| Nonalcoholic fatty liver disease(NAFLD)is a metabolic syndrome characterised by excessive fat accumulation in hepatocytes,which can progress to Nonalcoholic steatohepatitis(NASH)with symptoms of inflammation and fibrosis.The pathogenesis of NASH is complex,and disorders of the intestinal flora have been shown to be an important factor in the development of NASH.Helicobacter hepaticus(H.hepaticus),an important pathogenic bacterium in the liver and intestine,causes liver damage in susceptible animals.Studies have shown that H.hepaticus infection is closely related to the development of NAFLD,but the role and mechanism of H.hepaticus infection in the development and progression of NASH have not been elucidated.In this study,we investigated the effects of H.hepaticus infection on the disease process of NASH and related regulatory mechanisms by constructing NASH models in vivo and in vitro in combination with H.hepaticus infection.Experimental methods:In vivo experiments were conducted in which 80 C57BL/6 mice were randomly divided into four groups:control,H.hepaticus-infected,CDAHFD and H.hepaticus-infected CDAHFD groups.mice in the H.hepaticus-infected and H.hepaticusinfected CDAHFD groups were treated with H.hepaticus bacterial solution by gavage to ensure successful colonization by H.hepaticus.Mice in the CDAHFD and H.hepaticusinfected CDAHFD groups were fed a choline-deficient,L-amino acid-defined,high-fat diet.The remaining two groups were fed a normal diet for 12 weeks.Serum alanine transaminase(ALT)and aspartate aminotransferase(AST)levels,liver triglyceride(TG)and total cholesterol(TC)levels were measured every 3 weeks during the experiment.The liver sections were scored for NAS pathology using hematoxylin-eosin staining;collagen fibrils were quantified using Sirius red staining;the expression levels of liver inflammatory factors,fibrosis markers and factors related to bile acid metabolism were measured by real-time quantitative PCR;the protein expression of Farnesoid X receptor(FXR)and its downstream pathways in the liver were determined by Western blot.In vitro,human hepatoma cell Huh-7 cells were treated with 0.25 mM palmitic acid(PA)and 0.5 mM oleic acid(OA)for 24 h,followed by H.hepaticus for 24 h.The intracellular TG content was measured,and the expression of TNF-α,α-SMA and FXR mRNA expression in the cells were measured by realtime quantitative PCR,and the protein expression of FXR-CYP7a1 pathway was determined by Western blot.Experimental results:(1)In vivo experimental results showed that compared with mice in the CDAHFD group,H.hepaticus-infected CDAHFD mice had increased serum levels of ALT and AST,increased levels of TG and TC in liver tissues,increased inflammatory infiltration and liver collagen deposition,altered lipid deposition progress,and increased liver NAS scores.In addition,at the gene expression level,H.hepaticus infection increased the gene expression of pro-inflammatory factors IL-6,TNF-α and fibrogenic factors TGF-β andα-SMA,and inhibited the gene expression of adipogenic SREBP1c and FXR in CDAHFD mice;at the protein expression level,the expression of Collagen I and CYP7a1,the ratelimiting enzyme for lipid synthesis,was increased and the expression of FXR was decreased in the liver of H.hepaticus-infected CDAHFD group mice compared to the CDAHFD group mice.(2)The results of in vitro experiments showed a significant increase in cellular lipid droplet content after H.hepaticus action in PA-and OA-induced Huh-7 cells.The results of Western blot showed that the protein phosphorylation levels of JNK,STAT1 and p65 were significantly increased in PA-and OA-induced Huh-7 cells after H.hepaticus action.The expression of FXR was decreased and the protein expression of CYP7a1 was increased.The study concludes that H.hepaticus infection exacerbates the inflammatory response in NASH,promotes the development of liver fibrosis,exacerbates the impaired bile acid metabolism in the liver by inhibiting the FXR-CYP7a1 pathway,and thus accelerates the disease process in NASH. |
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