Effects Of Quercetin On Cholesterol Synthesis And Transport In Human L-02 Hepatocyte

Object: The purpose of this study was to investigate the formation of lipid droplets and expression of cholesterol rate-limiting enzyme HMG-CoA reductase and transported protein-ABCA1,ABCG5,ABCG8, detect the amount of cholesterol on human L-02 hepatocyte under the control of quercetin, to find out the effects of quercetin on cholesterol synthesis and transport.Methods: Use L-02 hepatocyte of normal human and the hepatocyte under fatty change after exposure to 50% fetal bovine serum for 24 hours as study objects. The experiment is divided into 8 groups: The first group is normal hepatocytes; the second group is fatty change hepatocytes;from group 3 to group 6, different amounts of quercetin is added to the fatty changed hepatocytes, to make the concentration of quercetin to be 10,20,40,80μmol/L respectively; Group 7 is the positive control group, i.e Probucol is added to the hepatocyte with fatty change to make the concentration to be 10μmol/L; In group 8, quercetin is added to the normal hepatocyte to make the concentration to be 20μmol/L. After each group is incubated for 24 hours, observe the formation of lipid droplets in the cells under oil red O dye. MTT—assay was used to observe the inhibitory effect of quercetin on cell growth of fatty change HL-02 cells;Detect the intracellular amount of total cholesterol(TC),free cholesterol(FC)and cholesterol ester(CE) through high performance liquid chromatography (HPLC) and calculate the ratio of intracellular CE to TC and the amount of FC in the medium. Then detect the HMG-CoA reductase and ABCA1,ABCG5,ABCG8 mRNA and ABCA1 protein expression of the hepatocytes of each group was detected by RT- PCR which decide the best concentration of quercetin that decreases the amount of intracellular cholesterol and make out the amount– effect relation curve. And expose the cell to quercetin at the best concentration for 6 h,12 h,24 h,48 h respectively, observe the formation of intracellular lipid droplets under oil red O dye. Detect the intracellular amount of TC and CE and calculate the ratio of intracellular CE to TC. Then detect the HMG-CoA reductase and ABCA1,ABCG5,ABCG8 mRNA's expression of the fatty changed hepatocytes, determine the best duration that quercetin decreases intracellular CE, and draw out the duration-effect relation curve. Using±s to represent experimental data, using SPSS13.0 statistical software to analyze the data, a P value of <0.05 is considered significantly important.Results: After exposing normal hepatocytes to 50% fetal bovine serum for 24 hours ,find out numerous lipid droplets in the luminal side of the cells ,which can be dyed to red with oil red O dye. what's more, intracellure CE increases dramatically through measurement of HPLC. Thus, fatty changed hepatocytes can be set up successfully.Then exposing the fatty changed hepatocytes to different concentrations of quercetin for 24 hours. The result displays: the growth of fatty change cells was inhibited ;oil red O dye shows the intracellular lipid droplets are much less than before exposure. Quercetin decreases the intracellular TC and CE of the fatty changed liver cells,while increase the amount of FC in the medium as well concentrationally dependently.What's more,it decreases the HMG-CoA reductase mRNA expression and increases the ABCA1,ABCG5 and ABCG8 mRNA expression concentrationally dependently at the same time; Then After mixing the quercetin of 40μmol/L with the fatty changed hepatocytes for 6,12,24,48 hours, the result says: the decrease of TC and CE in the cells and increase of FC in the medium by quercetin is dependent on the duration , at the same time the decrease of HMG-CoA reductase and increase of ABCA1,ABCG5 and ABCG8 mRNA's expression is dependent on the duration and the intracellular lipid droplets decreases duration dependently.Conclusions:1. Both the quantity of TC and CE increase in the fatty-changed human L-02 hepatocytes;2. Quercetin can decrease the quantity of TC and CE and increase the efflux of FC in the fatty-changed human L-02 hepatocytes;3. The possible mechanisms of quercetin decreasing the intracellur cholestol are related to upregulation of ABCA1,ABCG5,ABCG8 and downregulation of HMG-CoA reductase;...