Fat Metabolism Mechanism Of DRC3f Protecting Nonalcoholic Fatty Liver Disease Induced In Rats

Objective: Non-alcoholic fatty liver disease is characterized by hepatic lipid accumulation and hepatocyte steatosis.With the improvement of people's quality of life,the prevalence of NAFLD in China is increasing year by year.The present study suggests that NAFLD is caused by a variety of factors in parallel.Eating habits,genetic factors and environmental factors may jointly affect the fat metabolism,leading to hepatocellular inflammation,fibrosis,cirrhosis,even to hepatocellular carcinoma.DRC3 f,a sixteen amino-acid polypeptide fragments was detected in the serum of patients with chronic hepatitis B by time flight mass spectrometry in our previous study,which had a higher expression in less severe patients.Further study suggested that DRC3 f had a protective effect on immune liver injury and liver fibrosis.In previous experiments,DRC3 f was also found to improve lipid deposition induced by high-fat diet combined with carbon tetrachloride in rats serologically and pathologically.Since the molecular mechanism of these protective effects remained unclear,this study was ment to investigate the possible fat metabolism molecular pathways by which DRC3 f may improve rat's NAFLD.Methods: For it's relatively superior effect in previous study,DRC3 f 200?g/kg group(DRC3f group),together with normal control group(Ctrl group),high fat diet combined with carbon tetrachloride induced rat NAFLD group(CCl4+HFD group),western medicine positive control group(compound glycyrrhizin diluents,CG group)and traditional Chinese medicine positive control group(Jiangzhitongluo capsules diluents,JTSC group)were selected to be analyzed in this study.1 The relative mRNA expressions of fatty acid oxidative connected signaling pathway adiponectin/LKB1/AMPK/ACC/CPT1 and fatty acid production connected signaling pathway SREBP-1c/PNPLA3/FAS,combined with PPAR and LXR pathway indexes,inflammation and fibrosis indexs such as IL-6,TGF-?1,collagen1 and PCNA were determined by qRT-PCR.2 The protein levels of p-AMPK?/AMPK?,PI3 K and p-Akt/Akt in the liver tissue of Ctrl group,CCl4+HFD group,DRC3 f 200?g/kg group,CG group and JTSC group were determined by western-blot.Results:1 AMPK signaling pathway related gene and protein expressionThe relative expressions of AMPK?1 and AMPK?2 mRNA in CCl4+HFD group were significantly lower than those in the Ctrl group(P<0.001),and in DRC3 f group,CG group,JTSC group(P<0.01).Moreover,these indexes was higher in DRC3 f group than that in CG group and JTSC group(P<0.001).The protein level of p-AMPK?/AMPK? in CCl4+HFD group was decreased comparing to Ctrl group(P<0.01),while increased in DRC3 f group comparing to CCl4+HFD group(P<0.05).The relative expression of adiponectin and LKB1 mRNA in CCl4+HFD group decreased compared with Ctrl group(P<0.05).The expression of adiponectin and LKB1 mRNA in DRC3 f group were higher compared with the CCl4+HFD group(P<0.01),and than those in CG group and JTSC group(P<0.05).The relative expression of CPT1 mRNA in CCl4+HFD group was lower than that in Ctrl group(P<0.05),and the relative expression of ACC mRNA was increased(P<0.001).Compared with CCl4+HFD group,the relative expression of ACC mRNA was decreased in DRC3 f group,CG group and JTSC group(P<0.001),and the relative expression of CPT1 mRNA was increased(P<0.01).The relative expression of SREBP-1c,PNPLA3 and FAS mRNA in CCl4+HFD group increased compared with Ctrl group(P<0.001).Compared with the CCl4+HFD group,the expression of SREBP-1c,PNPLA3 and FAS mRNA in DRC3 f group,CG group and JTSC group were lower(P<0.01),but the expression level of SREBP-1c mRNA in DRC3 f group was higher than that in JTSC group(P<0.001).As for PNPLA3 and FAS mRNA,there were no significant difference throughout DRC3 f group,CG group and JTSC group.2 Expression levels of PPAR?,LXR?,PPAR?There was no significant difference in the expression of PPAR? and LXR? mRNA throughout these groups.Compared with Ctrl group,the relative expression of PPAR? mRNA was increased in CCl4+HFD group(P<0.001).The elevation of PPAR? mRNA was reversed in JTSC group(P<0.05),but not in DRC3 f group(P=0.118).3 Protein levels of insulin-related PI3K/Akt pathwayWestern blot analysis showed that there was no significant difference in the expression of PI3 K and p-Akt/Akt protein between these groups.4 Inflammation,fibrosis and cell proliferation-related factors IL-6,TGF-?1,collagen1,PCNA expression levelsThe relative expression of IL-6,TGF-?1 and collagen1 mRNA in CCl4+HFD group was higher than that in Ctrl group(P<0.001).Compared with CCl4+HFD group,the relative expression of IL-6 and TGF-?1 mRNA decreased in DRC3 f group,CG group and JTSC group(P<0.05).The expression level of collagen1 mRNA in DRC3 f group,CG group and JTSC group decreased compared with CCl4+HFD group(P<0.05).And the relative expression of collagen1 mRNA in DRC3 f group was lower than that in CG group and JTSC group(P<0.01).There was no significant difference in the expression of PCNA mRNA throughout these groups.Conclusions:1 DRC3 f protects against non-alcoholic fatty liver induced by high-fat diet combined with carbon tetrachloride may be due to the activation of AMPK by upregulating adiponectin/LKB1,the activation of AMPK regulates ACC/CPT1 and inhibits SREBP-1c/PNPLA3/FAS signaling to promote fatty acid oxidation and to inhibitis fatty acid synthesis.DRC3 f may improve the liver inflammation and may prevent fibrosis in the liver to a certain extent.2 The therapeutic effect of DRC3 f on fatty liver may be independent of PPAR?,LXR?,PPAR? and PI3K/Akt pathway.