The Effects Of Fndc5/Irisin Deficiency On Gut Microbiota And Depressive-like Behaviors In CUMS Mice

Objective:Depression is a common and serious mental disease,the main patho-physiological mechanism of which includes microbiome-gut-brain axis dysfunction.Irisin,an exercise-related myokine,is reported to reduce de-pression-like behaviors,but the related mechanism remains unclear.The objective of this study was to investigate the effects of Fncd5/irisin defi-ciency on the gut microbiota and depression-like behavior in depressed mice.To provide theoretical basis and practical reference for the combined treatment of depression with lifestyle-oriented adjuvant therapy and tradi-tional antidepressant drugs.Methods:C57BL/6N-Fndc5^em1cyagen mice were generated using the CRISPR-cas9 technique.CUMS program was used to construct the depres-sion model,and the period was 4 weeks continuously.The mice were di-vided randomly into four groups:control group,Fndc5/irisin knockout group,CUMS model group and Fndc5/irisin knockout mice treated with CUMS group.The depression symptoms were examined by SPT,FST and TST in the C57BL/6N-Fndc5^em1cyagen mice and the WT mice were treated with or without CUMS.After the behavioral test,mouse feces were collected.The 16S r RNA sequencing analysis was employed to assess the composition and species difference analysis of the gut microbiota.The LC-MS analysis was employed to detect the metabolic difference between the two groups.The functions of different metabolite were be predicted by KEGG topology analysis and KEGG enrichment analysis.Results:1.Compared with the CUMS group,the Fndc5/irisin knockout group showed more severe depression-like behavior after CUMS treatment.After 4 weeks of CUMS treatment,the results of the behavioral test showed that,compared with the control group,the results indicated that the immobility time in the FST and TST was significantly increased in the Irisin KO group(P_FST=0.0279,P_TST<0.0001),and the sucrose preference decreased with statistical significance(P_SPT=0.0241).In addition,mice in the CUMS group showed depressive-like behaviors,the immobility time in FST and TST was increased(P_FST=0.0031,P_TST=0.0007),and the su-crose preference was decreased(P_SPT<0.0001),compared with the control group.Compared with the Irisin KO group,the immobility time of FST and TST was significantly increased(P_FST=0.0096,P_TST=0.0008),and the sucrose preference was significantly increased(P_SPT<0.0001)in the Irisin KO-CUMS group.Moreover,when compared with mice in the CUMS group,the immobility time in the TST was increased(P_TST<0.0001),and the sucrose preference decreased significantly(P_SPT<0.0001),while the immobility time of the FST showed no significant difference in the Irisin KO-CUMS group.1.Fndc5/irisin knockout mediated alteration of the gut microbiota.16S r RNA sequencing results showed that,compared with the WT mice,the Alpha diversity analysis of gut microbiota in the C57BL/6N-Fndc5^em1cyagen mice showed a significant increase in the ACE index（P<0.01）,and the Simpson index decreased（P<0.05）.The PCo A analysis in Beta diversity analysis showed that the gut microbiota of the WT mice and the C57BL/6N-Fndc5^em1cyagen mice were separated from each other,and the difference was significant（P<0.01）.The NMDS analysis in Beta diversity analysis showed that the stress is 0.029<0.05,indicating that the data dimension reduction effect is very good.R=0.9867>0,indicating that the difference between groups was greater than the difference within groups,and had a high confidence（P=0.005）.The results indicated that there was a significant difference in gut microbiota composition between the WT mice and the C57BL/6N-Fndc5^em1cyagen mice（P<0.01）.At the phylum level,the two phyla with the highest relative abundance of gut microbiota in the WT mice and the C57BL/6N-Fndc5^em1cyagenmice were:Firmicutes,Bacteroidota.Compared with the WT mice,the C57BL/6N-Fndc5^em1cyagen mice had fewer Firmicutes and more Bac-teroidota.The Firmicutes/Bacteroidota ratio was regulated by Irisin.At the genus level,compared with the WT mice,the relative abundance of g＿＿Lactobacillus and g＿＿Bifidobacterium in the gut microbiota of the C57BL/6N-Fndc5^em1cyagen mice was significantly reduced.The relative abundance of g＿＿Lactobacillus and g＿＿Bifidobacterium were sig-nificantly decreased（P<0.01）after Fndc5/irisin knockout,while g＿＿Allo-baculum（P<0.01）,g＿＿Parabacteroides（P<0.05）and g＿＿Eubacte-rium＿xylanophlum＿group（P<0.05）were increased.Circos maps based on the genus level showed the composition of gut microbiota in the WT mice and the C57BL/6N-Fndc5^em1cyagen mice.The liner discriminative analysis（LDA）score conducted based on LEf Se analysis showed that the g＿＿Lactobacillus,g＿＿Bacillus and g＿Bifidobacterium were of great importance in the difference of microbi-ota in the WT mice,the g＿＿norank＿f＿＿Muribaculaceae,g＿＿nor-ank＿f＿＿norank＿o＿＿Clostridia＿UCG-014 and g＿＿Allobaculum were of great importance in the C57BL/6N-Fndc5^em1cyagen mice.PICRUSt2 analysis prompted the functional genes in the gut microbi-ota of the WT mice and the C57BL/6N-Fndc5^em1cyagen mice were found in the 6-phospho-beta-glucosidase pathway（K01223）and phosphoglycerate mutase pathway（K15634）are significant differences.2.The results of untargeted metabolomics analysis.PLS-DA showed that the sample metabolites in the WT mice and the C57BL/6N-Fndc5^em1cyagen mice had good aggregation and high repro-ducibility in respectively.Meanwhile,the aggregation range of sample me-tabolites between the two groups showed a completely separate state,sug-gesting that the characteristics of each sample metabolite in the WT mice and the C57BL/6N-Fndc5^em1cyagen mice were significantly dif-ferent.Volcano plot showed that compared with the WT mice,the metab-olites whose expression was significantly upregulated after Fndc5/irisin knockout including:N-Carbamoyl glucuronide lorcaserin,（-）-stercobilin;significantly downregulated metabolites including:N-Desmethylmifepris-tone（RU 42633）,Metabutethamine,Meperidinic acid glucuronide,Asp Ile Lys,Cinncassiol D2 glucoside,Ala Ile Gly Leu,3,4,5-trihydroxy-6-[（3-methyl-2-oxo-4-phenylbut-3-en-1-yl）oxy]oxane-2-carboxylicacid,Janthitrem C.The differential metabolites are mainly concentrated on the pathways of Arginine and proline metabolism pathway and the mTOR signaling pathway.3.The correlation between gut microbiota and metabolites after Fndc5/irisin knockout.Spearman correlation analysis heatmap shows that g＿＿Allobaculum were negatively correlated with 3,4,5-trihydroxy-6-[（3-methyl-2-oxo-4-phenylbut-3-en-1-yl）oxy]oxane-2-carboxylicacid（neg＿9114）,whereas g＿＿Allobaculum were positively correlated-with（-）-Stercobilin（neg＿818）and N-Carbamoyl glucuronide lorcaserin（neg＿8896）.The two-factor correlation network diagram indicated that the top 3 of total network nodes were（-）-Stercobilin（neg＿818）,N-Carbamoyl glucuronide lorcaserin（neg＿8896）and 3,4,5-trihydroxy-6-[（3-methyl-2-oxo-4-phenylbut-3-en-1-yl）oxy]oxane-2-carboxylic acid（neg＿9114）.The three central coefficients（degree＿centrality,closeness＿centrality,and betweenness＿centrality）was（-）-Stercobilin（neg＿818）,followed by N-Carbamoyl glucuronide lorcaserin（neg＿8896）,3,4,5-trihydroxy-6-[（3-me-thyl-2-oxo-4-phenylbut-3-en-1-yl）oxy]oxane-2-carboxylicacid（neg＿9114）.As for the microbiome,the largest value was g＿＿Allobaculum.Conclusion:1.Fndc5/irisin knockout mice showed more severe depressive-like be-haviors than normal mice after CUMS treatment.2.Fndc5/irisin deficiency reduced the diversity of gut microbiota in mice,Fndc5/irisin deficiency regulated the composition of gut microbiota in mice,which was specifically showed as a less population of Firmicutes,while had the greater population of Bacteroides,a regulation of the ratio of Firmicutes/Bacteroides was regulated by Irisin,and a significant decrease in the relative abundance of g＿＿Lactobacillus and g＿＿Bifidobacterium.3.Fndc5/irisin deficiency can significantly change the metabolic profile of gut microbiota in mice,the differential metabolites are mainly concentrated on the pathways of Arginine and proline metabolism pathway and affected the mTOR signaling pathway.