Phloridzin Ameliorates Non-alcoholic Fatty Liver Disease Via Inhibiting SREBP Regulated By MTOR

Nonalcoholic fatty liver disease (NAFLD) refers to the no long-term a large number of alcohol damage and other stable liver damage factors caused of excessive fat deposition in the liver cells as the main characteristics of clinical pathological syndrome, liver fatty change and NASH is most common in the clinical. NAFLD is increasingly drawing the attention of the researchers for its traits in potential harmfulness and sustainable affects to mainly organs.The liver cell lipid deposition is the precondition of NAFLD. mTOR/SREBP signaling pathways is the key way to regulate lipid metabolism, in the preliminary work, we confirmed through experiments in vivo and in vitro that phlorizin can significantly inhibit fatty degeneration and lipid synthesis in liver cells and we assumed the mechanism is associated with the activation of inhibiting mTOR, SREBPs protein. Based on this, this issue put forward "phlorizin inhibiting liver cell lipid deposition via regulating the signaling pathway activity of mTORC1/SREBP-1C, thus restrain the development of NAFLD as the scientific hypothesis. We study the influence of phlorizin after intervening AP2 transgenic mice and lipid change L02 cells and its mechanism, so we aim to provide reliable experimental basis for the prevention and treatment of NAFLD.ObjectiveThis study was to make a tentative discussion on the mechanism of the intervention of phloridzin to L02 liver cells, via vitro L02 people change fat liver cells model in vitro first, by means of mTOR inhibitors and activators medicine, and as the mTOR downstream protein SREBP Lentivirus transfection for medium. To determine whether phlorizin inhibiting liver cell lipid deposition via regulating the expression of mTORC1/SREBP-1C in L02 cells.Then we used the method of western blotting to detect L02 liver cells influence of mTOR/SREBP signaling pathways of protein expression after the intervention of phlorizin, including p38MAPK、p-p38MAPK、mTOR、p-mTOR、 GβL、Raptor、SREBP-1c、FAS, and many other proteins. Finally used aP2-SREBFlc primary transgenic mouse model to investigate the effect of phlorizin to nonalcoholic fatty liver disease in the side of glucolipid metabolism, which verify the mechanism of action of phlorizin in vivo experiments. Subsequently, we used phlorizin to intervene the AP2-SREBFlc primary transgenic mice of nonalcoholic fatty liver disease, and to investigate whether can phlorizin regular the over-expression of SREBFlc protein level in mice, and then improve lipid metabolism. Finally, we researched the mechanism of phlorizin to regular the signaling pathway of mTORCl/SREBP-1C in ap2-SREBP-lc mice liver tissue, to investigate the influence of phlorizin to NAFLD and its mechanism from in vivo and in vitro.MethodsL02 liver cell experimentsOleic acid was used to induce L02 fatty model of degeneration of liver cells combined with mTOR inhibitors medicine after intervening of phlorizin in different concentration. The number of lipid droplets and oil red 0 staining, intracellular TG, TC, AST, ALT index were detected.Oleic acid was used to induce L02 fatty mode of degeneration of liver cells combined with mTOR activators medicineafter intervening of phlorizin in different concentration. The number of lipid droplets and oil red 0 staining, intracellular TG, TC, AST, ALT index were detected.The influence of phlorizin to the level of mTOR、SREBP-1c、FAS mRNA in the over-expression of EF1A-SREBP-IRES-eGFP Lentivirus transfection L02 cells were detected.The preliminary research on the mechanismsWestern blot method was used to detect fat model of L02 liver cells in mTORCl/SREBP-1C signaling pathways of protein expression after the intervention of phlorizin, including p38MAPK, mTOR, SREBP-1C, FAS and its phosphorylation protein, etc.The animal experimentsAP2-SREBFlc transgenic mice, female,9 weeks of age, high triglycerides feed were divided into control group,AP2-SREBF1c transgenic mice model group, simvastatin group, phlorizin high and low dose group (120,60 mg/kg) in the principal of weight and blood sugar level equally.continuous dosing for 20 weeks. Measurements include:(1) general indicators:body weight, food intake, Lee’s index. (2) the glucolipid metabolism index:abdominal blood glucose, insulin tolerance, triglyceride, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), free fatty acids, insulin, oral glucose tolerance. (3) the histopathologic observation:liver tissueWestern blot method was used to detect AP2 transgenic mice liver tissue mTOR/SREBP signaling pathways protein expression.ResultsThe effects of phlorizin to L02 fatty model.After the phlorizin intervention, OD value of the oil red staining 0 dosage group and the TC, TG, AST, ALT levels were significantly lower than the model group (P<0.05or P<0.01). After Rapamycin combined with various doses of phlorizin intervention, oil red staining OD value compared with the model group also fell, but compared the decreased range of each dose group and phlorizin high dose group, there was no significant difference. After Leucine combined with various doses of phlorizin intervention, oil red staining OD values also fell, But the decreased range of each dose group was smaller than hlorizin high-dose single dose group.Phlorizin can significantly decrease the expression of mTOR and its downstream SREBP-lc、FAS mRNA in Rapamycin group and hlorizin high-dose group in EF1A-SREBP-IRES-eGFP Lentivirus transfection L02 cells.The effects of phlorizin to fatty model L02 liver cells and mTORC1/ SREBP-1C signaling pathways protein expression.After drug intervention, GβL, Raptor, p-mTOR, FAS, SREBP-1C protein expression significantly decreased combined with model group, p38MAPK protein expression increased significantly compared with model group (p<0. Olor p<0.05)The phlorizin effects of aP2-SREBF1c primary transgenic mice of NAFLD.Compared with aP2-SREBFlc primary nonalcoholic fatty liver disease transgenic mouse models group, in the simvastatin group and phlorizin group, weight of mice, fasting blood glucose, TG, TC, FFA, AST, ALT insulin levels, low density lipoprotein values were decreased, but high-density lipoprotein cholesterol is increased (P<0.05or P<0.01), liver biopsy in endothelial cells, fat cells have no hyperplasia, no obvious fatty liver. So phlorizin have the function of protecting the liver, preventing and treating the fatty liver.The effects of phlorizin to aP2-SREBFlc primary transgenic mice in mTORCl /SREBP-1C signaling pathways and protein expression.After drug intervention,GβL, Raptor,p-mTOR, FAS,SREBP-1C protein expression significantly decreased compared with model group, p38MAPK protein expression increased significantly compared with the model group (p<0.01 or p<0.05)ConclusionPhlorizin can regular the fasting blood glucose, blood fat, etc in aP2-SREBFlc primary nonalcoholic fatty liver disease transgenic mouse. Therebefore, phlorizin can inhibit liver cell lipid deposition via regulating the signaling pathway activity of mTOR/SREBP, thus restrain the development of NAFLD.