| Objective Although it has been reported that electroacupuncture stimulation can improve intestinal motility and regulate intestinal flora,there are few related studies,and the relationship between electroacupuncture stimulation,intestinal flora and intestinal motility is unclear.We established a constipation mouse model induced by loperamide,and observed the effects of electroacupuncture stimulation on intestinal motility and the composition,abundance and diversity of intestinal flora in constipation mice;The effect of Electroacupuncture on intestinal motility of constipated mice was observed by constructing a model of sterile mice transplantation(FMT)induced by loperamide,so as to verify whether electroacupuncture can change intestinal motility by regulating intestinal flora.At the same time,we will study the mechanism of electroacupuncture stimulation changing intestinal motility in constipated mice by regulating intestinal flora.Methods C57bl/6(male)mice of 6-8 weeks were induced into a constipation mouse model by intraperitoneal injection of loperamide 4mg/kg.The normal control group was intraperitoneally injected with normal saline.They were randomly divided into five groups:normal group,constipation group(Lop group),constipation pseudo stimulation group(Lop+SEA group),constipation low frequency electroacupuncture stimulation group(Lop+LEA group)and constipation high frequency electroacupuncture stimulation group(Lop+HEA group).Loperamide injection and electroacupuncture stimulation were carried out simultaneously.After 3 weeks,feces of each group of mice were collected to detect fecal water content,16 S r DNA amplicon sequencing technology was used to detect intestinal microflora of mice,and steel ball excretion test,whole gastrointestinal transit test and gastrointestinal transit test were carried out;Constipation sterile mice fecal bacteria transplantation model was established and randomly divided into 5 groups:antibiotic + constipation + normal mice fecal transplantation group(FMT+ Control),antibiotic + constipation + constipation mice fecal transplantation group(FMT+Lop),antibiotic + constipation + pseudoelectroacupuncture stimulated mice fecal transplantation group(FMT+ SEA),antibiotic + constipation + low frequency Electroacupuncture stimulated mice fecal transplantation group(FMT+LEA),Antibiotic + constipation + high frequency electroacupuncture stimulation mice fecal transplantation group(FMT+HEA).After 3 weeks,the fecal water content was detected,and the steel ball excretion test,gastrointestinal transit test and intestinal transit rate test were carried out.Ultra high performance gas chromatography mass spectrometry(GC-MS)was used to detect the contents of seven short chain fatty acids in the feces of mice in each group.LC-MS targeted metabonomics was used to detect bile acids in mouse feces.TDC and 5-HT in serum were detected by enzyme linked immunosorbent assay(ELISA).Gpr41,TGR5 and TPH1 in mouse colon were detected by Western blot;QRT PCR was used to detect the m RNA expression of gpr41,TGR5,TPH1,5-HT3 R,5-ht4 r and c-kit in colon tissues of mice in each group.Immunofluorescence technique(CO staining of c-kit and Ano1)was used to detect the expression level and distribution range of ICC in mouse intestinal muscle layer.Results(1)Compared with the constipated mice,the fecal water content of the low-frequency stimulation group increased,and the steel ball excretion time,total gastrointestinal transit time and intestinal transit rate decreased.(2)Shannon index decreased significantly in constipated mice and increased after electroacupuncture stimulation.NMDS showed that there were differences between the normal group and the constipation group,while the low-frequency stimulation group showed that the microbiota was close to the normal group.At the gate level,Bacteroides and Proteus decreased and firmicum increased after low-frequency stimulation.At genus level,low-frequency stimulation decreased the content of no rank muribaculaceae and parasutterella,and increased the content of Lactobacillus.(3)After fecal bacteria transplantation,compared with fmt+lop group,the fecal water content of fmt+lea group increased,and the steel ball excretion time,gastrointestinal transit time and gastrointestinal transit time decreased.(4)Compared with the control group,the contents of acetic acid and propionic acid in feces of mice in constipation group decreased,and the contents of some secondary bile acids also decreased,but low-frequency electroacupuncture stimulation could increase their contents.(5)The expression of gpr41,TPH1,TGR5,TDC,5-HT and their receptors 5-HT3 R,5-ht4 r decreased in constipated mice,and the expression of gpr41,TPH1,TGR5,TDC,5-HT increased after low-frequency electroacupuncture stimulation and fecal transplantation.After fecal bacteria transplantation,fmt+lea could also up regulate its expression compared with fmt+lop group.(6)The expression of c-kit and Ano1 in intestinal myometrium of mice in low frequency stimulation group was higher than that in constipation group.At the same time,after fecal bacteria transplantation,the expression of c-kit and Ano1 in intestinal myometrium of mice in fmt+lea group was also higher than that in fmt+lop group.Conclusion Low frequency electroacupuncture can improve intestinal motility of constipated mice by regulating intestinal flora.Low frequency electroacupuncture stimulation can increase the content of 5-HT and the number of ICC by increasing the content of some short chain fatty acids and bile acids,up regulating the expression of GPR41-TPH1 and TGR5-TDC. |
