| Objective This study is based on the TCM theory of “heart and small intestine being interior-exteriorly related” and “preventive treatment by acupuncture and moxibustion”.Firstly,the intestinal dysbacteriosis rat model was established.The intestinal flora,5-HT metabolism and related protein expression were observed,to explore the connection between intestinal flora,5-HT and myocardial cells,to clarify the correlation between intestinal flora and cardiovascular disease.Then,the I/R rat model was established,with intestinal flora and 5-HT as the breakthrough point,using electroacupuncture pretreatment at Neiguan and Zusanli.The intestinal flora,5-HT metabolism and related protein expression were observed,to study the effects of electroacupuncture pretreatment on 5-HT metabolism in I/R rats by regulating intestinal flora,and to provide experimental basis for treatment of myocardial ischemia diseases from the perspective of “heart and small intestine”.Methods 20 male Wistar rats were randomly divided into 2 groups: normal group and dysbacteriosis group,with 10 rats in each group.The rats in the dysbacteriosis group were given intragastric administration of lincomycin hydrochloride(5g/kg·BW),and the rats in the normal group were given intragastric administration of normal saline with the same volume,once a day for 7 days.The changes in morphology and body mass of rats in each group were observed.On the 8th day,the composition of intestinal flora was detected by quantitative PCR.The number and morphology of EC cells were observed by immunohistochemistry.The contents of 5-HT,Trp,5-HIAA and Kyn in ileum tissues and blood were detected by ELISA.The contents of 5-HT in cardiomyocytes were detected by ELISA.The protein expression levels of 5-HT2 BR and 5-HT4 R in cardiomyocytes were detected by western-blot.The m RNA expression levels of 5-HT2 BR and 5-HT4 R in cardiomyocytes were detected by quantitative PCR,and the data were statistically analyzed.40 male Wistar rats were randomly divided into 4 groups: sham group,ischemia/reperfusion(I/R)model group,Neiguan group and Zusanli group,with 10 rats in each group.Sham group: fixed once a day,20 min each time,for 7 days,and the surgery of threading without ligation of the left anterior descending coronary artery was performed on the 8th day.I/R model group: fixed once a day,20 min each time,for 7 days,and the surgery of ischemiareperfusion of the left anterior descending coronary artery was performed on the 8th day.Neiguan group: treated with electroacupuncture at Neiguan(PC6)once a day,20 min each time,for 7 days,and the surgery was the same as I/R model group on the 8th day.Zusanli group: treated with electroacupuncture at Zusanli(ST36),once a day,20 min each time,for 7 days,and the surgery was the same as I/R model group on the 8th day.During the surgery,the changes of ECG in each group were observed.After the surgery,the composition of intestinal flora was detected by quantitative PCR.The number and morphology of EC cells were observed by immunohistochemistry.The contents of 5-HT,Trp,5-HIAA and Kyn in ileum tissues and blood were detected by ELISA,as well as CK,CK-MB and c Tn T in blood.HE staining was used to observe the morphological structure of myocardial cells.Masson staining was used to observe the area of myocardial infarction.The contents of 5-HT in cardiomyocytes were detected by ELISA.The protein expression levels of 5-HT2 BR and 5-HT4 R in cardiomyocytes were detected by western-blot.The m RNA expression levels of 5-HT2 BR and 5-HT4 R in cardiomyocytes were detected by quantitative PCR,and the data were statistically analyzed.Result 1.Effects of dysbacteriosis on the 5-HT secretion of enterochromaffin cells in rats(1)changes in morphology and body mass of rats: after 1 week of lincomycin intervention,the rats in dysbacteriosis group had diarrhea and soft stool,accompanied by decreased food and water intake,depressed spirit,and lackluster hair.The disease activity index of dysbacteriosis group was significantly higher than that of normal group(P<0.05).(2)changes in intestinal flora composition: compared with normal group,the number of Escherichia coli,Bacteroidetes,Enterococcus and Peptostreptococcus in dysbacteriosis group increased,while the number of Lactobacillus and Bifidobacteria decreased(P<0.05,P<0.01).(3)morphological changes in ileum tissues: in normal group,intestinal mucosal epithelial cells were intact,glands were arranged in order,and goblet cells were abundant.In dysbacteriosis group,intestinal mucosal epithelial cells were destroyed,glandular arrangement was disordered,goblet cells decreased and disappeared.(4)changes in the number and morphology of EC cells: the number of EC cells in dysbacteriosis group was higher than that in normal group(P<0.01),and specific secretory granules were observed in the cytoplasm.(5)changes of 5-HT content in ileum tissues and blood: compared with normal group,the contents of 5-HT,Trp,5-HIAA and Kyn in ileum tissues and blood of the dysbacteriosis group increased(P<0.01,P<0.05).2.Effects of dysbacteriosis on 5-HT content and receptor expression in cardiomyocytes(1)changes of 5-HT content in cardiomyocytes: the content of 5-HT in cardiomyocytes of dysbacteriosis group was significantly higher than that of normal group(P<0.01).(2)changes of 5-HT receptor expression in cardiomyocytes: compared with normal group,the protein and m RNA expression of 5-HT2 BR 、 5-HT4 R in cardiomyocytes of dysbacteriosis group increased(P<0.01,P<0.05).3.Effects of electroacupuncture pretreatment on myocardial injury markers and morphology in I/R model rats(1)changes of ST segment value of ECG: compared the data of 20 min after ligation with before ligation: the value of ST segment in I/R model group,Neiguan group and Zusanli group increased(P<0.05).Compared the data of 20 min after ligation with 40 min after reperfusion,the value of ST segment in I/R model group,Neiguan group and Zusanli group fell back(P<0.01,P<0.05).At 40 min after reperfusion,compared with the sham group,the value of ST segment in I/R model group increased(P<0.05).Compared with I/R model group,the value of ST segment in Neiguan group and Zusanli group decreased(P<0.01).Compared with Zusanli group,the value of ST segment in Neiguan group decreased(P<0.01).(2)changes in myocardial injury markers: compared with sham group,the contents of CK,CK-MB and c Tn T in blood of I/R model group increased(P<0.01,P<0.05).Compared with I/R model group,the contents of CK,CK-MB and c Tn T in blood of Neiguan group and Zusanli group decreased(P<0.01).Compared with Zusanli group,the contents of CK-MB in blood of Neiguan group decreased(P<0.01).(3)morphological changes in cardiomyocytes: in the sham group,myocardial fibers were arranged neatly,with clear horizontal lines,complete cell structure and normal shape,without inflammatory infiltration or cavitation degeneration.In I/R model group,myocardial fibers were disordered,with partial fiber rupture,obvious cell structure destruction,inflammatory cell infiltration and cavitation degeneration.In Neiguan group and Zusanli group,myocardial fibers were slightly disorganized,with a small amount of fiber fracture,inflammatory infiltration,cells edema and necrosis.(4)changes in myocardial infarction area: compared with sham group,the myocardial infarction area in I/R model group increased(P<0.05).Compared with I/R model group,the myocardial infarction area of Neiguan group and Zusanli group decreased(P<0.05,P<0.01).Compared with Zusanli group,the myocardial infarction area of Neiguan group decreased(P<0.01).4.Effects of electroacupuncture pretreatment on 5-HT secretion of EC cells in I/R model rats by regulating intestinal flora(1)changes in the intestinal flora composition: compared with sham group,the number of Escherichia coli,Bacteroidetes,Enterococcus and Peptostreptococcus increased in I/R model group,while the number of Lactobacillus and Bifidobacteria decreased(P<0.05,P<0.01).Compared with I/R model group,the number of Escherichia coli,Bacteroidetes,Enterococcus and Peptostreptococcus in Neiguan group and Zusanli group decreased,while the number of Lactobacillus and Bifidobacteria increased(P<0.05,P<0.01).Compared with Zusanli group,the number of Lactobacillus in Neiguan group increased(P<0.05).(2)changes in the number and morphology of EC cells: compared with sham group,the number of EC cells in I/R model group increased(P<0.01),and specific secretory granules were observed in the cytoplasm.Compared with I/R model group,the number of EC cells in Neiguan group and Zusanli group decreased(P<0.05).(3)changes of 5-HT content in ileum tissues and blood: compared with sham group,the contents of 5-HT,Trp,5-HIAA and Kyn in ileum tissues and blood of I/R model group increased(P<0.01,P<0.05).Compared with I/R model group,the contents of 5-HT,Trp,5-HIAA and Kyn in ileum tissues and blood of Neiguan group and Zusanli group decreased(P<0.01,P<0.05).Compared with Zusanli group,the contents of 5-HT,5-HIAA in ileum tissues of Neiguan group decreased(P<0.05).5.Effects of electroacupuncture pretreatment on 5-HT content and receptor expression of cardiomyocytes in I/R model rats by regulating intestinal flora(1)changes of 5-HT content in cardiomyocytes: compared with sham group,the content of 5-HT in cardiomyocytes of I/R model group increased(P<0.01).Compared with I/R model group,the content of 5-HT in cardiomyocytes of Neiguan group and Zusanli group decreased(P<0.05).Compared with Zusanli group,the content of 5-HT in cardiomyocytes of Neiguan group decreased(P<0.01).(2)changes of 5-HT receptor expression in cardiomyocytes: compared with sham group,the protein and m RNA expression of 5-HT2 BR and 5-HT4 R in cardiomyocytes of I/R model group increased(P<0.01,P<0.05).Compared with I/R model group,the protein and m RNA expression of 5-HT2 BR and 5-HT4 R in cardiomyocytes of Neiguan group and Zusanli group decreased(P<0.05,P < 0.01).Compared with Zusanli group,the protein and m RNA expression of 5-HT2 BR and 5-HT4 R in cardiomyocytes of Neiguan group decreased(P<0.05,P<0.01).Conclusion(1)The model of lincomycin-induced dysbacteriosis in rats was successfully established,with the decrease of beneficial intestinal bacteria and the increase of conditioned pathogenic bacteria.Dysbacteriosis can further lead to the increase of EC cells in ileum tissues,the increase of 5-HT and related metabolites in ileum tissues and blood,and the increase of 5-HT and its receptor expression in cardiomyocytes.It was suggested that the changes of intestinal flora in rats may have a close relationship with cardiomyocytes by affecting the metabolism of 5-HT.(2)In I/R model rats,intestinal beneficial bacteria decreased and conditioned pathogenic bacteria increased,resulting in the increase of EC cells in ileum tissues,the increase of 5-HT and related metabolites in ileum tissues and blood,as well as the increase of 5-HT and its receptor expression in cardiomyocytes.It was suggested that myocardial ischemia reperfusion in rats may cause changes in the dysfunction of intestinal flora,further leading to the abnormal metabolism of 5-HT,which may induce or aggravate myocardial injury.(3)Electroacupuncture pretreatment at Neiguan or Zusanli could significantly reduce the content of myocardial injury markers in the I/R model rats,improve the degree of myocardial damage,and reduce the area of myocardial infarction.It was suggested that electroacupuncture pretreatment at Neiguan or Zusanli had protective effect on the structure and function of myocardial tissue in I/R rats,and the effect of Neiguan is better than Zusanli.(4)Electroacupuncture pretreatment at Neiguan or Zusanli could significantly improve the disorder of intestinal flora in I/R rats,inhibit the secretion of 5-HT by EC cells,thus causing the decrease of 5-HT and related metabolites in ileum tissues and blood,as well as the decrease of 5-HT and its receptor expression in cardiomyocytes.It was suggested that electroacupuncture pretreatment at Neiguan or Zusanli can effectively reduce myocardial ischemia reperfusion injury in I/R rats,and the mechanism may be related to regulating intestinal flora and influencing 5-HT metabolism. |
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