Effect Of Huoxue Wentong Decoction On TLR4 Signaling Pathway And Gut Microbiota After Myocardial Infarction

Myocardial infarction(MI)is myocardial necrosis caused by persistent ischemia and hypoxia of coronary artery,which is one of the most important causes of death from cardiovascular disease.Modern medical treatment of MI is increasingly mature,but there are still restenosis after interventional therapy,reperfusion arrhythmia,no-reflow phenomenon,drug resistance and other problems.Previous studies by our group have found that Huoxue Wentong Decoction(HXWT),a traditional Chinese medicine compound,has the effects of reducing inflammatory response,reducing myocardial apoptosis,promoting angiogenesis,and improving myocardial ischemia,but because the traditional Chinese medicine compound has the characteristics of multiple components,multiple targets,and multiple pathways in the treatment of MI,the above studies are only part of the mechanism of action of this prescription,and its pharmacological components and targets of action have not been clarified and still need further study.In view of this,based on the theory of the gut-heart axis,this study investigated whether HXWT can improve the injury of MI by mediating TLR4 signaling pathway and gut microbiota homeostasis through animal experiments and bioinformatics techniques.Objective:1.To investigate the protective effect of HXWT on cardiac injury after MI based on TLR4 signaling pathway2.To explore the relationship between gut microbiota and MI based on the gut-heart axis,and to explore whether HXWT can reduce the damage of MI by regulating gut microbiota homeostasis.Methods:1.Thirty male SD rats were randomly divided into sham operation group(Sham),model group(Model)and HXWT group(HX).MI was modeled after adaptive feeding,the coronary artery in the sham group was only threaded without ligation,the coronary artery in the HX group was ligated,and the HX group was treated with HXWT for 4 weeks after the end of modeling.At the end of treatment,cardiac injury and TLR4-related pathway expression after MI were evaluated by Elisa,TTC staining,HE staining,Masson staining,echocardiography,and RT-PCR.2.Use high-throughput sequencing technology to analyze the changes of gut microbiota in rats,use non-targeted metabolomics to detect intestinal metabolites in rats,combined with bioinformatics means,explore the mechanism of HXWT mediating gut microbiota in the treatment of MI.Results:1.The results of Elisa experiment showed that the contents of IL-6,TNF-α,and LPS in the serum of rats in the Model group were significantly increased compared with those in the sham group(P<0.01);compared with those in the Model group,the HX group could significantly reduce the contents of IL-6 and TNF-α in the serum of rats with MI(P<0.01)and reduce the LPS level(P<0.05).2.The results of HE staining showed that the myocardial cells in the Model group showed infarction,disorganized structure,myocardial fiber rupture and necrosis,and a large number of inflammatory cell infiltration.At the same time,the terminal ileum tissue was slightly poor,and some villi were damaged and broken;the myocardial cell structure in the HX group was basically normal,the infarction was smaller than that in the Model group,the intestinal epithelial structure in the mucosal layer of the terminal ileum tissue was better,and the intestinal villi and lamina propria were detached.3.The results of Masson staining showed that the myocardial fibers of rats in the Model group were disorganized and thickened,showing a large number of blue collagen fiber deposition;the myocardial fibers of rats in the HX group were slightly disorganized,showing blue collagen fiber deposition,and the collagen fibers were reduced compared with those in the Model group(P<0.01).4.The results of TTC staining showed that the myocardial infarct size was reduced in the HX model group(P<0.05).5.Echocardiographic results showed that EF and FS were significantly decreased(P<0.01),LVEDS was increased(P<0.01),and LVEDD tended to increase(P>0.05)in the Model group compared with the sham group.Compared with the Model group,EF and FS were increased(P<0.01),and LVEDS and LVEDD tended to decrease(P>0.05)in the HX group.6.The results of RT-PCR showed that compared with the sham group,the contents of TLR4,Myd88,and NF-kB in the myocardium of rats in the model group increased(P<0.05),and the contents of NF-kB,TLR4,and Myd88 in the ileum tissue tended to increase,but they were not statistically significant.Compared with the Model group,the contents of TLR4,Myd88,and NF-kB in the myocardium of rats in the HX group were reduced(P<0.05),and the contents of TLR4,Myd88,and NF-kB in the ileal tissue tended to be reduced,but they were not statistically significant.7.The results of community structure showed that compared with the sham group,the ratio of Firmicutes/Bacteroidetes(F/B)decreased(P<0.05),the relative abundance of Lactobacillus_reuteri decreased(P<0.05),and the relative abundance of Bacteroides and Prevotellaceae_NK3B31_group increased(P<0.01,P<0.05)in the Model group;compared with the Model group,the F/B value increased(P<0.05),and the relative abundance of Bacteroides and Prevotellaceae_NK3B31_group decreased(P<0.05)in the HX group.8.Metabolomics results showed that 16 metabolites including α-aminoadipic acid,a-tocopherol,5-methyluridine,glucosaminic acid,and erythronicacid were significantly expressed in the sham group;9 metabolites including tridecanol,L-isoleucine,5-hydroxy-hydantoin,melibiose,and sebaceous acid were significantly expressed in the Model group;and 11 metabolites including acetylornithine deacetylase,guanine,daidzein,and(r)3-hydroxybutyric acid were significantly expressed in the HX group.Metabolic pathway analysis indicated that the cAMP signaling pathway was the most significant metabolic pathway.9.Bioinformatics results showed that STAT3,PTGS2,TP53,IL6,MAPK1,VEGFA,TNF,CASP3,FOS,and MYC1 were hub genes for HXWT in the treatment of MI.Metabolite-reaction-enzyme-gene network diagram showed that arachidonic acid metabolism was the pathway of HXWT mediating gut microbiota in the treatment of MI.Conclusion:1.HXWT plays a role in the treatment of MI by down-regulating TLR4 signaling pathway expression and inhibiting the inflammatory response.2.HXWT may improve the progression of MI by regulating the balance of Firmicutes/Bacteroidetes and reducing the number of pathogenic bacteria such as Bacteroides and Prevotella.3.cAMP signaling pathway and arachidonic acid metabolism may be the action pathway of HXWT mediating gut microbiota in the treatment of MI.