PGC-1α在槲皮素通过雌激素样作用减轻FFA诱导肝细胞脂肪变性中的机制

Quercetin protects against free fatty acid-induced hepatocyte steatosis by an estrogen-like effect involving PGC-1α

目的 探讨槲皮素(quercetin,Que)对游离脂肪酸(free fatty acids,FFA)诱导肝细胞脂肪变性的雌激素样保护作用及机制。方法 CCK-8法检测细胞活力,油红O染色观察脂质蓄积,雌激素以不同方式干预HepG2细胞,确定药物最佳干预方式为后处理方式。以后处理方式将HepG2细胞暴露于不同浓度的雌激素和槲皮素,确定药物最佳干预浓度。细胞随机分为4组:对照组(Control)、模型组(FFA)、阳性药雌激素对照组(E2)和槲皮素组(Que)。GPO-PAP法测定甘油三酯(triglyceride,TG)含量;DCFH-DA法检测活性氧(reactive oxygen species,ROS)水平;酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)蛋白含量;实时定量PCR(Real-time PCR)检测肝细胞中过氧化物酶体增殖物激活受体γ共激活因子-1α(peroxisome proliferator-activated receptorγcoactivator-1α,PGC-1α)、过氧化物酶体增殖剂激活受体α(peroxisome proliferator-activated receptorα,PPARα)、肉碱棕榈酰转移酶Iα(carnitine palmitoyl transferase 1α,CPT1α)和乙酰辅酶A羧化酶1(acetyl-CoA carboxylase,ACOX1)mRNA表达。结果 确定1μmol/L雌激素和30μmol/L槲皮素后处理用于后续实验。与对照组相比,模型组红色脂滴显著增多、TG含量明显增加(P<0.001),PGC-1α、PPARα、CPT1α和ACOX1 mRNA的表达均下降(P<0.05),ROS水平升高,TNF-α蛋白含量增多(P<0.01);与模型组相比,雌激素组和槲皮素组均可明显降低红色脂滴和ROS水平,雌激素组(P<0.05)和槲皮素组(P<0.05)均使TG含量降低,雌激素组(P<0.001)和槲皮素组(P<0.01)均使PGC-1α的表达明显上调,雌激素组(P<0.001)和槲皮素组(P<0.01)使TNF-α蛋白含量均明显减少,并显著上调PPARα(P<0.01)、CPT1α(P<0.001)和ACOX1(P<0.001)的表达水平。槲皮素组与雌激素组相比,上述指标差异均无统计学意义。结论 槲皮素通过诱导PGC-1α表达,激活脂肪酸β氧化、减轻氧化应激以及抑制炎性反应,发挥雌激素样作用,最终改善肝细胞脂肪变性。

Objective To investigate the estrogen-like protective effect and mechanism of quercetin on free fatty acids(FFA)-induced hepatocyte steatosis.Methods Cell viability was detected by CCK-8 assay,and lipid accumulation in hepatocytes was observed by Oil Red O staining.Following administration of various estrogen intervention paradigms to HepG2 cells,the optimal drug intervention was determined to be post-treatment.HepG2 cells were exposed to different concentrations of estrogen and quercetin in a post-treatment manner to determine the optimal concentration of drug intervention.Cells were randomly divided into four groups:Control,model(FFA),positive drug estrogen control(E2),and quercetin(Que).Triglyceride(TG) levels were determined using the glycerol phosphate oxidase peroxidase(GPO-PAP) method.Reactive oxygen species(ROS) levels were detected using DCFH-DA.Protein contents of tumor necrosis factor α(TNF-α) were measured by enzyme-linked immunosorbent assay.Expression levels of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α),peroxisome proliferator-activated receptor α(PPARα),carnitine palmitoyl transferase 1α(CPT1α),and acetyl-CoA carboxylase(ACOX1) were measured by Real-time PCR.Results It was determined that 1 μmol/L estrogen and 30 μmol/L quercetin post-treatment of HepG2 cells was optimal for subsequent experiments.Compared with the control group,numbers of red lipid droplets and TG contents were significantly increased(P<0.001);expression levels of PGC-1α,PPARα,CPT1α and ACOX1 mRNA were significantly decreased(P<0.05);ROS levels were increased;and TNF-α protein levels were significantly increased(P<0.01) in the FFA group.Compared with the FFA group,both the estrogen and quercetin group displayed significantly reduced levels of red lipid droplets and ROS.Moreover,both the estrogen group(P<0.05) and quercetin group(P<0.05) exhibited significantly decreased TG contents,and both the estrogen group(P<0.001) and quercetin group(P<0.01) exhibited significantly upregulated expression of PGC-1α.In addition,both the estrogen group(P<0.001) and quercetin group(P<0.01) displayed significantly decreased TNF-α protein contents and significantly upregulated expression levels of PPARα(P<0.01),CPT1α(P<0.001),and ACOX1(P<0.001).There was no significant difference in the above indicators between the quercetin group and estrogen group.Conclusions Quercetin exerts an estrogen-like effect by inducing expression of PGC-1α,activating fatty acid β oxidation,reducing oxidative stress,and inhibiting inflammatory response,ultimately improving hepatic steatosis.