A Study Of Acupuncture Regulating Gut Microbiota To Promote Intestinal Motility In FC Mice Via TLR2 Signaling Pathway

ObjectiveTo reveal the mechanism of acupuncture regulating gut microbiota to promote intestinal motility in FC mice via TLR2 signaling pathway.Methods48 healthy female C57BL/6 mice,including 24 wild-type mice and 24 TLR2^-/-mice.Wild-type mice were randomly divided into control group（NC group）,functional constipation model group（FC group）,functional constipation model+electroacupuncture group（EA group）.TLR2^-/-mice were randomly divided into control group（KO group）,functional constipation model group（KO+FC）,functional constipation model+electroacupuncture group（KO+EA group）.FC model was established with compound diphenoxylate suspension by gavage at the dose of 1.0 mg/ml for 14 days in FC group,EA group,KO+FC group and KO+EA group.Mice in EA group and KO+EA group were electroacupunctured at ST25（tianshu）and ST37（shangjuxu）at the same side,alternate left and right every day.Select sparse and dense waves,30 min every day,five days as a course,two courses totally,and mice rest for 2 days between two courses.Experiment 1 The expulsion time of the first black stool,the fecal numbers,the fecal weight and the fecal moisture content in 8 hours,were calculated before and after modeling and after EA treatment.The gastric emptying rate and the small intestine propulsion rate were calculated after EA treatment.And HE stain was used to observe the colon morphology,the arrangements of the submucosal and myenteric plexus.Experiment 2 After EA treatment,colonic contents were collected.16S rRNA high-throughput sequencing was performed to explore the changes in intestinal microbiota diversity,species structure,composition,and different species at different taxonomic levels（phylum,class,order,family,and genus）.Experiment 3 After EA treatment,orbital blood sampling and colon were collected.Transmission Electron Microscopy（TEM）was used to detect the cellular ultrastructure of EGC.Double immunofluorescence labeling was used to detect the colocalized expression of GFAP and TLR2.Immunohistochemistry,WB and RT-PCR were used to detect the expressions of TLR2,NF-κB and GDNF in the colon.Immunohistochemistry and RT-PCR were used to detect the expression of MyD88 in the colon.ELISA was used to detect the expression of TNF-αin the colon and serum.Spearman analysis was performed to detect the correlation between TLR2 signaling pathway and different species of intestinal flora.ResultsExperiment 1 Effect of EA on intestinal motility1.Before modeling,there was no significant difference among the expulsion time of the first black stool,fecal numbers,fecal weight,fecal moisture content,gastric emptying rate and the small intestine propulsion rate in each group（P>0.05）.After modeling,the expulsion time of the first black stool in FC group and EA group were significantly longer compared with the NC group（P<0.01）,and the fecal numbers,fecal weight and fecal moisture content in 8 hours were significantly fewer（P<0.01）.In KO+FC group and KO+EA group,the expulsion time of the first black stool was significantly longer（P<0.01,P<0.05）,and the fecal numbers,fecal weight,fecal moisture content were significantly fewer（P<0.01,P<0.01,P<0.01,P<0.01,P<0.05,P<0.01）.After EA treatment,the expulsion time of the first black stool of EA group was significantly shorter（P<0.01）,and the fecal numbers,fecal weight,fecal moisture content were significantly increased（P<0.01）compared with the FC group.Above indexes showed no significant difference between KO+EA group and KO+FC group（P>0.05）.2.The gastric emptying rate and the small intestine propulsion rate of FC group were significantly decreased compared with NC group（P<0.01,P<0.05）.Which in EA group were significantly increased compared with FC group（P<0.01）.These two indexes of KO+FC group were significantly lower than which of the KO group（P<0.01）.And there was no significant difference between KO+EA group and KO+FC group about these two indexes（P>0.05）.3.A clear delineation of submucosal and myenteric plexus were observed in NC group and KO group.A fuzzy contours of plexus were observed in FC group and the opposite situation was observed in EA group.Compared with KO group,there was a fuzzy contours of plexus in KO+FC group and KO+EA group.Experiment 2 Effect of EA on intestinal microbiota.1.Diversity and richness of intestinal microbiotaCompared with the NC group,the richness of intestinal microbiota in FC group had no significant change（P>0.05）,and the diversity had an increasing trend,but there was no statistical difference（P>0.05）.Compared with the FC group,the richness of the EA group had no significant change（P>0.05）,while the diversity was significantly reduced（P<0.01）.The richness showed no significant difference（P>0.05）,while the diversity showed significant increase（P<0.01）in KO+FC group compared with KO group.The diversity and richness showed no significant difference（P>0.05）in KO+EA group compared with KO+FC group.Both indexes showed a significant decrease in KO group compared with NC group（P<0.01）.2.PCoA and NMDS analysis Intuitive differences of the composition of intestinal microbiota were observed between FC group and NC group,EA group and FC group,KO+FC group and KO group.A similar composition of intestinal microbiota was observed between KO+EA group and KO+FC group,NC group and KO group.3.Species relative abundanceAt the level of phylum:Compared with the NC group,the relative abundance of Firmicutes and Desulfobacteria in the FC group decreased,Bacteroidetes,Verrucobacterium,Actinobacteria,Patescibacteria,Campylobacter,Deferrobacteria and Proteobacteria increased.After EA treatment,the relative abundance of Firmicutes increased and the relative abundance of Bacteroidetes,Actinobacteria,Patescibacteria,Campylobacte and Deferribacteres decreased.Compared with KO group,all the phylum showed an increase,after EA treatment only few phylum species showed a converse change,most phylum showed no significant change.At the level of genus:Compared with the NC group,the relative abundance of no.rank.f.Muribaculaceae,Akkermansia,Candidatus.Saccharimonas,Bacteroides,Alistipes,Rikenellaceae.RC9.gut.group,Helicobacter,Odoribacter,Lachnospiraceae.NK4A136.group and unclassified.f.Lactobacillale were significant higher,while the relative abundance of Staphylococcus,Lactobacillus,Streptococcus,unclassified.c.Bacill and Faecalibacterium were significant lower in FC group.After EA treatment,the relative abundance of most above genus species showed a converse change.Compared with KO group,the relative abundance of no.rank.f.Muribaculaceae,Lactobacillus,Akkermansia,Candidatus.Saccharimonas,Bacteroides,Alistipes,Rikenellaceae.RC9.gut.group,Helicobacter,Odoribacter,Lachnospiraceae.NK4A136.group,unclassified.f.Lactobacillales and Streptococcus were significant higher and the relative abundance of Staphylococcus,unclassified.c.Bacilli and Faecalibacterium were significant lower.After EA treatment,only few genus species showed a converse change,most genera showed no significant change.4.Lefse analysisFirmicutes was significantly enriched in NC group and KO group,Firmicutes and Patescibacteria were significantly enriched in FC group,Bacteroidetes was significantly enriched in KO+FC group,Bacteroidetes and Verrucomicrobia were significantly enriched in KO+EA group.The relative abundance of different species between groups:compared with the NC group,the relative abundance of phylum Firmicutes,class Bacilli,order Verrucomicrobia,family Akkermansia and genus Staphylococcus were lower,while the relative abundance of phylum Bacteroidetes,Patescibacteria,class Bacteroidetes,order Erysipelotrichales and genus Bacteroidetes were higher in FC group.Compared with the FC group,above flora showed a converse change in EA group.Compared with the KO group,above flora showed a same change with FC group in KO+FC group.Compared with the KO+FC group,no significant change was observed in KO+EA group.Experiment 3 Effect of EA on EGC and TLR2-NF-κB-GDNF signaling pathway1.EGC and TLR2 signaling pathwayCompared with the NC group,the morphology of EGC was damaged,and the expression of GFAP,co-expression of GFAP and TLR2,the expression of TLR2 and NF-κB showed a significant increasement in the FC group（P<0.01,P<0.01,P<0.05,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01）,the expression of MyD88,TNF-αand GDNF also showed a significant increase（P<0.01）in the FC group.Compared with the FC group,the morphology of EGC was better,the expression of GFAP,the co-expression of GFAP and TLR2,the expression of TLR2and NF-κB showed a significant decrease（P<0.05,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01,P<0.01）,the expressions of MyD88,TNF-αand GDNF showed a significant increase（P<0.01）in the EA group.Compared with the KO group,the morphology of EGC was damaged,the expression of GFAP showed a significant increase（P<0.05）,the co-expression of GFAP and TLR2,the expression of TLR2 showed no significant change（P>0.05）,the expression of MyD88,NF-κB and TNF-αshowed a significant increase（P<0.01,P<0.01,P<0.01,P<0.05,P<0.05）in the KO+FC group.Compared with the KO+FC group,no significant change was observed in KO+EA group.2.Correlation analysisThere was a significant positive correlation between TLR2 and Patescibacteria（r=0.56）,there was a significant negative correlation between MyD88 and phylum Firmicutes（r=-0.66）,class Bacilli（r=-0.65）and genus Staphylococcus（r=-0.51）,there was a significant positive correlation between MyD88 and phylum Bacteroidetes（r=0.69）,class Bacteroidia（r=0.69）and genus Bacteroides（r=0.756）.There was a significant negative correlation between NF-κB and phylum Firmicutes（r=-0.88）,class Bacilli（r=-0.88）and genus Staphylococcus(r=-0.764,and a significant positive correlation between NF-κB and phylum Bacteroidetes（r=0.83）,class Bacteroidia（r=0.83）,genus Bacteroides（r=0.881）and order Erysipelotrichales（r=0.52）.There was a positive correlation between TNF-αand genus Bacteroides（r=0.55）.There was a positive correlation between GDNF and phylum Firmicutes（r=0.82）,class Bacilli（r=0.79）,order Verrucomicrobia,family Akkermansia（r=0.58）and genus Staphylococcus（r=0.80）.There was a negative correlation between GDNF and phylum Bacteroidetes（r=-0.78）,class Bacteroidia（r=-0.78）,genus Bacteroides（r=-0.66）and order Erysipelotrichales（r=-0.59）.Indicating that there was a significant correlation between TLR2-NF-κB-GDNF signaling pathway and intestinal microbiota.Conclusions1.EA can improve intestinal motility in FC mice.2.EA can regulate the disturbed intestinal microbiota in FC mice.3.EA may promote intestinal motility by improving intestinal microbiota,inhibiting EGC,regulating TLR2-NF-κB-GDNF signaling pathway.The effect of EA on TLR2^-/-mice was not obvious,which indicated the important role of TLR2-NF-κB-GDNF signaling pathway in EA treat FC.