| Objective Our study was designed to assess the effects of inulin（INU）and metformin（MET）on atherosclerosis（AS）in ApoE-/-mice,and further investigate the underlying mechanisms via gut microbiome and atherosclerotic inflammation.Methods Eight-week male ApoE-/-mice were randomly divided into control diet（CON）group,AS model group,AS+INU group and AS+MET group.During 12 weeks of treatment,body weights（BW）of all mice was monitored weekly and food intake was recorded every 2 days.After the treatments,associated indicators were investigated,including:（1）Assessment of the effects of INU and MET on AS: Face oil red O staining,oil red O staining,Masson’s staining and HE staining were used to assess aorta root injury in AS.（2）Measurement of lipid indicators in AS: Plasma concentrations of cholesterol（TC）,triglyceride（TG）,low density lipoprotein（LDL）and high density lipoprotein（HDL）in four groups of mice were detected by automatic biochemical analyzer.（3）Analysis of gut microbiota: 16S rRNA hypervariable sequencing was adopted to detect changes of the diversity and abundance of gut microbiota among diverse groups.（4）Analysis of gut microbiota-derived short chain fatty acids（SCFAs）: SCFA contents in each group were measured using gas chromatographymass spectrometry（GC-MS）.（5）Detection of lipoplysaccharide（LPS）in plasma: Limulus amebocyte lysate test was used to measure plasma LPS levels in diverse groups.（6）Determination of inflammatory cytokines: Micro-samples multiple indicators flow cytometry cytometric bead array（CBA）was selected to determine concentrations of pro-inflammatory tumor necrosis factor（TNF）-α,interleukin（IL）-1β,IL-6,IL-17 A,as well as anti-inflammation IL-10.（7）Correlation analysis: Spearman correlation analysis was used to analyze the correlation among metabolic indicators,gut microbiota and inflammation.Results（1）Pathological detection results of oil red O staining,Masson’s staining and HE staining indicated that dietary INU and MET intervention could separately attenuate the lipid acceleration of plague,fibrosis and vascular lesion.（2）Furthermore,dietary fiber INU treatment showed no influence in BW（P>0.05）,but MET intervention could significantly altered the BW from week 7 to 12（P<0.05）.Meanwhile,food intake showed no significant difference among diverse groups（P>0.05）.（3）Plasma TC,TG,and LDL were notably decreased in INU or MET treatment（P<0.05）,but HDL showed no significant change（P>0.05）.（4）To further investigate the underling mechanism of the effectiveness,16S rRNA sequencing were performed and found that long-term dietary fiber INU or MET administration could modulate the gut microbiota by significantly reducing Alistipes,Intestinimonas,Bilophila,Oscillibacter,Negativibacillus（P<0.05）and intriguingly increasing the relative abundance of Faecalibaculum（P<0.05）in INU treatment;and by significantly reducing Proteobacteria,Firmicutes/Bacteroides（F/B）ratio,Romboutsia and increasing Akkermansia and Bifidobacteria in MET treatment.（5）Gut microbiota derived SCFAs including acetic acid,propionic acid and valeric acid were significantly increased with INU or MET intervention（P<0.05）;butyrate acid was notably increased in MET treatment（P<0.05）,but showed increased tendency without significance in INU treatment（P>0.05）.（6）Abnormal elevated LPS levels in plasma were reduced with INU or MET treatment respectively（P<0.05）.（7）Long-term dietary INU or MET administration significantly suppressed proinflammatory TNF-ɑ,IL-1β,IL-6 and L-17A（P<0.05）,but showed no significant influence in IL-10（P>0.05）.（8）Spearman correlation analysis showed that Bacteriodes,Bifidobacteria,Akkermansia were positively correlated with SCFAs（acetic acid,propionic acid,butyric acid and valeric acid）,and negatively associated with pro-inflammatory indicators.Conversely,Fermicutes,F/B ratio,Proteobacteria and Romboutsia were negatively correlated with SCFAs,but positively with the above pro-inflammatory indicators.Conclusion Dietary INU and MET ameliorate AS via suppressing inflammation and modulating gut microbiota in mice. |
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