The Study On The Relationship Between Trimethylamine Oxide And Atherosclerosis And The Mechanism Of Nuanxinkang On Endothelial Cells

ObjectiveCoronary atherosclerotic heart disease is the main cause of death from heart disease in China.Atherosclerosis is the most important pathological basis of coronary heart disease.Despite the intervention and treatment of the risk factors of atherosclerosis and the full reconstruction of blood circulation in patients with coronary heart disease,the mortality rate of coronary heart disease is still high,which shows that there are still unknown risk factors of atherosclerosis.Trimethylamine oxide(TMAO),a metabolite of intestinal flora,is currently a hot spot in clinical research.It is generally considered as a new target for the occurrence and development of coronary artery atherosclerosis,but more clinical studies are needed to confirm it.Traditional Chinese medicine believes that the syndrome of qi deficiency and blood stasis is the most important syndrome TYPe and pathological state of coronary heart disease.Warm Heart Kang is an important research achievement of the Heart Failure Laboratory of Guangzhou University of Traditional Chinese Medicine.The research results indicate that Warm Heart Kang plays a role in the mouse model of heart failure through endothelial inflammatory response,and currently there is no research on the mechanism of coronary heart disease.This study aims to further clarify the relationship between TMAO and coronary heart disease of Qi deficiency and blood stasis syndrome,and preliminarily explore the interventional effect of Warxinkang on TMAO mediated endothelial damage.MethodsFirstly,this experiment reviewed and analyzed the relationship between qi deficiency and blood stasis syndrome and coronary artery atherosclerosis.Further more,we studied the relationship between serum TMAO,endothelial inflammatory factors and coronary artery atherosclerosis.Finally,a model of TMAO injury to umbilical vein endothelial cells was established in cell experiments to observe the effect of different doses of Wanxinkang on TMAO mediated endothelial injury.part ⅠA retrospective analysis of 354 patients with acute coronary syndrome of Qi deficiency and blood stasis syndrome hospitalized in the Department of Cardiology,Shunde Hospital,Guangzhou University of Traditional Chinese Medicine from January 2019 to December 2020 was conducted.The general data of all patients,blood lipids,carotid intima-media thickness,Crouse score,Gensini score,blood stasis score,Qi deficiency syndrome,etc.were collected,and the above indicators were statistically analyzed.part Ⅱ199 patients with acute coronary syndrome of Qi deficiency and blood stasis syndrome hospitalized in the Department of Cardiology,Shunde Hospital,Guangzhou University of Traditional Chinese Medicine from January 2021 to December 2022,and 60 healthy controls were collected.General information,blood lipids,carotid intima-media thickness,Crouse score,Gensini score,blood stasis score,and Qi deficiency syndrome were collected from all patients,and blood samples were collected to detect serum TMAO and endothelial inflammatory factor IL-1β NO,ET-1,etc.,and statistically analyze the above indicators.part ⅢThe model of umbilical vein endothelial cells injured by TMAO was established to observe the possible mechanism of Nuanxinkang improving endothelial function.The human umbilical vein blood endothelial cell line is from the Heart Failure Research Institute of Guangzhou University of Traditional Chinese Medicine.It is cultured in vitro,subcultured and resuscitated,and observed and identified under the microscope after indirect fluorescence immunoassay staining.The model of HUVECs injured by TMAO was established by CCK-8 method.and the level of IL-1β,ICAM-1 and MMP-9 in endothelial cells was detected by ELISA.ROS level of endothelial cell mitochondria was detected by immunofluorescence,and MitoTracker Green was used to detect mitochondrial quality.Extract the serum containing Nuanxinkang from mice,detect the concentration of Nuanxinkang on endothelial cells by CCK-8 method,and detect the level of IL-1β of Nuanxinkang on endothelial cell inflammation by ELISA.The effects of ICAM-1 and MMP-9,the level of mitochondrial ROS in endothelial cells were detected by immunofluorescence,the quality of mitochondria was detected by MitoTracker Green,and the internal effects of Nuanxinkang on mitochondrial ROS and TMAO were detected by SIRT3-SOD2-mtROS signal pathway.The above test indicators were statistically analyzed.Resultspart Ⅰ1.The more Qi deficiency symptoms,the higher the blood stasis score[24(19,35)vs 43(31,56)vs 66(59,74)vs 68(62,76),P<0.001].2.Blood stasis score was positively correlated with BMI(P<0.001),LDL-C.TNI,CK,CK-MB,LDH,HBDG(P<0.001),and IMT,Gensini score,and Crouse score,risk factors of atherosclerosis imaging(P<0.001).3.The more serious atherosclerosis,the higher blood stasis score[21(19,23)vs.38(31,46)vs.67(61,75),P<0.001].4.The ROC characteristic curve was used to analyze the ability of blood stasis score to identify severe atherosclerosis in ACS patients.The results showed that the best evaluation threshold value of blood stasis score to identify severe atherosclerosis was 53.5 points,at this time,the sensitivity was 91.6%,and the specificity was 95.7%(AUC:0.974,95%CI:0.959-0.988,P<0.001).5.Based on 53.5 points,it is divided into high blood stasis score and low blood stasis score.The number of smokers and BMI in patients with high blood stasis scores are higher than those with low blood stasis scores.The biochemical indicators of myocardial damage such as TNI,CK,CK-MB,LDH,and HBDH in patients with high blood stasis score were higher than those in patients with low blood stasis score.LDL-C,IMT,Gensini and Crouse scores of atherosclerosis imaging risk factors in patients with high blood stasis score were higher than those in patients with low blood stasis score.The number of Qi deficiency syndromes in patients with high blood stasis scores is more than that in patients with low blood stasis scores.6.Gensini score is positively correlated with BMI,TNI,BNP,CK,CK-MB,LDH and HBDH,biochemical markers of myocardial damage,CHOL,LDL-C and IMT,Crouse score,risk factors of atherosclerosis imaging.7.Single factor Logistic regression was used to analyze the risk factors of severe atherosclerosis.The results showed that the factors related to severe atherosclerosis were smoking,blood stasis score,TNI,CK-MB,CK,LDH,HBDH,CHOL,LDL-C,and palpitations,fatigue,spontaneous sweating,pale face,etc.with statistical significance.Then,we included the significant single factor into the multivariate logistic regression analysis.The results showed that the factors related to severe atherosclerosis were blood stasis score(OR=1.261,95%CI=1.186,1.341,P<0.001),fatigue(OR=5.214,95%CI=1.726,15.754,P<0.001),and pale face(OR=13.431,95%CI=4.050,44.541,P<0.001).part Ⅱ1.Atherosclerotic patients with coronary heart disease have higher BMI and higher myocardial injury markers BNP,CK,CK-MB,LDH and HBDH than healthy people.The atherosclerotic risk factors,LDL-C,IMT and Crouse scores of atherosclerotic imaging risk factors were also higher than those of healthy people.Inflammatory factor IL-1β The level of NO is higher in healthy individuals,but lower in healthy individuals.2.In blood lipids CHOL,LDL-C,imaging risk factors Crouse score,inflammatory factor IL-1β Level,serum TMAO level,blood stasis score,and the proportion of qi deficiency syndrome fatigue,pale face,etc.,in patients with severe atherosclerosis,the above indicator level is higher than that in patients with moderate atherosclerosis>that in patients with mild atherosclerosis.In terms of inflammatory factor NO level,patients with severe atherosclerosis<patients with moderate atherosclerosis<patients with mild atherosclerosis.Both were statistically significant.3.Serum TMAO level and BMI,blood lipids CHOL,LDL-C,TG,atherosclerosis imaging risk factors IMT,Crouse score,Gensini score,and endothelial inflammatory factor IL-1β、The level of ET-1 is positively correlated,and negatively correlated with the level of NO.The serum TMAO is positively correlated with the TCM blood stasis score.The more Qi deficiency syndromes,the higher the serum TMAO.Both were statistically significant.4.Endothelial inflammatory factor IL-1β It is positively correlated with BMI,blood lipid LDL-C,TG,and atherosclerosis imaging risk factors Gensini score,Gensini score.It is positively correlated with TCM blood stasis score.The more qi deficiency symptoms,the more IL-1β The higher the level.Both were statistically significant.5.After adjusting for confounding factors(including gender,age,BMI,diabetes,hypertension,low-density lipoprotein),TMAO has a total effect on Gensini score(β=3.990,95%CI:3.137-4.782,P<0.001)and direct effects(β=2.254,95%CI:1.437-3.166,P<0.001)。On the path of indirect effects,TMAO vs.IL-1β(β=12.228,95%CI:8.452-15.866,P<0.001)and IL-1β Integral to Gensini(β=0.142,95%CI:0.100 to 0.178,P<0.001)have significant effects,indicating that IL-1β There is a partial mediating effect on the relationship between TMAO and atherosclerosis Gensini score(β=1.736,95%CI:0.979-2.558,P<0.05)。IL-1β It explains 43.5%of the damage of TMAO on atherosclerosis.6.The ROC characteristic curve was used to analyze the ability of serum TMAO to recognize severe atherosclerosis in ACS patients.The results showed that the best evaluation threshold value of serum TMAO to recognize severe atherosclerosis was 4.2356pg/ml,at this time,the sensitivity was 91.5%,and the specificity was 75.2%(AUC:0.887,95%CI:0.841-0.933,P<0.001).7.patients with high TMAO concentration and low TMAO concentration were divided by 4.2356pg/ml.patients with high TMAO concentration had significant differences in BMI,lipid CHOL,LDL-C,TG,imaging risk factors IMT,Crouse score,Gensini score,and inflammatory factor IL-1β The levels of the above indicators in patients with high TMAO concentration are higher than those in patients with low TMAO concentration in terms of levels,ET-1 levels,etc.The NO level in patients with high TMAO concentration was lower than that in patients with low TMAO concentration.In terms of TCM symptoms,in terms of blood stasis scores and the proportion of fatigue and pale complexion in patients with Qi deficiency syndrome,the levels of the above indicators in patients with high TMAO concentration are higher than those in patients with low TMAO concentration.Both were statistically significant.part Ⅲ1.The appropriate concentration of TMAO acting on HUVEC is 600μmol/L.TAMO intervention on HUVEC has no obvious effect on cell viability for 24 hours,and cell viability decreases significantly after 48 hours.2.There is no statistical difference between the effect of different proportions of Nuanxinkang medicated serum on the activity of HUVEC cells and the control group.20%of Nuanxinkang medicated serum intervened HUVEC,and the results showed that there was no significant difference in the cell viability of endothelial cells at different time points.3.TMAO(600μmol/L)interferes with endothelial cells for 24 hours,inflammatory factor IL-1β、The concentrations of MMP-9 and ICAM-1 were significantly higher than those of normal HUVEC group(P<0.001).4.The effect of TMAO on endothelial cell inflammation may be related to ROS.The number of mitochondria in HUVEC group with TMAO intervention was significantly lower than that in normal HUVEC group(P<0.001).5.TMAO group inflammatory factor IL-1β、The levels of MCP-1,MMP-9 and mitochondrial ROS were significantly higher than those of the control group(P<0.001);However,the inflammatory factor IL-1 in Nuanxinkang+TMAO+HUVEC group β、The levels of MCP-1,MMP-9 and mitochondrial ROS were significantly lower than those of TMAO+HUVEC group(P<0.001).The results also suggested that Nuanxinkang could significantly improve the number of active mitochondria(P<0.05).6.This subject tested the effect of Nuanxinkang on the protein level of SIRT3-SOD2-mtROS signal pathway.After TMAO intervention,the expression of SIRT3 protein was significantly lower than that of the control group(P<0.001),while the intervention of Nuanxinkang made the expression of SIRT3 protein higher than that of TMAO group(P<0.001).The ratio of Ac-SOD2/SOD2 in the Warm Heart Kang group is significantly higher than that in the TMAO group(P<0.001).Because SOD2 is mainly activated by converting to Ac-SOD2,and this process is related to SIRT3,we detected the protein expression level of SOD2 and Ac-SOD2.The results showed that the ratio of Ac-SOD2/SOD2 in TMAO group was significantly lower than that in control group(P<0.001).Conclusion1.The blood stasis score may be an objective quantitative indicator of coronary artery atherosclerosis in the syndrome of qi deficiency and blood stasis.When the blood stasis score is≥53.5 points,severe atherosclerosis may occur in the coronary artery.TCM blood stasis score,fatigue and pale face are the risk factors of severe coronary atherosclerosis.2.Serum TMAO may be a risk factor for coronary heart disease.When serum TMAO≥ 4.2356pg/ml,severe atherosclerosis may occur in coronary artery.Patients with high TMAO concentration have higher blood lipids,more severe atherosclerosis,and more severe qi deficiency and blood stasis in traditional Chinese medicine.At this time,the endothelial inflammatory factor IL-1 β、ET-1 are higher,but NO is lower.3.Serum TMAO may be mediated by the endothelial inflammatory factor IL-1 βInjury of vascular endothelium,leading to atherosclerosis of vascular endothelium.4.TMAO down-regulation of SIRT3-SOD2-mtROS signaling pathway and increased inflammatory response of atherosclerotic endothelial cells may be one of its atherogenic mechanisms.5.Nuanxinkang can improve TMAO mediated endothelial damage and upregulate the SIRT3-SOD2-mtROS signaling pathway to reduce IL-1 β It may be one of the anti atherosclerotic mechanisms of the drug.