| Coronary atherosclerotic heart disease(CHD)is an ischemic heart disease caused by stenosis or obstruction of coronary artery lumen.Its pathological basis is the fibrous fatty lesions of coronary artery wall.Recent studies have shown that intestinal flora participates in the occurrence and development of cardiovascular diseases through multiple pathways such as regulating human immune response,lipid metabolism,and carbohydrate metabolism.The previous research of the research group showed that trimetlylamine oxide(TMAO),a metabolite of intestinal flora,can accelerate the progress of atherosclerosis vascular inflammation.The Tanyu Tongzhi Decoction has a good effect on treating hyperlipidemia and atherosclerosis by improving the level of TMAO and blood lipids,reducing vascular inflammation damage.In order to further explore the mechanism of prevention and treatment of CHD with phlegm and blood stasis syndrome,this study used a high fat and high choline diet combined with ligation of the left anterior descending coronary artery to establish an animal model of CHD with phlegm and blood stasis syndrome.Taking intestinal flora and intestinal barrier function as the entry points,the effects of phlegm and blood stasis on cardiac function,blood lipids,aortic plaque area,platelet activation markers,inflammatory factors The intervention effect of intestinal tight junction protein was analyzed.The characteristics of intestinal flora and the changes of intestinal barrier function in mice with CHD of phlegm and blood stasis syndrome were analyzed,and the mechanism of optimizing intestinal flora metabolism and preventing and treating CHD of phlegm and blood stasis syndrome was explored.Objective:1 To observe the protective effect of Tanyu Tongzhi Decoction on mice with CHD of phlegm and blood stasis syndrome.2 To observe the effect of Tanyu Tongzhi Decoction on regulating intestinal flora and intestinal barrier function in CHD with phlegm and blood stasis syndrome.Methods:Seventy-five SPF grade male ApoE-/-mice were fed a high fat and high choline diet for 12 weeks,and were randomly divided into model group,metoprolol group,low,medium,and high dose groups after ligation of the left anterior descending coronary artery at the end of the 8th week.Thirty C57BL/6J mice were fed with regular feed for 12 weeks.At the end of the 8th week,C57BL/6J mice were randomly divided into control group and sham operation group.The sham operation group received coronary artery threading without ligation.After the operation,mice in each medication group were gavaged for 4 weeks,while the other groups were gavaged with purified water for control.Echocardiography was used to compare the cardiac function of mice in each group,HE staining was used to observe the pathological changes of myocardial tissue and ileum tissue,aortic gross oil red O staining was used to measure the percentage of plaque lesion area,blood biochemical analysis was used to detect the changes in blood lipid levels of mice in each group,and enzyme linked immunosorbent assay was used to detect Platelet factor 4(PF4),P-selectin(CD62P),Interleukin-6(IL-6),Tumour necrosis factor-α(TNF-α),Lipopolysaccharide(LPS)levels and levels of Intercellular adhesion molecule-1(ICAM-1)and Vascular endothelial cell growth factor(VEGF)in aortic tissue were measured by liquid chromatography mass spectrometry.Serum TMA and TMAO levels were measured,and mouse feces were taken for 16S rRNA sequencing and analysis of intestinal flora abundance and diversity,Protein immunoblotting was used to detect the expression levels of tight junction proteins Occludin and Claudin-1 in ileum tissue.Result:1 The results of echocardiography showed that compared with the sham operation group,the left ventricular ejection fraction(LVEF),left ventricular short axis shortening(LVFS),cardiac output(CO),and left ventricular mass(LV Mass)in the model group were significantly decreased(p<0.01),Compared with the model group,the LVEF,LVFS,CO,and LV Mass of mice in the middle and high dose groups of the Tanyu Tongzhi Decoction and the metoprolol group increased significantly(p<0.05),The LVEF and LVFS of mice in the low dose group of Tanyu Tongzhi Decoction increased significantly(p<0.05),while CO and LV Mass showed an upward trend.2 The HE staining results of myocardial tissue showed that compared with the sham operation group,the myocardial infarction area of the model group mice became significantly thinner,with myocardial fibers breaking and necrosis,and a large number of inflammatory cells infiltrating;The myocardial fiber integrity,necrosis,and inflammatory infiltration in the myocardial infarction area of mice in each dose group of Tanyu Tongzhi Decoction and metoprolol group were significantly improved.3 The results of aortic gross oil red O staining showed that compared with the sham operated group,the lipid deposition in the aorta of the model group mice was significantly increased(p<0.01),Compared with the model group,the degree of atherosclerotic plaque lesion in the aorta of mice in each dose group of Tanyu Tongzhi Decoction and metoprolol group was significantly reduced(p<0.01).4 The results of serum lipid level detection showed that compared with the sham operation group,the levels of total cholesterol,triglycerides,and low density lipoprotein cholesterol in the serum of the model group mice were significantly increased(p<0.01),while the levels of high density lipoprotein cholesterol were significantly decreased(p<0.01),Compared with the model group,the levels of TC,TG and LDL-C in the serum of mice in each dose group of Tanyu Tongzhi Decoction were significantly decreased(p<0.05),while the levels of HDL-C were significantly increased(p<0.01).Only TG,LDL-C levels were significantly decreased in the metoprolol group of mice(p<0.01).5 The detection results of platelet activation markers and inflammatory factors showed that compared with the sham operation group,the model group mice had PF4,CD62P,TNF-α,The levels of IL-6,LPS and the expressions of ICAM-1 and VEGF in the aorta were significantly increased(p<0.01),Compared with the model group,mice in the middle and high dose groups of Tanyu Tongzhi Decoction have PF4,CD62P,and TNF-α,The levels of IL-6,LPS,ICAM-1,VEGF were significantly decreased(p<0.05),and the levels of PF4,TNF-α in the low dose group of Tanyu Tongzhi Decoction were significantly decreased,The levels of IL-6,LPS,ICAM-1 and VEGF decreased significantly(p<0.05),and the level of CD62P showed a downward trend,but there was no significant statistical difference.The mice in the metoprolol group had TNF-α,The levels of IL-6,LPS,ICAM-1 and VEGF decreased significantly(p<0.05).6 The results of TMA and TMAO detection showed that the serum levels of TMA and TMAO in the model group were significantly higher than those in the sham operation group(p<0.01),Compared with the model group,the levels of TMA and TMAO in serum of mice in each dose group of Tanyu Tongzhi Decoction and metoprolol group were significantly decreased(p<0.01).7 The results of intestinal flora sequencing analysis showed that compared with the sham operation group,the abundance of intestinal microflora in the model group significantly decreased,the ratio of Firmicutes to Bacteroides in the intestinal microflora in the model group increased,the abundance of Clostridium XlVa and Desulfovibrio significantly increased(p<0.05),and the abundance of Prevotella significantly decreased(p<0.01),The abundance of Alloprevotella and Lactobacillus decreased;Compared with the model group,the abundance of the high dose group with Tanyu Tongzhi Decoction increased significantly,and the distribution of the flora in the Tanyu Tongzhi Decoction group was closer to the sham operation group.The ratio of Firmicutes to Bacteroides decreased,the abundance of Clostridium_XlVa significantly decreased(p<0.05),the abundance of Prevotella significantly increased(p<0.05),and the abundance of Alloprevotella and Lactobacillus increased,but there was no statistical difference.8 The HE staining results of ileum tissue showed that compared with the sham operation group,the model group mice showed a decrease in goblet cells in ileum tissue,partial loss of crypt structures,and significant infiltration of inflammatory cells in the mucosal layer and submucosa;The number of goblet cells in the ileum of mice in both the Tanyu Tongzhi Decoction group and the metoprolol group was relatively normal,the abnormality of crypt structure was improved,and the inflammatory infiltration in the mucosa and submucosa was significantly reduced.9 The results of intestinal tight junction protein detection showed that compared with the sham operation group,the expression levels of Occludin and Claudin-1 proteins in the ileum of the model group were significantly decreased(p<0.01),Compared with the model group,the levels of Occludin and Claudin-1 protein expression in each dose group of Tanyu Tongzhi Decoction and metoprolol group significantly increased(p<0.05).Conclusion:1 ApoE-/-mice using high-fat and high choline diet combined with ligation of the left anterior descending coronary artery can lead to atherosclerotic plaque formation,decreased cardiac function,lipid metabolism disorder,platelet activation and increased levels of inflammatory factors,indicating that this method can simulate the pathological characteristics of myocardial ischemia combined with Congee,which is consistent with the pathological changes of CHD with phlegm and blood stasis syndrome.2 The levels of TMA and TMAO in mice with CHD of phlegm and blood stasis syndrome are significantly increased,the abundance of intestinal flora is significantly decreased,and the proportion of Firmicutes/Bacteroides is significantly increased,among which the abundance of pathogenic bacteria Clostridium and Desulfovibrio is increased,while the abundance of beneficial bacteria Prevotella,Alloprevotella and Lactobacillus is decreased and the changes in flora are related to TMAO levels,dyslipidemia,platelet activation Inflammatory reactions are closely related;Tanyu Tongzhi Decoction can reduce the levels of TMA and TMAO in mice,downregulate the ratio of Firmicutes to Bacteroides,increase the abundance of beneficial bacteria such as Prevotella,Alloprevotella and Lactobacillus and reduce the abundance of pathogenic bacteria such as Clostridium_XlVa.3 The process of phlegm stasis syndrome in CHD is accompanied by changes in intestinal inflammation and permeability,leading to intestinal barrier damage,Tanyu Tongzhi Decoction can alleviate the inflammatory reaction of intestinal tissue,upregulate the expression levels of intestinal tight junction proteins Occludin and Claudin-1,and protect the normal structure and function of the intestinal barrier.4 Tanyu Tongzhi Decoction can improve the cardiac function of mice with CHD of phlegm and blood stasis syndrome,alleviate myocardial tissue damage,regulate blood lipid levels,inhibit platelet activation and inflammatory reactions,delay the formation of plaques and thereby prevent the occurrence and development of CHD of phlegm and blood stasis syndrome.The above effects may be achieved by regulating the structure of intestinal flora,inhibiting the production of TMAO,reducing the inflammatory reaction of intestinal tissue and protecting the intestinal barrier. |
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