Basic Research Of Gut Microbiota And Its Metabolites In The Prevention And Treatment Of Nonalcoholic Steatohepatitis

Objective:Nonalcoholic steatohepatitis(NASH)is an emerging public health problem with an increasing incidence and prevalence globally;effective therapeutic strategies for NASH are lacked.Recent years,cumulative data indicates that gut microbiota plays a pivotal role in the onset and progression of NASH.Hence,this study aims to investigate that whether gut microbiota-targeted therapeutic strategies will be an useful and new breakthrough for the prophylaxis or treatment of NASH and the internal mechanisms.Methods:Specified pathogen-free(SPF)male C57BL/6 mice were randomly divided into five groups.The control group was fed with standard chow for 16 weeks,the high-fat diet(HFD)group was fed with HFD for 16 weeks,the HFD+FMT group was fed with HFD for16 weeks and treated with fecal microbiota transplantation(FMT)for the latter 8 weeks,the HFD+CB was fed with HFD for 16 weeks and treated with Clostridium butyricum B1(CB)for the latter 8 weeks,the HFD+NaB group was fed with HFD for 16 weeks and treated with sodium butyrate(NaB)for the latter 8 weeks.Liver was stained with hematoxylin and eosin(HE),oil red O or Masson,respectively.Gut microbiota was analyzed by an Illumina MiSeq platform at baseline and at 16 weeks after treatment.The expressions of inflammation-,metabolism-or immune-associated genes in liver or epididymal fat tissue or the terminal ileum were detected by realtime-PCR or western blot or immunohistochemistry.The levels of metabolism-associated hormones,serum glucose and liver enzymes in serum were measured.The concentrations of short-chain fatty acids in cecal content were determined by high performance liquid chromatograph.CD4~+T cells were isolated from the spleen of mice and human hepatoma cell line(HepG2)was used in vitro for further mechanism investigations.Results:After 16 weeks,compared with the control group,the metabolic disturbance was occurred in the HFD group and the incidence rate of steatohepatitis in the HFD group was100%.After 8 weeks intervention such as FMT,CB or NaB,the metabolic indeces in each intervention group were significantly improved compared with the HFD group,and the liver histopathology was significantly attenuated compared with the HFD group.The mRNA levels of inflammation-associated genes in liver and epididymal fat tissue were obviously increased and the intrahepatic immune was more imbalanced in the HFD group compared with the control group.However,the inflammation-associated genes in liver and epididymal fat tissue were significantly downregulated in each intervention group compared with the HFD group,and the intrahepatic immune was also corrected in each intervention group.The gut microbiota analysis showed that the overall structure of the gut microbiota was significantly disrupted by 16 weeks HFD and was deviated from the control group;each intervention shifted the overall composition of the HFD-disrupted gut microbiota toward that of the control mice,and elevated the abundances of the beneficial bacteria Christensenellaceae and Lactobacillus and so on.The concentration of butyrate in the cecal content showed no significant difference between the control and HFD group,but was significantly increased in each intervention group.In vitro experiments showed that NaB could regulate the differentiation of CD4~+T cells and enhanced the GLP-1 receptor expression of hepatocyte via the inhibition of histone deacetylase(HDAC).Conclusions:Gut microbiota-targeted strategies including FMT,CB and NaB,could attenuate HFD-induced steatohepatitis mainly or partly via improving HFD-induced gut microbiota disturbance,correcting the imbalanced inflammation and immune microenvironment,strengthening the intestinal mucosal barrier,promoting the secretion of gut-derived hormone GLP-1 and enhancing liver reactivity to GLP-1.