Inulin Ameliorates Non-alcoholic Fatty Liver Disease Via Modulating Gut Microbiome And Inflammation

Objective To investigate the effects and mechanisms of inulin(INU)on gut microbiome and liver inflammation in mice with non-alcoholic fatty liver disease(NAFLD).Methods C57BL/6 mice were randomly allocated to the following four groups:normal diet group(ND),high-fat diet group(HFD),normal diet with INU group(ND-INU)and HFD with INU group(HFD-INU).During the experiment,body weights(BWs)of all mice were monitored weekly and food intake was recorded every 2 days.The faeces of mice were collected after 14 weeks.16S rRNA hypervariable sequencing was used to detect the diversity and abundance of gut microbiota among diverse groups.The amount of fecal short chain fatty acids(SCFAs)in each group was determined using gas chromatographymass spectrometry(GC-MS).At the end of the experiment,all mice were anesthetized and the plasma and tissue samples were collected for detecting the following indications.(1)Plasma concentrations of alanine aminotransferase(ALT),aspartate aminotransferase(AST),cholesterol(TC)and triglyceride(TG)in four groups of mice were detected by automatic biochemical analyzer.(2)ELISA kits were used to measure plasma insulin(INS)and plasma/liver tissue interleukin(IL)-1β,IL-18;CBA method was selected to detect the levels of interleukin(IL)-6,IL-10,tumor necrosis factor(TNF)-αin mice.(3)Limulus amebocyte lysate test was used to determine the levels of lipopolysaccharide(LPS)in plasma and liver tissue.(4)Changes of liver morphology and structure of mice were observed by HE staining among 4 groups.(5)Immunohistochemistry and flow cytometry were used to detect the proportions of hepatic macrophages(Mψ)in diverse groups.(6)The percentages of monocytic myeloid-derived suppressor cells(M-MDSCs)in peripheral blood,liver and spleen were detected by flow cytometry.(7)The expressions of liver Nod-like receptor protein 3(NLRP3),ASC,caspase-1and NF-κB protein in each group were measured by western blot.(8)Spearman correlation analysis was used to analyse the correlation between plasma and liver inflammatory factors,fecal(SCFAs)and intestinal flora.Results1.The effects of INU on gut microbiota and SCFAs in mice.16S rRNA sequencing and analysis revealed that compared with ND group,an obviously increased abundance of Firmicutes,Proteobacteria and a decreased Bacteroidetes were observed at the phylum level in HFD group.We also found that HFD induced significant reductions of Bifidobacterium,Akkermansia as well as an augmentation of Blautia compared with those in ND group in genus level.Nevertheless,compared to HFD group,the relative abundance of Proteobacteria,Blautia were decreased and the proportions of Bifidobacterium,Akkermansia and Ileibacterium were increased in HFD-INU group.In addition,the results of GS-MS showed that the amounts of fecal SCFAs(containing acetic acid,propionic acid andbutyric acid)in HFD group were decreased compared with those in ND group.However,the reductions of SCFAs in HFD group were obviously elevated with INU administration.2.The effects of INU on routine parameters in NAFLD mice.The BW of HFD group was significantly increased compared with that in ND group.Additionally,the concentrations of ALT,AST,TG,TC,fasting blood glucose as well as homeostatic model assessment of insulin resistance(HOMA-IR)in HFD group elevated noticeably compared to ND group.However,compared to HFD group,BW in HFD-INU group was notably decreased.Meanwhile,INU treatment ameliorated blood lipid,aminotransferase levels and insulin resistance in NAFLD mice.3.The effects of INU on inflammation and hepatic damage in mice with NAFLD.The pro-inflammatory indications(including LPS,IL-1β,IL-18,IL-6 and TNF-α)in HFD group enhanced compared with ND group.Feeding of NAFLD mice with INU dramatically decreased the above pro-inflammatory cytokines.Moreover,anti-inflammatory IL-10 was decreased in HFD group without significant difference,but notably boosted in HFD-INU group.HE staining of the liver sections showed that INU also effectively alleviated steatosis and steatohepatitis of NAFLD compared to HFD group.The ratios of hepatic Mψs and TLR4~+Mψs were increased in HFD group.However,the proportions of Mψs and TLR4~+Mψs in HFD-INU group were respectively lower than those in HFD group.Compared with HFD group,feeding INU to NAFLD mice also prminently boosted the proportion of M-MDSCs in liver,peripheral blood and spleen.Western blot showed that HFD feeding could significantly increase the expressions of NLRP3,ASC,caspase-1 and NF-κB in liver.However,after INU intervention the mentioned proteins were dramatically decreased in NAFLD mice.4.Spearman correlation analysis showed that the abundances of beneficial bacteria including Bacteroidetes,Akkermansia and Bifidobacterium exhibited positive correlations with SCFAs and IL-10,respectively.However,these beneficial bacteria were negatively correlated with metabolic and pro-inflammatory indicators(TG,TC,ALT,AST,LPS,IL-18,IL-1β,TNF-α,IL-6).Reversely,the abundance of Proteobacteria,Blautia and Ileibacterium were negatively correlated with SCFAs,but positively associated with the above mentioned metabolic and pro-inflammatory indicators.Conclusion Dietary INU ameliorates NAFLD via modulating gut microbiota and suppressing inflammation in mice.