| Objectives:With the development of the economy and society which are accompanied by changes in diet and lifestyle,non-alcoholic fatty liver disease(NAFLD)has become a global public health problem.Recent studies have shown that disorders of the intestinal flora can be closely linked to metabolic diseases such as NAFLD.Guava leaves have pharmacological activities including the ability to lower blood sugar,body weight and blood lipids as well as anti-oxidative and antibacterial properties.Previous research from our research group showed that guava leaf extract(GLE)can effectively reduce body weight and insulin resistant type 2 diabetes in mice,and up-regulate the liver PI3-K/Akt signaling pathway.The above-mentioned activities of GLE suggest that it has potential for the prevention and treatment of NAFLD.This is a novel idea which needs to be further investigated.In this study,a high-fat diet was used to simulate NAFLD in mice and this animal model was used to study the regulation of the intestinal flora of NAFLD mice by GLE.In addition,the effects of GLE on the pathways related to bile acid metabolism and the metabolites of the intestinal flora were explored.The role of intestinal flora in GLE’s improvement of NAFLD and the potential molecular mechanism involved provides a basis for its use as a potential clinical treatment of this disease.Method:Five-week-old male C57BL/6J mice were used to establish a diet-induced NAFLD mouse model,and they were randomly divided into a normal diet group(NC group,n=6)and a high-fat diet group(n=18).They were fed with ordinary diet(5% fat)and high-fat diet(60% fat),respectively,for 10 weeks,after which the latter group of mice were randomly divided into 3 further groups of 6: a low-dose GLE intervention group(LD group,GLE 819mg/kg·d),a high-dose GLE intervention group(HD group,GLE 1638mg/kg·d)and a model group(HFD group,given the same volume of normal saline).After 8 weeks of intervention,blood,liver tissue,intestinal tissue and cecal contents were collected.Detection indicators: 1.Performed HE,Masson,Oil Red O and glycogen staining to observe liver pathological changes and lipid deposition.2.Determined the body weight,blood lipids and biochemistry of mice in each group.3.ELISA method was used to measure serum TNF-α,IL-6 and fasting insulin levels,and insulin resistance index(HOMA-IR)and insulin sensitivity index(ISI)were calculated.4.16 S r DNA high-throughput sequencing method was used to analyze the flora of the cecal content.5.RT-PCR and western blotting methods were used to analyze key molecules of lipid synthesis in the liver(sterol regulatory element binding protein-1c,SREBP-1c;fatty acid synthase,FAS;acetyl-Co A carboxylase,ACC;HMG-Co A reductase,HMG-Co Ar)and key molecules of the bile acid pathway(farnesoid X receptor,FXR;small heterodimer partner,SHP;cholesterol 7α-hydroxylase,CYP7A1;G-protein-coupled receptor 5,TGR5)and the terminal ileum tissue expression of FXR and fibroblast growth factor 15(FGF15.6.Liquid chromatography-mass spectrometry(LC-MS)was used to determine the content and composition of bile acids in the mouse liver.7.Gas chromatography-mass spectrometry/mass spectrometry(GC-MS/MS)was used to determine the content and composition of short-chain fatty acids(SCFAs)in the cecal contents.SPSS 22.0 software was used for statistical analysis of the experimental data obtained.One-way ANOVA and the LSD method were used for paired comparison between groups.The components of bile acids were analyzed by orthogonal partial least square discriminant analysis.The Spearman method was used to analyze the correlation between intestinal flora and bile acids.The results are expressed as means ±standard deviations and P < 0.05 was considered statistically significant.Result:1.After 8 weeks of GLE intervention,the body weight and serum TNF-αlevels in the HD groups were lower than those in the HFD group(P < 0.05),and liver cell steatosis,ballooning,lobular inflammation and liver fibrosis were improved.HOMA-IR,serum AST,LDL-C,TC,TG and non-HDL-C of HD group mice were significantly lower than those in the HFD group(P < 0.05).Compared with the HFD group,the SREBP-1c and ACC m RNA and the m RNA and protein expression of HMG-Co Ar were significantly down-regulated in the mice livers of the LD and HD groups(P <0.05).2.High-dose GLE significantly changed the β diversity of the intestinal flora of NAFLD mice(P <0.05).Compared with the HFD group,the abundance of types of intestinal Firmicutes,Lactobacillus and Lactobacillus in the LD and HD groups was significantly reduced(P < 0.05).Compared with the NC group,the HFD group had significantly reduced concentrations of SCFAs including acetic,propionic and butyric acids in the cecum of mice(P < 0.05).The proportion of SCFAs of the LD and HD groups had significantly higher amounts of acetic and butyric acids than those of the HFD group(P < 0.05).3.Compared with HFD group,the total amount of serum bile acids in the HD group was significantly reduced(P < 0.05)and the levels of CA,UDCA,ω-MCA,α-MCA and β-MCA in liver were significantly increased.In addition,the intestinal flora was correlated with most of the bile acid levels in the liver(P < 0.05).4.The expression levels of protein and m RNA of TGR5,FXR and CYP7A1 in the HFD group were significantly lower than those in the NC group.These molecules in the liver of the HD group were significantly up-regulated(P < 0.05).At the end of the ileum,the expression levels of FXR and FGF15 protein in the HFD group was lower than that of the NC group,and their expression in the HD group was significantly higher than that of the HFD group(P < 0.05).Conclusion:GLE can improve liver cell steatosis,ballooning,lobular inflammation and liver fibrosis caused by high-fat diet.It can also protect liver function,lower blood lipids,reduce inflammation,improve insulin resistance and inhibit SREBP-1c and its downstream target genes such as FAS and ACC as well as reduce fatty acid synthesis.At the same time,GLE intervention significantly changed the structure of the intestinal flora of NAFLD mice with a reduction in the abundance of Firmicutes and an increase of intestinal SCFAs.This was accompanied by a reduction in the accumulation of serum bile acids and increased the content of hydrophilic bile acids in the liver.Activation of TGR5,FXR and CYP7A1 in the liver and FXR and FGF15 in the terminal ileum were also observed.Therefore,one possible mechanism of GLE in the treatment of NAFLD is to regulate the metabolism of bile acids and SCFAs by targeting the intestinal flora and activating bile acid metabolism. |
