| ObjectiveTo investigate the change about PPAR-a/HO-1signal transduction pathway in HepG2cell steatosis,and explore the possible mechanism of non-alcoholic fatty liver disease(NAFLD).MethodsA steatotic hepatocytes model was established by treating HepG2cells with oleic acid,the cells were treats with different concentrations of fenofibrate(a selective PPAR-a agonist),and a specific PPAR-a antagonist MK886(10μmol/L),for24hours.The HepG2cells were randomly divided into6groups:(1)Normal control group(2)Model group:OA0.2mmol/L(3)FF(5μmol/L) Model group:OA0.2mmol/L+FF5μmol/L(4)FF(10μmol/L) Model group:OA0.2mmol/L+FF10μmol/L(5)FF(50μmol/L) Model group:OA0.2mmol/L+FF50μmol/L(6)MK-886(10μmol/L) Model group:OA0.2mmol/1+FF10μmol/L+MK-88610μmol/LThe relative activity of cells were detected by MTT method, and drawn the cells growth curve according to the MTT results. Significant fat accumulation was documented by Oil Red O staining, and intracellular triglyceride (TG) levels was detected by triglyceride enzymatic assay. The level of Malondialdehyde (MDA) and Superoxide Dismutase (SOD) were assayed by thibabituric acid method, xanthine oxidase method. The expressions of PPAR-a and HO-1mRNA were detected by real-time PCR methods, both PPAR-a and HO-1protein expressions were detected by immunocytochemistry.Results1.The cells growth curve showed that the cells proliferation keep activity, when the concentration of OA<0.2mM, and cells proliferation could deceased markedly if the concentration of OA>0.2mM. The Oil Red O staining results showed that, compared with the normal control group, orange lipid droplets of varying sizes in the cells were observed by treating them24hours, and the cells steatosis obviously. Compared with the model group, the lipid droplets were decreased, and began smaller gradually in FF(5,10,15mM) model group, but no difference was observed between MK-886group and model group.2.Intracellular TG assay results:Compared with the normal control group, the TG deposition of HepG2in the model group increased markedly, and TG content was379.98±23.19mg/g protein(P<0.01); Compared with the model group, in FF group, they decreased TG gradually(P<0.01); No obvious difference was observed between MK-886and model group(P>0.05).3.MDA and SOD assay results:Compared with the normal control group, the elevation of MDA content was detected(P<0.01), however, SOD reduced remarkably(P<0.01). Compared with the model group, FF decreased MDA content significantly(P<0.01),while the SOD activity significantly increased(P<0.01); No obvious difference was observed between MK-886and model group(P>0.05).4.RT-PCR showed that PPAR-aand HO-1mRNA decreased significantly as compared with that in normal control group(P<0.01). Compared with the model group, mRNA expression of PPARa increased in FF model groups sequentially(P<0.01), at the same time point, expression of HO-1mRNA in FF model groups was similar to PPAR-a mRNA(P<0.01,r=0.993). But in MK-886group, changes of expression of PPAR-a and HO-1mRNA were no obvious compared with those in model group(P>0.05).5.Immunocytochemistry results:In normal control group, the positive staining of PPAR-a protein in nucleus showing some buffy or brown particles,and the positive staining of HO-1protein in cytoplasm showing some buffy or brown particles. Image-Pro Plus6.0analysis and statistical results showed that the expression of PPAR-a and HO-1protein in the model group was decreased compared with the normal control group, respectively(P<0.01). Compared with the model group, Expression of PPAR-a protein increased in FF model groups sequentially(P<0.01), at the same time point, expression of HO-1protein in FF model groups was similar to PPAR-a protein(P<0.01,r=0.997), the expression of PPAR-a and HO-1protein between MK-886group and model group were no different (P>0.05).Conclusion1.The NAFLD cell model was successfully established with0.2mM OA for24hours to HepG2cells.2.The dynamic changes about PPAR-a/HO-1signal transduction pathway in HepG2cell steatosis was significantly.3.Activation of PPAR-a prevents OA-induced HepG2cells steatosis, and HO-1might involve in it as downstream effector of PPAR-a;Specific PPAR-a antagonist MK-886could block that mentioned above. |
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