A Study Of Gut Microbiota In Experimental Autoimmune Myocarditis

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Analysis the composition of gut microbiota in mice with experimental autoimmune myocarditisObjective To investigate the changes of the composition of gut mircrobiota in mice with experimental autoimmune myocarditis.Methods Male BALB/c mice,6-8 weeks old,18-20 g were used.A mouse model of experimental autoimmune myocarditis(EAM)was induced by mouse alpha-cardiac myosin heavy chain(MyHC-?)sequence(MyHC-?614–629: acetyl-SLKLMATLFSTYAS).Mice were randomly divided into two groups(normal control group and EAM group).Heart,spleen and fecal specimens of two groups of mice were collected on the 21 st day after immunization.After fecal DNA was extracted,16 Sr RNA sequencing was used to analyze the differences of gut mircrobiota between the two groups.Results 1.The body weight of mice with autoimmune myocarditis was significantly lower than that of control group(24.74±0.67 vs.20.89±1.27,P=0.015).The weight of heart and spleen in mice with myocarditis were significantly increased compared with normal mice.HE slices showed a large number of inflammatory cell infiltration in the heart tissue of mice with myocarditis,but no inflammatory cells were found in the heart tissue of normal mice.PCR results showed that m RNA expression levels of TNF-?,IL-1? and MCP-1 inflammatory factor were significantly increased in myocarditis mouse heart tissue.These results suggest that MyHC-? induces autoimmune myocarditis mouse model successfully.2.The composition of gut mircrobiota in mice with experimental autoimmune myocarditis changed significantly.Compared with normal mice,the gut mircrobiota of experimental autoimmune myocarditis mice showed species differences at each level from Phylum to Genus.At the Phylum level,the experimental autoimmune myocarditis mice showed a decrease in Bacteroidetes(P=0.018)and an increase in Firmicutes(P=0.036)compared with normal mice.At the Class level,the experimental autoimmune myocarditis mice showed a decrease of Bacteroidia(P=0.007)and the increase of Epsilon Proteobacteria(P=0.005)and Clostridia(P=0.037)compared with normal mice.At the Order level,experimental autoimmune myocarditis mice mainly showed Bifidobacteriales(P= 0.021),Bacteroidales(P=0.009)decreased and Lactobacillales(P=0.044),Clostridiales(P=0.032)and Campylobacterales(P=0.009)increased.At the Family level,the experimental autoimmune myocarditis mice showed a decrease of Bifidobacteriaceae(P=0.028)and Helicobacteraceae(P=0.010),Lactobacillaceae(P=0.020)and Streptococcaceae(P=0.027)increased.At the Genue level,the main manifestations of experimental autoimmune myocarditis mice were Bifidobacterium(P=0.026)decreased and Parabacteroides(P=0.047),Lactobacillus(P= 0.021),Lachnospira(P=0.045),Roseburia(P=0.049),Ruminococcus(P=0.016),Flexispira(P=0.007),Helicobacter(P=0.029)increased.3.The F/B ratio(Firmicutes/Bacteroidetes),which measures gut mircrobiota dysbiosis,was elevated in the experimental autoimmune myocarditis group(?2 times the normal group)(P<0.05).4.The gut mircrobiota richness and diversity index of experimental autoimmune myocarditis mice were higher than that of normal mice,and the Shannon index was statistically significant(P=0.0095).5.Uni Frac analysis showed that the composition of intestinal microbiota was different between the normal group and the experimental autoimmune myocarditis group.6.LEf Se analysis comparison.The relative abundance of 12 taxa in the experimental autoimmune myocarditis group was increased by LEf Se analysis,and the relative abundance of 4 was lower than that of the control group.7.Further use PICRUSt to predict microbial community function.We found 29 significant differences in functional pathways through predictions,and these functional differences analysis provided ideas for next experiments.Conclusion Compared with normal mice,the composition of gut mircrobiota in experimental autoimmune myocarditis mice changed significantly.Mainly manifested in the reduction of Bacteroides and the increase of the Firmicutes at the Phylum level.At the same time,the F/B ratio increased and the diversity of gut mircrobiota was higher than that of normal mice.These results indicate that there is gut mircrobiota dysbiosis in EAM mice.Part ? Effect of fecal microbiota transplantation on mice with experimental autoimmune myocarditisObjective To explore the effects of fecal microbiota transplantation on experimental autoimmune myocarditis in mice and its possible mechanisms.Methods Male BALB/c mice,6-8 weeks old,18-20 g were used.A mouse model of experimental autoimmune myocarditis(EAM)was induced by mouse alpha-cardiac myosin heavy chain(MyHC-?)sequence(MyHC-?614–629: acetyl-SLKLMATLFSTYAS).Mice were randomly divided into three groups: normal control group,experimental autoimmune myocarditis group(EAM group)and fecal microbiota transplantation group(EAM+FMT group).The bacterial flora was extracted from the normal mouse fecal samples,and the mice in the fecal microbiota transplantation group were intragastrically administered with 200 ul/time,3 times a week for 2 weeks.The weight of mice in each group was recorded daily.Cardiac echocardiography was performed on the 21 st day after initial immunization.Specimens of peripheral blood,heart and spleen of each group were collected.HE and Immunohistochemistry were performed;Real-time PCR was used to detect the expression of IFN-?,IL-4 and IL-17 A in the heart;flow cytometry was used to detect the proportion of Th1,Th2,Th17 and Tregs in peripheral blood,heart and spleen.Results 1.The weight of mice in the EAM group and EAM+FMT group was lower than that in the normal control group,however,there was no difference in body weight between the EAM group and EAM+FMT group.The heart weight of the mice in EAM+FMT group was lighter than that in the EAM group,and the heart/weight ratio was reduced in EAM+FMT group.2.The results of echocardiography showed that compared with the control group,the heart function of the EAM group was significantly changed,and the EF% and FS% were significantly decreased.The heart function of the EAM+FMT group was slightly improved compared with the EAM group,both EF% and FS% were higher than those in the EAM group,but did not reach statistical significance.3.HE staining of the heart showed that the inflammatory cell infiltration range was reduced,the inflammation score was decreased,and the necrotic area was decreased in EAM+FMT group.Immunohistochemistry showed that CD3(T cells)and CD68(macrophages)positive cells in myocardial tissue were reduced in EAM+FMT group.4.Real-time PCR analysis of cardiac tissue showed that the expression of IFN-? m RNA in myocardium was significantly decreased in EAM+FMT group compared with the EAM group,while the expression levels of IL-4 and IL-17 A m RNA were not statistically different between the EAM group and fecal microbiota transplantation group.5.Flow cytometry showed that the proportion of CD4~+IFN-?~+ cells in the spleen and heart of the EAM+FMT group was lower than that in the EAM group(P<0.05);The proportion of CD4~+IL-17~+ cells in the peripheral blood,spleen and heart were no significant difference between the two groups.Compared with the EAM group,the proportion of CD4~+IL-4~+ cells in the spleen and heart increased slightly in the fecal microbiota transplantation group,but did not reach statistical difference;6.Flow cytometry showed that the percentage of CD4~+CD25~+Foxp3~+Treg cells in peripheral blood and spleen of EAM group were lower than that of control group,and the proportion of Treg in spleen was decreased statistically.In the fecal microbiota transplantation group the proportion of CD4~+CD25~+Foxp3~+Treg cells in peripheral blood and spleen were higher than that of EAM group,and the proportion of Treg in spleen increased statistically,p<0.05.The proportion of CD4~+CD25~+Foxp3~+Treg cells in the heart was not significantly different among the three groups.Conclusion Fecal microbiota transplantation can alleviate the inflammatory reaction in myocarditis mice to a certain extent,mainly in reducing inflammatory cell infiltration in myocardium and partly improving cardiac function.The possible mechanism is to reduce the proportion of CD4~+IFN-?~+ cells in the spleen and heart and increase the proportion of CD4~+CD25~+Foxp3~+Treg in the spleen.Part ? Analysis the composition of gut microbiota after fecal microbiota transplantation in mice with experimental autoimmune myocarditisObjective To investigate the composition of gut microbiota in mice with experimental autoimmune myocarditis after fecal microbiota transplantation.Methods Male BALB/c mice,6-8 weeks old,18-20 g were used.A mouse model of experimental autoimmune myocarditis(EAM)was induced by mouse alpha-cardiac myosin heavy chain(MyHC-?)sequence(MyHC-?614–629: acetyl-SLKLMATLFSTYAS).Mice were randomly divided into two groups: experimental autoimmune myocarditis group(EAM group)and fecal microbiota transplantation group(EAM+FMT group).The bacterial flora was extracted from the normal mouse fecal samples,and the mice in the fecal microbiota transplantation were intragastrically administered with 200 ul/time,3 times a week for 2 weeks.Fecal specimens were collected from two groups on the 21 st days after the initial immunization,fecal DNA was extracted,and 16 S r RNA sequencing was used to analyze the difference of the composition of gut microbiota between the two groups.Results 1.We found that the composition of the gut microbiota in the EAM group and the EAM+FMT group were different from Phylum to Genus levels.The abundance of Bacteroidetes was relatively increased after FMT at the Phylum level,which was significantly different from that of EAM group(P=0.0276).At the same time,the abundance of Firmicutes was slightly lower than that of the EAM group(P=0.6868).The F/B ratio of the EAM+FMT group was decreased,although the difference between the two groups did not reach significance(P=0.0744).At the Class level,the proportion of Bacteroidia in the EAM+FMT group was higher than that in the EAM group(P=0.033),while the proportion of Clostridia was slightly lower than that in the EAM group(P=0.67).At the same time,we observed a significant decrease in Deferribacteres in the EAM+FMT group(P=0.026).At the Order level,the proportion of Bacteroidales in the EAM+FMT group was higher than that in the EAM group(P=0.03),while the abundance of Clostridiales,Lactobacillales,and Campylobacterales were decreased in EAM+FMT group,and the proportion of Campylobacterales between two groups was statistically significant(P<0.05).At the Family level,the proportion of Helicobacteraceae and Lactobacillaceae in EAM+FMT group was lower than that in EAM group(P=0.035 and P=0.66,respectively).At the same time,we found that the proportion of Prophyromonadaceae decreased in the EAM+FMT group(P=0.02).At the Genue level,the proportion of Parabacteroides in the EAM+FMT group was slightly lower than that in the EAM group,while the proportion of Flexispira and Ruminococcus were increased in EAM ~+ FMT group,but did not reach statistical difference.2.We found that FMT treatment did not further increase the richness and diversity of the bacteria in myocarditis mice.On the contrary,the trend of the Shannon index decreased in the EAM+FMT group compared with the EAM group.In the principal component analysis,we observed a clear separation of microbiota clusters between the two groups,indicating that there were differences in intestinal microbial composition between the EAM and EAM+FMT groups.Conclusion After the fecal microbiota transplantation treatment,the proportion of Bacteroidetes was increased and the abundance of Firmicutes was lowered in EAM mice,and the F/B ratio was decreased also.At the same time,the diversity of the gut microbiota was partially restored.These results demonstrate that fecal microbiota transplantation could reshape the gut microbiota dysbiosis in EAM mice.