The Mechanism Study Of PYC Regulating Plin5 Expression And Lipid Accumulation In HepG2 Cell By PI3K/PPAR? Pathway

Objective:Non-alcoholic fatty liver diseases?NAFLD?are a clinicopathic syndrome,which characterized by excessive lipid accumulation in the liver in the absence of alcohol consumption and other well-defined factors of liver injury.The pathological process of NAFLD ranges from non-alcoholic fatty liver?NAFL?to non-alcoholic steatohepatitis?NASH?to liver cirrhosis,and in some cases even progress to drive hepatocellular carcinoma.Lipid droplet storage protein 5?Plin5?is a newly discovered protein of PAT?Perilipin1?ADRP?Tip47?family,which is highly expressed in oxidative tissues such as heart,liver,skeleton muscle and brown adipose tissues.Studies show that Plin5 is closely associated with lipid accumulation.Plin5 is highly expressed in the liver of NAFLD patients and obese mice.Over-expression Plin5 in mice promotes triglycerides accumulation in the liver,which suggests that Plin5 is involved in the formation of NAFLD.However,how does the Plin5 participate in the process of lipid accumulation is not very clear.Pycnogenol?PYC?is a French coastal pine bark extract with strong anti-inflammatory and anti-oxidant effects.Our previous studies found that PYC can suppress LPS-induced Plin2 expression in macrophages.In vivo,PYC can be found to improve atherosclerosis and alleviate nonalcoholic fatty liver and promote browning of white fat in ApoE^-/-mice.However,the effect of PYC on the expression of Plin5 in NAFLD has not been investigated.Our study intends to establish a NAFLD model in vitro and to observe the expression of Plin5 in hepatic steatosis.Furthermore,we intend to explore the possible mechanism of Plin5 expression under PYC stimulation in HepG2cells.Methods:HepG2 cells were treated with different concentrations of OA for different time.The expression of Plin5 and PPAR?was detected by RT-PCR and western blot.Then HepG2 cells were treated with the PPAR?inhibitor GW6471,PPAR?inhibitor GSK0660,PPAR?inhibitor GW9662,p38MAPK inhibitor SB203580,the expression of Plin5 was detected by RT-PCR and western blot.HepG2 cells were then treated with PI3K inhibitor LY294002,Plin5 and PPAR?expression were detected by RT-PCR and western blot.The lipid accumulation was determined by Oil Red O staining.Then different dosages of PYC were used to treat OA-stimulated HepG2 cells,the content of intracellular triglyceride was determined by GPO-POD method.Plin5 and PPAR?expression were detected by RT-PCR and Western blot.Results:1.OA promotes the expression of Plin5 in HepG2 cells in a dose-and time-dependent manner.HepG2 cells were treated with different concentrations of OA?solvent control,200?M,400?M?and different time?0h,12h,24h?,the expression of Plin5 was increased in a dose-and time-dependent manner.2.PPAR?mediates the OA-induced Plin5 expression in HepG2 cells.HepG2 cells were treated with indicated PPARs family inhibitors,GW6471 significantly decreased Plin5expression at the mRNA level?P<0.05?,the other two inhibitors have no effects on the expression of Plin5.The Plin5 protein expression was also significantly decreased under GW6471 treatment?P<0.05?.HepG2 cells were treated with different concentrations of OA?solvent control,200?M,400?M?,the expression of PPAR?was also increased in a dose-dependent manner.3.PI3K mediates the OA-induced Plin5 expression in HepG2 cells.HepG2 cells were treated with p38MAPK inhibitor SB203580,there was no significant difference in Plin5protein expression between the OA+SB203580 group and the OA group?P>0.05?.However,HepG2 cells were treated with LY294002,the expression of Plin5 was significantly decreased in OA+LY294002 group compared with OA group?P<0.05?.4.PI3K mediates the OA-induced Plin5 expression by regulating PPAR?in HepG2 cells.HepG2 cells were treated with LY294002,the expression of PPAR?was significantly decreased in OA+LY294002 group compared with OA group?P<0.05?.5.OA promotes lipid accumulation by PI3K/PPAR?pathway in HepG2 cells.The intracellular lipid accumulation was increased as the ever-increasing OA concentrations.However,HepG2 cells were treated with GW6471 or LY294002,the intracellular lipid accumulation was significantly reduced both in OA+GW6471 and OA+LY294002 group?P<0.05?.6.PYC suppresses OA-induced lipid accumulation and reduces triglyceride contents in HepG2 cells.HepG2 cells were treated with different doses of PYC,the intracellular lipid accumulation was much lower in OA+PYC₁₀0 and OA+PYC₅₀0 group than those in OA group?P<0.05?,and the intracellular triglyceride content was significantly decreased?P<0.05?.7.PYC down-regulate the expression of Plin5 and PPAR?in HepG2 cells.HepG2 cells were treated with different doses of PYC,the expression of Plin5 and PPAR?was significantly decreased in the OA+PYC₁₀0 and OA+PYC₅₀0 group?P<0.05?.Conclusions:1.OA promotes Plin5 expression and lipid accumulation by PI3K/PPAR?pathway in HepG2 cell.2.PYC may alleviate lipid accumulation and decrease intracellular TG content via down-regulating Plin5 expression by PI3K/PPAR?pathway in HepG2 cell.