Effect Of Curc-mPEG454,a PEGylated Curcumin Derivative,on Hepatocellular Steatosis

BackgroundNon-alcoholic fatty liver disease(NAFLD)is the most common chronic liver disease in China,but there is no specific drug for NAFLD therapy.Our previous studies had confirmed that the PEGylated curcumin derivative(Curc-mPEG454)could significantly reduce the body weight gain and serum triglyceride(TG)levels in high-fat diet(HFD)-fed mice.However,the mechanism by which Curc-mPEG454 regulated lipid and fatty acid metabolisms in the liver is not fully clear.It also remains unclear that whether it has a similar effect on human hepatocellular steatosis.This study used bioinformatics methods to analyze the liver mRNA sequencing data to explore whether Curc-mPEG454 regulates key genes related to lipid metabolism.Then a vitro HepG2 cells steatosis model induced by free fatty acid(FFA)was used to investigate the effect of Curc-mPEG454 on human hepatocyte steatosis.Methods1.RNA sequencing and identification of differentially expressed genes(DEGs): Total RNA from liver tissues of mice,including control group,HFD group and Curc-mPEG454 50 mg/kg treatment group,were extracted and sequenced.The gene expression levels were determined using the fragments per kilobase of exon per million fragments mapped(FPKM)method.The DEGs between three groups were screened using the NOISeq method with cut-off thresholds of fold change > 2 and probability > 0.7.2.GO and KEGG pathway enrichment analyses of DEGs: The R package “clusterProfiler” was used to conduct GO and KEGG analyses for DEGs.The Padj < 0.05 was chosen as cut-off criteria.3.Cell model and grouping: Human hepatoma cell line HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum.Cells were divided into four groups: control group,FFA model group and Curc-mPEG454 group(12.5 μM and 25 μM).After pretreatment for 2 h with Curc-mPEG454,cells were stimulated by 1mM FFA for another 24 h.4.Detection of intracellular lipid content: The intracellular lipid content was evaluated by Oil red O staining and intracellular TG content determination.5.Detection of gene expression levels: The mRNA and protein levels of PPARγ,PPARα,CD36,FABP,FATP,ME1,SCD,ACSL1,CYP4A11,ACOT2,ACOX1,ACADM were detected by Real time PCR and Western blot.6.Data statistics: All experimental data were expressed as mean ± standard deviation(SD).Comparisons between groups were analyzed by software SPSS 19.0 one-way ANOVA and Bonferroni test.P < 0.05 was considered to be statistically significant.Results1.Identification of DEGs: HFD led to 1319 DEGs compared with control group,including 638 upregulated and 681 downregulated.And after Curc-mPEG454 treatment,there were 511 genes changed compared to the HFD group,including 136 upregulated and 375 downregulated.2.GO analysis: DEGs induced by HFD were significantly enriched in fatty acid metabolic process(include 50 DEGs),and 21 genes among the 50 DEGs participated in long-chain fatty acid metabolic process,including CD36,FATP4,ACOT and CYP2E1.Besides,in treatment group we found that fatty acid metabolic process was significantly regulated by Curc-mPEG454,including 37 genes such as CD36,PPARα,PPARγ,CYP4A10,ACOT2,ACOT3 and ACADM.3.KEGG pathway analysis: DEGs induced by HFD and Curc-mPEG454 were highly enriched in PPAR signaling pathways,and mainly participated in lipid synthesis(ME1,SCD1),fatty acid transport(CD36,FATP,FABP)and fatty acid oxidation(CYP4A10,ACOX1,ACADM),indicating that PPAR signaling pathway is closely related to the progression of NAFLD.4.Curc-mPEG454 significantly improved lipid accumulation in HepG2 cells: Oil red O staining showed that 1mM FFA significantly increased the intracellular lipid droplets,while the number of lipid droplets was obviously decreased by Curc-mPEG454 in a dose dependent manner.Furthermore,the TG content decreased by 19.0% at 12.5μM and 27.5% at 25μM(p<0.05)in Curc-mPEG454 group compared with FFA group.5.Curc-mPEG454 significantly down-regulated the expression of genes related to fatty acid uptake and lipid synthesis in PPAR signaling pathway: FFA significantly increased mRNA and protein levels of genes related to fatty acid uptake(CD36,FABP,FATP4),lipid synthesis(ME1,SCD)and fatty acid oxidation(CYP4A11,ACOX1,ACADM);while Curc-mPEG454 significantly down-regulated the expression of genes related to fatty acid uptake(CD36,FABP5,FATP2),lipid synthesis(ME1,SCD,ACSL1)and fatty acid oxidation(CYP4A11,ACOT2,ACADM).6.Curc-mPEG454 significantly down-regulated the expression of transcription factors PPARγ and PPARα in PPAR signaling pathway: PPARγ mainly regulated fatty acid uptake and lipid synthesis.Its mRNA and protein levels increased in FFA group,while significantly decreased in Curc-mPEG454 group(p<0.01).PPARα mainly regulated the expression of fatty acid oxidation-related genes.And the protein level of PPARα had no significantly change in FFA group,but decreased in Curc-mPEG454 group.ConclusionsIn vitro and in vivo experiments confirmed that Curc-mPEG454 could significantly down-regulate PPARγ and fatty acid metabolism key genes(CD36,FABP,FATP,ME1,ACSL1),thereby reducing hepatocellular fatty acid uptake and lipid synthesis,reducing intracellular TG accumulation and improving hepatocellular steatosis.The lipid-lowering effect of Curc-mPEG454 was not associated with the transcription factor PPARα.