Effects Of Lycium Barbarum Polysaccharide On The Level Of Oxidative Stress And The Expression Of UCP2 In Nonalcoholic Fatty Liver Cells

Objective:Nonalcoholic fatty liver disease is one of the major chronic liver diseases in the world,but the mechanism of dietary prevention and research is still under continuous exploration.The main purpose of this study was to establish the cell model of nonalcoholic fatty liver(NAFLD),to observe the effect of Lycium barbarum polysaccharide(LBP)on lipid deposition,oxidative stress levels and UCP2 expression levels in NAFLD cells,studied the possible mechanism of LBP to improved oxidative stress,which can provide theory basis for the clinical application of LBP to prevent NAFLD.Methods:The research used RPMI1 640 medium containing 10%fetal bovine serum to established the experimental model of L02 in vitro and NAFLD cell model was induced with 1mM(oletic acid:palmitate=2:1)free fatty acid(FFA)mixture in combination for 24 hours.CCK-8 method was used to determine the optimal concentration.The experimental cells were divided into five groups:1)the normal control group,2)NAFLD model group:the terminal concentration of FFA in the culture medium was 1mM,3)NAFLD model group+30μg/ml LBP:the final concentration of FFA in the medium was 1 mM,and the final concentration of LBP in the medium was 30μg/ml,4)NAFLD model group+100μg/ml LBP:The final concentration of FFA in the medium was 1mM,and the final concentration of LBP in the medium was 100μg/ml,5)NAFLD model group+300μg/ml LBP:The final concentration of FFA in the medium was 1mM,and the final concentration of LBP in the medium was 300 μg/ml,after cultured 48h according to the training requirements of each experimental group,the cells in each group were collected for testing the related experimental indexes.Staining was used to observed intracellular lipid droplets by oil red"O",GPO-PAP enzyme method was applied to determine the content of triglyceride(TG),colorimetry method was applied to determine the content of superoxide dismutase(SOD),glutataione(GSH),catalase(CAT),malonydialdehyde(MDA),RT-PCR technology and Western Blot were used to detect the expression of UCP2 mRNA and protein in L02 cells in each group.Results:1.Selection of LBP intervention dosage:30,100 and 300μg/ml LBP were selected as low,medium and high dose group.2.Oil red "O" dyeing results:Under the inverted microscope,few lipid droplets were found in the normal L02 cell group,while a large number of granulated lipid droplets were made into orange red in the model cells,which were distributed on the edge of the intracellular membranes,and the LBP intervention group could significantly reduce the red dye drops in the cells.3.The content of TG in intracellular:compared with model group,the level of TG was significantly decreased(P<0.05)in high concentration of LBP,but low concentration and medium concentration of LBP has no significantly effect on the content of TG(P>0.05).Compared with normal group,the level of TG was significantly increased(P<0.05)in model group and low concentration of LBP,low concentration and medium concentration has no significantly effect on the content of TG(P>0.05).4.Activity determination of MDA、SOD、CAT and GSH in cells:compared with model group,the level of MDA was significantly decreased(P<0.05)in normal group、low and high concentration of LBP,low concentration of LBP has no significantly effect on the level of MDA(P>0.05).Compared with normal group,the level of MDA was significantly increased(P<0.05)in low and medium concentration of LBP,high concentration has no significantly effect on the level of MDA(P>0.05),compared with high group,the level of MDA was significantly increased(P<0.05)in low and medium concentration of LBP.Compared with model group,the level of SOD、CAT、GSH was significantly increased(P<0.05)in normal group and high concentration of LBP,low and medium concentration of LBP has no significantly effect on the level of MDA(P>0.05).Compared with normal group,the level of SOD、CAT、GSH was significantly increased(P<0.05)in low concentration of LBP,medium and high concentration of LBP has no significantly effect on the level of SOD、CAT、GSH(P>0.05).5.UCP2 expression level in different groups of cells:compared with model group,the expression of UCP2 mRNA and UCP2 protein was significantly decreased(P<0.05)in normal group、medium and high concentration of LBP,the expression of UCP2 mRNA was significantly decreased(P<0.05)in low concentration of LBP,but low concentration of LBP has no significantly effect on the expression of UCP2 protein(P>0.05).Compared with normal group,the expression of UCP2 mRNA and UCP2 protein was significantly increased(P<0.05)in medium and high concentration of LBP,the expression of UCP2 mRNA was significantly increased(P<0.05)in low concentration of LBP,but low concentration of LBP has no significantly effect on the expression of UCP2 protein(P>0.05).Compared with low concentration of LBP,the expression of UCP2 protein was significantly decreased(P<0.05)in medium and high concentration of LBP,the expression of UCP2 mRNA was significantly decreased(P<0.05)in high concentration of LBP,but medium concentration of LBP has no significantly effect on the expression of UCP2 mRNA(P>0.05).Compared with medium concentration of LBP,the expression of UCP2 mRNA and UCP2 protein was significantly decreased(P<0.05)in high concentration of LBPConclusion:LBP can improve the level of oxidative stress in NAFLD cells,and may prevent NAFLD by down-regulating UCP2 expression.