fla-CN In Vitro

Obesity has been the most common metabolic disease worldwide.It has been shown to be correlated with a series of chronic diseases,including type 2 diabetes,insulin resistance,hyperlipoidemia,fatty liver and hypertension.We previously investigated the anti-diabetic chemical constituents in Chinese herb potentilla chinensis and demonstrasted the anti-diabetic role of compound tiliroside.We synethetized a series of derivatives and found that flavonoid derivative Fla-CN exhibited marked effect of anti-diabetes and anti-obesity.Based on these,in this project,we aimed to investigate the role and the molecular mechanism of Fla-CN in the regulation of lipid metabolism using mouse preadipocyte cell line 3T3-L1 and human hepatoma carcinoma cell line Hep G2,so as to provide experimental evidence for the screening of active drug and the identification of drug target.In part one,we investigated the inhibitory effect of Fla-CN on 3T3-L1 preadipocytes differentiation and its molecular mechanism.Fla-CN could markedly inhibit the differentiation of 3T3-L1 preadipocytes and reduce the contents of intracellular lipids.Fla-CN mainly inhibited the early phase of 3T3-L1 preadipocytes differentiation.Fla-CN could increase the protein phosphorylations of AMPK during 3T3-L1 preadipocytes differentiation.This inhibitoryeffect was partially blocked by Compound C.Fla-CN also upregulated miR-27a/b.Fla-CN inhibited adipocyte differentiation via activating AMPK and up-regulating miR-27a/b in 3T3-L1 cells.Fla-CN could significantly inhibit the expression of related mRNA,transcription factors and adipocyte specific protein during 3T3-L1 preadipocytes differentiation,including PPAR?,C/EBP?,SREBP-1c,FAS and aP2.In part two,we investigated the effect of Fla-CN on 3T3-L1 adipocytes lipolysis.Fla-CN could markedly promote the intracellular triglycerides hydrolyzation of adipocytes to reduce the contents of intracellular triglyceride and increase the levels of extracellular glycerols and free fatty acids.Firstly,Fla-CN could increase the protein phosphorylations of AMPK and ACC in adipocytes.Compound C partially blocked the promotion effect of Fla-CN on the intracellular triglycerides hydrolyzation of adipocytes.Secondly,Fla-CN down-regulated miR-29b/c by activating AMPK in adipocytes.Finally,Fla-CN could up-regulate the related mRNA levels of intracellular triglycerides hydrolyzation of adipocytes and increase the protein expressions of HSL and ATGL in adipocytes.In part three,we investigated the effect of Fla-CN on high glucose induced lipogenesis of HepG2 cells and its molecular mechanism.Flavonoid derivative Fla-CN could effectively inhibit high glucose induced lipogenesis of Hep G2 cells and reduce the intracellular lipid contents in a dose-dependent manner.MiR-122 mimic/inhibitor transfection combined with AMPK activator/inhibitor treatment proved that AICAR had no influence on miR-122 expression,suggested that activating AMPK had no effect on expression of miR-122.Fla-CN reduced the levels of miR-122 in HepG2 cells.After treating with Compound C,which blocked the activation of AMPK,Fla-CN still reduced the expression level of mi R-122,suggesting that the suppression role of Fla-CN on the expressions of miR-122 was independent of AMPK activation.Oil O Red staining showed that upon the treatment of Compound C,Fla-CN showed reduced inhibitory effect on the lipogenesis in HepG2 cells.Therefore,Fla-CN inhibited high glucose induced lipogenesis in HepG2 cells by activating AMPK and reducing the levels of miR-122.Fla-CN reduced the protein and mRNA expressions of lipogenesis related genes during high glucose induced lipogenesis of HepG2 cells,including PPAR?,C/EBP?,SREBP-1c,and FAS.Additionally,Fla-CN could dose-dependently increase the mRNA and protein expressions of fatty acid oxidation related genes during this process,including PPAR?,CPT-1? and PGC-1?.In conclusion,by using AMPK activator/inhibitor treatment and miRNAs(miR-27a/b,miR-29b/c and miR-122)mimics/inhibitors transfection,we for the first time demonstrated that Fla-CN inhibited 3T3-L1 preadipocytes differentiation by up-regulating miR-27a/b and activating AMPK;Fla-CN could reduce the expressions of miR-29b/c via activating AMPK,resulted in enhanced lipolysis of adipocytes.Fla-CN inhibited high glucose induced lipogenesis of HepG2 cells by activating AMPK and reducing the expressions of miR-122.