| ObjectiveTo explore the molecular mechanism of curcumin and liraglutide-mediated attenuation of hepatic steatosis by isobaric tags for relative and absolute quantitation(iTRAQ)proteomics and MiSeq sequencing technology respectively in rats.Methods(1)Twenty-four SD rats were randomly divided into 2 groups,eight for normal diet(ND)group fed with normal diet,and sixteen for high-fat diet(HFD)group fed with high-fat diet.After twelve weeks,rats with HFD were randomly divided into 2 groups,HFD group(n= 8)only give a high-fat diet,HFD + curcumin(HFD +C)group(n= 8)fed with high-fat diet plus intragastric curcumin(200mg/kg/d).All rats were anesthetized and sacrificed after 16 weeks.Oil red O staining was used to detect hepatic adipose deposits.Liver triglyceride and serum alanine aminotransferase(ALT),aspartateaminotransferase(AST),estradiol levels were detected through enzyme linked immunosorbent assay(ELISA)in all rats.Protein levels of liver were measured by iTRAQ-based proteomics,and the mRNA levels of 17-beta-dehydrogenase 2(17β-HSD2),peroxisome proliferator-activated receptor gamma coactivator-1α(PGC-1α)and peroxisome proliferators-activated receptors a(PPARa)in liver were assayed by real-time quantitative polymerase chain reaction(RT-qPCR).(2)Twenty-four SD rats were randomly divided into 2 groups,eight for normal diet(ND)group fed with normal diet,and sixteen for high-fat diet(HFD)group fed with high-fat diet.After twelve weeks,rats with HFD were randomly divided into 2 groups,HFD group(n= 8)only give a high-fat diet,HFD +liraglutide(HFD +L)group(n= 8)fed with high-fat diet plus subcutaneous injected with liraglutide(0.4mg/kg/day).All rats were anesthetized and sacrificed after 16 weeks.Oil red O staining was used to detect hepatic adipose deposits.Liver triglyceride and serum ALT,AST tumor necrosis factor alpha(TNF alpha),lipopolysaccharide(LPS),and diamine oxidase(DAO)levels were detected through ELISA in all rats.Electron microscope was used to detect the intestinal pathology,and changes of intestinal flora were maesued by MiSeq sequencing.ResultsThe lipid deposition and triglyceride in liver were significantly reduced after curcumin treatment(P<0.05).Curcumin treatment significantly reduced serem levels of ALT(P<0.05),and AST(P<0.05),and significantly increased serum estradiol(P<0.05).iTRAQ-based proteomics showed that the protein levels of glutathione S-transferase a2,Ras-related protein Rab 13,retinol dehydrogenase in liver of rats were higher in NC and HFD +C group than HFD group,while protein levels of phosphatase 1γ and 17β-HSD2 were lower in NC and HFD +C group than HFD group.RT-qPCR showed that compared to rats in HFD group,mRNA levels of 17β-HSD2 were lower(P<0.05),and PGC-1 a,PPARa were higher(P<0.05)in liver of NC and HFD +C group,respectively.The Lipid deposition and triglyceride in liver,and serum levels of ALT,TNF-a,LPS,DAO were markedly decreased within liraglutide treatment(P<0.05).Compared with NC group,decreased intestinal flora richness(as indicated byα-diversity analysis)were found in HFD group and HFD +C group,and there were no significant species diversity among these three groups.β-diversity analysis showed that there were significant differences of gut microbiota structure among the three groups.100 OTUs had significant differences in the three groups,and about 78 OTUs of them were significantly associated with seven metabolic abnormalities and inflammatory markers.In NC group and HFD +L group,23 OTUs positively correlated with metabolic abnormalities and inflammatory markers were markedly lower than those of in HFD group(P<0.05),and 5 OTUs negatively related with metabolic abnormalities and inflammatory markers were significantly higher than those of in HFD group(P<0.05).Conclusions(1)The inhibited gene expression of 17β-HSD2 in liver might contributes to the curcumin-mediated attenuation of hepatic steatosis in high-fat diet rats.(2)Liraglutide-mediated attenuation of hepatic steatosis in high-fat diet rats might resulted from weight loss and decreased serum flammation levels through modulation of gut microbiota. |
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