| Objective:The prevalence of non-alcoholic fatty liver disease(NAFLD)is increasing,which has become the most common chronic liver disease.NAFLD include simple hepatic steatosis,non-alcoholic steatohepatitis,liver fibrosis and hepatocellular carcinoma according to its severity.It is now widely believed that the mechanism of NAFLD involves dietary,genetics and gut microbiota,they affect fat metabolism,inflammation and fibrosis as “multi-hit”,thus cause liver toxicity.In previous study,a biomarker eukaryotic peptide chain releasing factor3b(e RF3b)peptide was detected in the serum of patients with chronic hepatitis,liver cirrhosis and hepatocellular carcinoma by using flight mass spectrometry,the expression of eRF3b was different in these patients.It was found that eRF3b have a protective effect on immune liver injury and liver fibrosis.eRF3b was found to reduce lipid deposition but also to improve serological and histopathology indexes induced by high-fat diet combined with carbon tetrachloride in rats.The specific molecular mechanism of these protective effects was not clear,in this study,fat metabolism of eRF3b improving liver injury in this rat model will be explored by examined AMPK pathway,PPAR/LXR pathway and PI3K/Akt pathway to provide experimental basis for the treatment of fatty liver disease.Methods:1 The change of lipid metabolism pathway gene levels:The relative mRNA levels of AMPK pathway indexes such as AMPK?1,AMPK?2,LKB1,adiponectin,ACC,CPT1,SREBP-1c,FAS,PNPLA3 and PPAR and LXR pathway indexes such as PPAR?,PPAR?,LXR? andinflammation,fibrosis and cell proliferation indexes such as IL-6,TGF-?1,collagen1 and PCNA in frozen liver tissue were determined by q RT-PCR of control group,model group,eRF3b 100?g/kg group,200?g/kg group,400?g/kg group,western medicine(compound glycyrrhizin)group,traditional Chinese medicine(Jiangzhi Tongluo soft capsules)group.2 The change of lipid metabolism-related factor protein levels:The protein levels of p-AMPK?/AMPK?,PI3 K and p-Akt/Akt in the liver tissue of control group,model group,eRF3b 100?g/kg group,200?g/kg group,400?g/kg group,compound glycyrrhizin group,Jiangzhi Tongluo soft capsules group were determined by Western-blot.Results:1 The relative mRNA and protein expression of AMPK? in each groupThe relative expression of AMPK?1 and AMPK?2 mRNA in model group were lower than those in control group(P<0.01)and were higher in eRF3b 100?g/kg group,200?g/kg group,400?g/kg group,compound glycyrrhizin group and Jiangzhi Tongluo soft capsules group than those in model group by q RT-PCR(P<0.01).The levels of AMPK?1 and AMPK?2mRNA in eRF3b 200?g/kg group were increased compared with compound glycyrrhizin group and Jiangzhi Tongluo soft capsules group(P<0.05).The protein level of p-AMPK?/AMPK? in model group was decreased compared with control group(P<0.01),and it was increased in the eRF3b 200?g/kg group by Western-blot(P<0.05).2 The relative mRNA expression of AMPK upstream genes in each groupThe relative expression of adiponectin and LKB1 mRNA were lower in model group than those in control group(P<0.05),and the adiponectin and LKB1 mRNA were increased in eRF3b 200?g/kg group(P<0.01).3 The relative mRNA expression of fatty acid oxidation genes in each groupCompared with control group,the relative expression of ACC mRNA level was higher and CPT1 mRNA level was lower in model group(P<0.001).In eRF3b 100?g/kg group,200?g/kg group,400?g/kg group,compoundglycyrrhizin group and Jiangzhi Tongluo soft capsules group,ACC mRNA levels were decreased and CPT1 mRNA levels were increased and the expression of ACC mRNA in eRF3b 400?g/kg group was lower than that in compound glycyrrhizin group(P<0.05).4 The relative mRNA expression of fatty acid synthesis genes in each groupThe relative levels of SREBP-1c,FAS,PNPLA3 mRNA in model group were higher than those in control group(P<0.05),and were lower in e RF3b100?g/kg group,200?g/kg group,400?g/kg group,compound glycyrrhizin group and Jiangzhi Tongluo soft capsules group(P<0.05),and the expression of SREBP-1c mRNA in eRF3b 200?g/kg group and 400?g/kg group were lower compared with compound glycyrrhizin group(P<0.05).5 The relative mRNA expression of PPAR/LXR pathway genes in each groupThere was no significant difference of PPAR? and LXR? mRNA in control group,model group,eRF3b 100?g/kg group,200?g/kg group,400?g/kg group,compound glycyrrhizin group and Jiangzhi Tongluo soft capsules group(P>0.05).PPAR? mRNA level in model group was higher than that in control group(P<0.001),and it was lower in the eRF3b 200?g/kg group and the Jiangzhi Tongluo soft capsules group(P<0.05).6 The protein level of PI3K/Akt pathway in each groupWestern blot analysis showed that there was no significant difference in the expression of PI3 K and p-Akt/Akt protein between these groups(P>0.05).7 The relative mRNA expression of inflammation,fibrosis and cell proliferation genes in each groupThe levels of IL-6,TGF-?1 and collagen1 mRNA in the model group were higher than those in the control group(P<0.01).Compared with the model group,IL-6 and TGF-?1 mRNA levels were decreased in e RF3b100?g/kg group,200?g/kg group,400?g/kg group,compound glycyrrhizin group and Jiangzhi Tongluo soft capsules group(P<0.05);collagen1 mRNA levels in eRF3b 100?g/kg group,200?g/kg group,compound glycyrrhizingroup and Jiangzhi Tongluo soft capsules group were decreased(P<0.01).The expression of IL-6 and collagen1 mRNA in eRF3b 200?g/kg group were lower than those in compound glycyrrhizin group and Jiangzhi Tongluo soft capsules group(P<0.01).There was no significant difference of PCNA gene expression between these groups(P>0.05).Conclusions:1 eRF3b may protect fatty liver injury in rat induced by high fat diet combined with carbon tetrachloride by activating the AMPK pathway and reducing the expression of PPAR? in the liver to improve lipid metabolism and reduce the inflammatory response and fibrosis.2 The mechanism by which eRF3b upregulated the AMPK pathway may be due to the increase of adipoenctin and LKB1 to activate AMPK,which in turn increased fatty acid oxidation and reduced fatty acid synthesis,thereby reducing intrahepatic fat accumulation.3 The therapeutic effect of eRF3b on fatty liver may be independent of PPAR?,LXR? and PI3K/Akt pathway. |
PDF Full Text Request