Effect Of Pomegranate Polyphenols On Cholesterol Efflux In Hepatocytes And Its Molecular Mechanism By PPARγ-ABCA1 / CYP7A1 Pathway

In this paper, human hepatocyte L02 was selected as experimental cell and the model of steatotic hepatocyte L02 in vitro was constructed. Purified pomegranate peel polyphenols (PPPs), punicalagin (PC) which is the main ingredient of PPPs and pomegranate ellagic acid (PEA) which is the product of PC absorbed were chosen as the tested polyphenols to study the effects’ of pomegranate peel polyphenols on cholesterol level for hepatocyte L02 under fatty change. Real-time PCR (RT-PCR) was used to analyse the expression levels of PPARγ mRNA, ABCA1 mRNA and CYP7A1 mRNA and explore the effects of pomegranate peel polyphenols on cholesterol efflux in human hepatocyte L02 via PPARγ-ABCA1/CYP7A1 pathway. In addition, to construct interference expression plasmid of PPARγ gene and LXRa gene, to establish L02 cell line with stable transfected recombinant plasmids, and to reduce the expression of PPARγ and LXRα in human L02 cell line.The main research findings we had found are as follows:(1) To establish the model of steatotic hepatocyte L02, we used oil red O staining to observe the lipid droplets in L02 cells and the contents of TC,TG,AST,ALT were measured by kits. According to the results, we could find that when hepatocyte L02 was incubated with RPMI-1640 contained 50% fetal bovine serum for 24h, more lipid droplets and less dameage to hepatocyte L02 cells. In other words, the most effective time (24h incubation) was chosen for further study.(2) TC contents in each group were measured to compare the effects of PPPs,PC and PEA on intracellular cholesterol. The data showed that all reagents tested in different concentrations dramatically decreased TC contents. Furthermore, the inhibitory effect of reagents tested strengthened with the increasing of concentration, indicating that all polyphenols tested were similar to lovastatin on lipid-lowering. This observation suggested that PPPs, PC and PEA possessed lipid-lowering effects and enhanced cholesterol efflux. Moreover, PEA was more effective, followed by PPPs.(3) The relative mRNA expression of PPARγ and its target genes ABCA1, CYP7A1 could be up-regulated by PPPs, PC and PEA as well as exhibited a dose-dependent manner. Furthermore, at the same doses PEA was more effective in the regulation of PPARγ mRNA, ABCA1 mRNA and CYP7A1 mRNA and next is PPPs. This was consistent with the lipid-lowering effects result above. Therefore, our present data suggest that PPPs, PC and PEA can regulate upstream the expression of PPARγ, ABCA1 and CYP7A1 to activate PPARγ-ABCA1/CYP7A1 pathway, leading to increased cholesterol efflux in L02 cells.(4) Targeting PPARγ and LXRα gene sequence, a plasmid expression vectors coding for miRNA were constructed. The successful construction was confirmed by DNA sequencing.The vectors were transfected into L02 cells by lipofectamine 2000. Transfection of PPARγ-miRNA, LXRα-miRNA plasmids remarkably down-regulated PPARγ and LXRα expressions in L02 cells both at mRNA level and protein level. The stable transfected L02 cell lines were constructed successfully.