The Improvement And Related Mechanism Research Of Quercetin On FFA-induced Hepatocyte Steatosis

Objective:In this study,the Hep G2 hepatocyte steatosis model was constructed with mixed free fatty acids(FFA).The changes of hepatocyte steatosis and lipid accumulation in each group were observed to explore the role of quercetin(Que)on hepatocyte steatosis.Methods:1.The model of FFA-induced hepatocellular steatosis was constructed and the Hep G2 cells were treated with quercetin for 24 h.After quercetin intervention treatment,we used the CCK-8 assay to determine cell activity and oil red O staining to observe the lipid droplet changes in hepatocytes,which together determine the optimal treatment and concentration of quercetin.2.According to the random principle,We divided Hep G2 cells into 3 groups:control(Control)group,model(FFA)group and quercetin(Que)group.For the control group,we cultured cells with normal culture medium;FFA group was intervened with 1mmol/L mixed FFA solution(oleic acid: palmitic acid=2:1)for 24 h;Que group was post-treated with 30 μmol/L quercetin for 24 h on the basis of FFA group.All indicators were tested 48 h after corresponding treatments of cells.3.After Oil Red O staining,we observed the changes of lipid droplets in hepatocytes by microscope.4.The triglyceride(TG)in hepatocytes was determined using GPO-PAP method.Results:1.The 1 mmol/L of the FFA solution(oleate/palmitate,2:1 ratio)induced hepatocellular steatosis;The number of red lipid droplets was the least in cells posttreated with 30 μmol/L of quercetin and did not affect the normal proliferation of Hep G2 cells,so the post-treatment of cells with 30 μmol/L of quercetin was used for subsequent experiments.2.Oil red O staining showed that the red lipid droplets of hepatocytes were significantly increased and larger in the FFA group.After the intervention of quercetin,the red lipid droplets of hepatocytes were significantly reduced and smaller than those of in the FFA group.3.TG content can reflect the degree of lipid accumulation in hepatocytes.Compared with the TG content of 0.39±0.03 mmol/gprot in the Control group,the TG content in the FFA group was significantly increased to 2.17±0.31 mmol/gprot(P < 0.01).Compared with the FFA group,the TG content was significantly decreased to 1.12 ±0.16mmol/gprot after quercetin intervention(P < 0.05).Conclusion:Quercetin may ameliorate FFA-induced hepatocyte lipid accumulation and steatosis.Objective:FFA-induced hepatocellular steatosis model was used to observe the effects of quercetin and estrogen on FFA-induced lipid accumulation and steatosis in hepatocytes.The effects of quercetin and estrogen on the expression of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α),the expression of peroxisome proliferator-activated receptor α(PPARα),the expression of uncoupling protein 2(UCP2),reactive oxygen species(ROS)production,inflammatory factor tumor necrosis factor alpha(TNF-α)production and the expression of the carnitine palmitoyl transferase1α(CPT1α)in hepatocytes were compared to explore the similar protective mechanisms that may be involved.Methods:1.The establishment of hepatocellular steatosis model and the selection of the optimal treatment and concentration of quercetin were based on Part 1.After estrogen intervention treatment,we used the CCK-8 assay to determine cell activity and oil red O staining to observe the lipid droplet changes in hepatocytes,which together determine the optimal treatment and concentration of estrogen.2.According to the random principle,We divided Hep G2 cells into 4 groups:control(Control)group,model(FFA)group,positive drug estrogen control(E2)group and quercetin(Que)group.For the control group,we cultured cells with normal culture medium;FFA group was intervened with 1 mmol/L mixed FFA solution(oleic acid:palmitic acid=2:1)for 24 h;E2 group was FFA intervention for 24 h + 1 μmol/L estrogen post-treatment for 24 h;Que group was FFA intervention for 24 h + 30 μmol/L quercetin post-treatment for 24 h.All indicators were tested 48 h after corresponding treatments of cells.3.After Oil Red O staining,we observed the changes of lipid droplets in hepatocytes by microscope.4.The TG in hepatocytes was determined using GPO-PAP method.5.The level of ROS in hepatocytes was detected using DCFH-DA.6.The content of TNF-α in hepatocytes was measured using ELISA.7.The expression levels of PGC-1α,PPARα,UCP2 and CPT1α m RNA in hepatocytes were measured by RT-PCR.Results:1.Relative to the FFA group,1 μmol/L and 10 μmol/L estrogen post-treatment groups can reduce the accumulation of red fat droplets,combined with CCK-8 results,1μmol/L of estrogen significantly improved cell activity,so the post-treatment of cells with 1 μmol/L of estrogen was selected for subsequent experiments.2.The Oil red O staining results showed that the larger number of red lipid droplets in hepatocytes were accumulated in the FFA group,which were attenuated by the intervention of estrogen and quercetin.3.TG content can reflect the degree of lipid accumulation in hepatocytes.FFA group can significantly increase the content of TG in hepatocytes(P < 0.001),which was decreased by 34.38% after estrogen intervention(P < 0.05)and was decreased by45.16% after quercetin intervention(P < 0.05);No significant difference was noted within the quercetin group versus the estrogen group.4.CPT1α expression can reflect mitochondrial fatty acid β-oxidation.The expression level of CPT1α m RNA in hepatocytes was decreased in the FFA group(P <0.05),which was increased by 5.91times(P < 0.001)after estrogen intervention and was up-regulated by 5.64 times(P < 0.001)after quercetin intervention;No significant difference was noted within the quercetin group versus the estrogen group.5.PGC-1α plays a crucial role in activating mitochondrial fatty acid β-oxidation in hepatocytes.The expression of PGC-1α m RNA in hepatocytes was decreased in the FFA group(P < 0.05),which was increased by 2.01 times after estrogen intervention(P <0.001)and was up-regulated by 1.68 times after quercetin intervention(P < 0.01);No significant difference was noted within the quercetin group versus the estrogen group.6.PPARα is an important regulatory factor that mediates PGC-1α activation of mitochondrial fatty acid β oxidation.The expression level of PPARα m RNA in hepatocytes was decreased in the FFA group(P < 0.05),which was increased by 3.51times(P < 0.01)after estrogen intervention and was up-regulated by 3.35 times(P < 0.01)after quercetin intervention;No significant difference was noted within the quercetin group versus the estrogen group.7.UCP2 is an important regulatory factor that mediates PGC-1α reduction in mitochondrial ROS production.The expression of UCP2 m RNA in hepatocytes was decreased in the FFA group(P < 0.05),which was increased by 1.93 times after estrogen intervention(P < 0.001)and was up-regulated by 2.61 times after quercetin intervention(P < 0.001);No significant difference was noted within the quercetin group versus the estrogen group.8.DCFH-DA can reflect the change of ROS fluorescence intensity after incubating hepatocytes.Fluorescence microscopy revealed that the green fluorescence signal of ROS in the FFA group was significantly enhanced,which were weakened in the estrogen group and the quercetin group.In addition,the TNF-α protein content in hepatocytes was increased in the FFA group(P < 0.05),which was decreased by 29.88% after estrogen intervention(P < 0.001)and was decreased by 23.22% after quercetin intervention(P <0.01);No significant difference was noted within the quercetin group versus the estrogen group.Conclusion:Quercetin may upregulate PPARα expression and upregulate UCP2 to reduce ROS and TNF-α production in hepatocyte possibly by driving PGC-1α expression,which together activate mitochondrial fatty acid β-oxidation,it exerts estrogen-like hepatoprotective effects and ultimately alleviates lipid accumulation and steatosis in hepatocytes.