| ObjectiveCoronary heart disease(CHD)ranks first in the incidence of cardiovascular disease,and it is the primary threat to public health.Traditional Chinese medicine(TCM)hold edge in improving CHD-related symptoms,improving quality of life,and reducing the incidence of cardiovascular adverse events,which is of great significance to reduce the social burden.Studies have shown that CHD and intestinal flora are closely related,and TCM has a regulatory effect on intestinal flora,but the research on specific correlation between intestinal flora and CHD and the role of TCM is still insufficient.Therefore,the objectives of this study are:1.Systematically evaluating the clinical randomized controlled(RCT)study of TCM on the regulation of intestinal flora of CHD,and preliminarily exploring the regulatory effect of TCM on the intestinal flora of CHD;2.To use Mendelian random(MR)method,taking 211 kinds of intestinal flora as exposure factors and coronary heart disease as outcome variables,and studying the causal association effect of intestinal flora on the risk of CHD from the perspective of genetics through the whole genomics database;3.Observing the regulatory effect of Shexiang Baoxin Pills(SBP)on the intestinal flora and metabolites of rats with CHD through preliminary animal experiments,combined with genomics and metabolomics,as well as with bioinformatics and experimental research to explore the mechanism.Methods1.A systematic review of traditional Chinese medicine in regulating intestinal flora in the treatment of patients with coronary heart diseaseFormulate search strategies to comprehensively search Chinese biological journals,Wanfang Data Knowledge Service Platform,VIP Chinese Journal Service Platform,CNKI Chinese Journal Service Platform,Pubmed,Embase,Web of Science and other databases until March 2023.Literature was screened in strict accordance with established inclusion and exclusion criteria.The screening was done independently by two review authors.After preliminary screening and full-text browsing screening,relevant information of literature was extracted,and systematic evaluation of the final full-text literature was carried out.Risk of bias was assessed according to the Cochrane Risk of Bias assessment tool.Methodological information,bias and other information were assessed qualitatively for each literature.RevMan 5.3 software was used for data entry and meta-analysis of the outcome of interest.2.Association between intestinal flora and coronary heart diseaseThis part used a Mendelian randomized(MR)method,based on a publicly accessible GWAS database(http://www.ebi.ac.uk/gwas),to explore possible causal relationships between gut microbiota and coronary heart disease using the "TwoSampleMR" package in R software(version 4.0.2).Inverse variance weighted(IVW),MR-Egger regression,weighted median method,Simple mode and Weighted mode were all used for Mendelian random analysis.To control data quality,sensitivity analysis such as leave-one-out,MRPRESSO,etc.were performed.3.A systematic review of traditional Chinese medicine in regulating intestinal flora in coronary heart diseaseRats in the blank group were fed normally,and rats in the model group were injected with ISO for 3 consecutive days after 8 weeks of high-fat feeding to induce coronary heart disease.The rats successfully modeled were randomly divided into model group,statin group,SBP group and combined group.After 8 weeks of continuous administration intervention,the cardiac function of each group was evaluated by animal echocardiography,and the serum LDH,CK-MB,HDL,LDL,TC,TG and FFA were then detected.The pathological changes of myocardium and intestine in rats were observed.Changes in intestinal microbiota structure and microbiota distribution were detected by 16S r genomics,and metabolic changes in stool samples were detected by LC-MS metabolomics.The pharmacological network and the Kyoto Genome Encyclopedia(KEGG)signaling pathway enrichment analysis were applied to study the active ingredients and core target proteins of SBP.Experimental methods such as Western blot and Elisa were used to verify the potential mechanism.Results1.A systematic review of traditional Chinese medicine in regulating intestinal flora in in the treatment of patients with coronary heart disease(1)Compared with Western medicine alone,the traditional Chinese increased Bifidobacteria and Lactobacillus in patients with coronary heart disease,and the difference was statistically significant(P<0.05).(2)Several studies have reported that the traditional Chinese medicine reduces harmful bacteria such as firmicutes in patients with coronary heart disease compared with Western medicine alone,while increasing the relative abundance of beneficial bacteria such as Bacteroidetes.2.Association between intestinal flora and coronary heart disease(1)At the genomic significance level,MR analysis did not indicate the causal association of intestinal flora on coronary heart disease.(2)At relatively lenient threshold levels,the result from IVW suggests at genus level,Fusicatenibacter(OR=1.187,95%CI:1.044-1.349),and Oxalobacter(OR=1 086,95%CI:1.018-1.158)may have a positive causal relationship with the risk of coronary heart disease.At family level,Clostridiales vadin BB60 group(OR=0.888;95%CI:0.844 to 0.911)may have a negative causal relationship with the risk of coronary heart disease,and sensitivity analysis verified the robustness of the results.3.Effect and mechanism of SBP on intestinal flora of rats with coronary heart disease(1)Echocardiography showed that compared with the blank group,LVEF and LVFS of model group decreased(P<0.05).The LVEF and LVFS of the combined group the increased than the model group(P<0.05).There were no significant changes in LVEF and LVFS in the SBP group and statin group(P>0.05).(2)CK-MB and LDH of the model group were increased(P<0.01)compared with the blank group.Compared with the model group,in the SBP group and the combined group,the level of CK-MB was reduced(P<0.05),while in the combined group,the level of LDH was also reduced(P<0.05).(3)Compared with the blank group,LDL was increased(P<0.05)and TG was decreased(P<0.05)in the model group.There were no significant differences in HDL,TC and FFA levels between the groups.(4)pathological Observation:obvious cardiomyocyte damage in the model group compared with the blank group was observed in myocardial tissue,and SBP and atorvastatin calcium alone or in combination with Atorvastatin calcium could improve pathological morphological damage.Compared with the blank group,intestinal villi atrophy,incomplete structure and disordered arrangement were observed in the model group compared with the blank group.Compared with the model group,the intestine tissue morphology of rats in SBP group,statin group and combined group was more complete than that in the model group,and the damage was milder.(5)Genomics research:the structural changes at the phylum level were more obvious,and the intestinal flora of each group mainly consisted of Firmicutes,Bacteroidetes and Proteobacteria.Compared with the blank group,the relative abundance of Firmicutes,Proteobacteria were increased and Bacteroidetes decreased in the model group.Compared with the model group,the relative abundance of Firmicutes in the combined group decreased,the relative abundance of Firmicutes and Proteobacteria in the statin group decreased,and the relative abundance of Bacteroidetes in the statin group increased,tending to revert to the distribution of the blank group.Bifidobacterium(Bifidobacteriales)was the main signature species in the SBP group,while in the combined group,the Bacteroidaceae was the main signature species.(6)LS-MS/MS metabolomics:After the intervention of SBP or/and statin,and differential metabolites mainly include amino acids,peptides,fatty acyl,etc.Spearman analysis suggested correlation between intestinal flora and metabolome.(7)Network pharmacology:144 active ingredients were screened from SBPs,with the core target of KEGG enrichment analysis found that the key pathway of SBP was lipid and atherosclerotic signaling pathway,with PPARy,TRL4,NF-κB,MMP 9,TNF-α,IL-6 and other important targets involved in the pathway.(8)The Western Blot experiment showed that the expression of NF-κB protein was significantly increased in the model group compared with the blank group(P<0.05).Compared with the model group,the combined group could significantly decrease the expression of NF-κB protein,and the difference was statistically significant(P<0.05).In combined group,TLR4 protein expression was significantly reduced than the model group(P<0.05);Compared with the blank group,the ZO-1 protein decreased(P<0.05)and the expression of MMP9 protein increased(P<0.05)in the model group.compared with the model group SBP combined with statin significantly increased rat ZO-1 while reduced MMP9(P<0.05).(9)The results of ELISA showed that the IL-6 in the model group was significantly increased(P<0.01)compared with the blank group.In the statin group and the combined group IL-6 level was decreased(P<0.01)compared with the model group.Conclusion1.TCM treatment may increase intestinal probiotics such as Bifidobacteria,Lactobacillus and Bacteroidetes in patients with coronary heart disease,and reduce harmful bacteria such as Firmicutes.2.Intestinal flora may have a causal effect on CHD,Fusicatenibacter and Oxalobacter may increase the risk of CHD,while Clostridiales vadin BB60 group may be a protective factor against coronary heart disease.3.SBP can regulate the intestinal flora and its metabolites in rats with CHD,when combined with atorvastatin,could upregulate ZO-1 and downregulate MMP9 to protect intestinal barrier function then maintain intestinal homeostasis,and may inhibit the expression of TLR4/NF-κB protein,then decrease the inflammatory factor such as IL-6 in the downstream to exerting cardioprotective effects. |
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