Investigation Of The Relationship Between Trimethylamine Oxide And Coronary Heart Disease

Background:In recent years,an ever-increasing number of studies have regarded Trimethylamine oxide(TMAO),a metabolite of intestinal flora,as a risk factor for coronary heart disease and involved in the development of the disease,but the mechanism is obscure.The aim of this study was to explore the influence of TMAO on the transport of low density lipoproteins(LDL)across the vascular endothelial cells and the relationship with the development of coronary heart disease,the stenosis severity of coronary artery and the prognosis of patients.Methods:Part1:TMAO with different concentration gradients was co-cultured with human coronary artery endothelial cells(HCAECs)for 48 h.The cell viability was detected by MTT assay,and the migration ability of HCAECs was assessed by scratch assay and Transwell assay.The distribution of red fluorescently labeled Human Dil-Low Density Lipoprotein(Human Dil-LDL)in coronary endothelial cells in control group and experimental group(TMAO group)was observed by fluorescence microscopy.The process of LDL transcellular transport was assessed according to the fluorescence intensity changes of Human Dil-LDL.The effect of TMAO on LDL transporter receptor proteins was assessed by comparing the expression of LDL receptor(LDLR)and scavenger receptor class B type Ⅰ(SR-B1)in coronary endothelial cells under basal status and after LDL stimulation by Western Blot.Part2:A total of 120 consecutive inpatients with suspected diagnosis of coronary heart disease in the Department of Cardiovascular Medicine,Dongguan Hospital,Southern Medical University,from January 2020 to September 2020 were included.74 patients with eventual definite diagnosis of coronary artery disease were deemed to be the experimental group.46 patients were considered as the control group by excluding the diagnoses of coronary heart disease.And the general clinical data of all enrolled patients were collected.The plasma TMAO of all patients were measured by Enzyme-linked immunosorbent assay(ELISA).Gensini scores were used to score coronary artery stenosis in patients with experimental group.General clinical characteristics and plasma TMAO levels were compared between the two groups.Multivariate logistic regression was used to analyze the risk factors of coronary heart disease.The ROC curve was used to assess the predictive ability of TMAO for coronary heart disease.The correlation between plasma TMAO levels and the degree of coronary artery stenosis was analyzed through Spearman correlation coefficient.The enrolled patients were followed up for 1 year,and the relationship between plasma TMAO level and prognosis of patients with coronary heart disease was analyzed by Cox regression.Results:Part1:The MTT assay suggested that TMAO at low concentration(100 μmol/L)had a pro-proliferative effect on coronary endothelial cells,and TMAO at high concentration(>1,000 μmol/L)had an inhibitory effect,and the LD50 was reached at a concentration of 100,000 μmol/L.The scratch assay indicated an increase in endothelial cell healing area in the TMAO group compared with the control group over the same time(P=0.0164).Similarly,the Transwell assay showed a significant increase in the number of endothelial cells penetrated in the TMAO group compared with the control group during the same time(P=0.0018).For the study of the effect of TMAO on trans-cellular transport of LDL,we found that LDL was granularly distributed in coronary endothelial cells,and the results suggested that the amount of LDL was reduced in the TMAO group compared with the control group(P<0.0001),but the CCK8 cell activity assay suggested that the number of cells was increased in the TMAO group compared with the control group(P=0.0001).After pretreatment with TMAO,the expression of LDL-R decreased in the TMAO+LDL group compared with the LDL group(P=0.043),while the expression of SR-B1 also decreased(P=0.0008).Part2:Plasma TMAO levels were elevated in the coronary heart disease group(P<0.05).Logistic regression analysis did not reveal a correlation between plasma TMAO and coronary artery disease.ROC curves suggested that the predictive value of plasma TMAO combined with troponin T(AUC:0.834,95%CI:0.761-0.906)was greater than that of troponin T combined with BNP(AUC:0.812,95%CI:0.734-0.889).Spearman correlation analysis showed a correlation coefficient of-0.147 between plasma TMAO levels and Gensini score(P=0.213).COX regression analysis suggested no statistically significant relationship between prognosis and TMAO in patients with coronary heart disease(P=0.747).Conclusion:Part1:(1)TMAO can promote the proliferation and migration ability of coronary endothelial cells at low concentrations of 100(μmol/L)and cause coronary endothelial cell damage at high concentrations.(2)TMAO inhibits intracellular transport of LDL in coronary endothelial cells.(3)TMAO inhibits LDL-R and SR-B1 transport pathways when stimulated in combination with LDL.Part2:(1)Patients with coronary heart disease have higher plasma TMAO levels.(2)TMAO can assist in the diagnosis of coronary heart disease with troponin T or BNP.(3)There was no significant relationship between plasma TMAO and the degree of coronary atherosclerotic stenosis and the prognosis of patients with coronary heart disease.