The Expression Of TRPA1 In Peripheral Blood Monocytes In Patients With Coronary Heart Disease And The Relationship Associated With The Severity Of Coronary Artery Stenosis

BackgroundCoronary heart disease（CHD）is a cardiovascular disease that seriously endangers human health,and atherosclerosis is the main pathological basis.The current study suggests that oxidative stress is involved in long-term chronic inflammation of blood vessels under the action of various risk factors such as blood glucose,lipid metabolism disorders and hypertension.In the early stage of atherosclerosis,monocytes in peripheral blood adhere to the surface of vascular endothelium,then pass through the endothelial cell layer,migrate into the subendothelial space,convert to macrophages,and engulf oxidized low-density lipoprotein（ox-LDL）to form a foam cells.The formation of foam cells is a key link in the occurrence and development of atherosclerosis.Therefore,monocytes are important target cells in the process of atherosclerosis.In recent years,domestic and foreign research found that transient receptor potential channel（TRP channel）located on the cell membrane is a type of ion channel superfamily that is permeable to a variety of ions such as calcium and sodium and is widely distributed in the cardiovascular system.According to the homology of TRP sequence,the TRP ion channels in mammals can be divided into seven subfamilies:TRPC,TRPM,TRPV,TRPA,TRPML,TRPP and TRPN.Its function is closely related to the formation of atherosclerosis,including research more widely as TRPV1,part of foreign research confirmed that activation of vascular smooth muscle cells of TRPV1,reduce the cellular uptake of oxidized low density lipoprotein,reduce the formation of myogenic foam cells,ultimately preventing atherosclerosis.TRPA1 is often co-expressed with TRPV1,and the increase of Ca²⁺concentration in the cytoplasm eventually activates the TRPV1 channel that is stimulated by the sensory stimulation of TRPA1 and TRPV1 isomerization channels.So does direct activation of TRPA1 play a more important role in the development of atherosclerosis?At present,domestic and foreign studies have found that monocyte TRPAl participates in a variety of physiological and pathological processes such as body pain response,inflammatory reaction,oxidative stress and vascular tension.So,TRPA1 in monocytes involved in the formation of atherosclerosis is of great significance,its expression and the degree of coronary artery disease whether there is a relationship?At present,there are few researches on it,and its mechanism deserves our further study.ObjectiveIn this experiment,we isolated and extracted peripheral blood monocytes in patients with coronary heart disease,TRPA1 in patients with coronary heart disease and non-coronary heart disease in patients with monocyte protein expression and mRNA transcription level change,analysis of TRPA1 m RNA in different lesions vessel count group,different Gensini score group,hypertension group and non-hypertension group,DM group and non-DM group.The correlation between TRPAl mRNA expression in monocytes and Gensini score of coronary artery disease was analyzed.Materials and MethodsThis study was divided into CHD group and non-CHD group according to the results of coronary angiography.The peripheral blood monocytes were isolated and extracted by collecting peripheral blood of patients,and the peripheral blood monocytes were identified by flow cytometry.The expression of TRPAl protein in monocytes was detected by immunofluorescence and Western Blotting.The transcription level of TRPAl mRNA in monocytes was detected by RT-PCR.According to coronary angiography coronary vessel count,coronary artery disease patients were divided into single vessel disease group,double vessel disease group and multivessel disease group.Quantitative analysis of coronary artery stenosis by coronary artery disease Gensini score Coronary heart disease patients were divided into Gensini score<30 points group,30⁶0 points group and>60 points group.To analyze the difference of expression of TRPAl mRNA in monocytes between hypertensive group and non-hypertensive group,DM group and non-DM group,different vascular lesion count group and different Gensini score group.The relationship between the level of TRPA1 mRNA expression of monocytes in coronary heart disease and the Gensini score both and BNP were analyzed by Spearman correlation.Results1.There were 39 patients in the CHD group,18 men and 21 women,36 in non-CHD group,18 in male and 18 in women.There was no significant difference in gender,age,smoking,hypertension,diabetes,body mass index（BMI）,uric acid,serum creatinine,triglyceride,LDL-C and total cholesterol in CHD group and non-CHD group.2.Peripheral blood monocytes were isolated by density gradient centrifugation and identified by flow cytometry.The purity of monocytes was 86.1%.3.The expression of TRPA1 protein in monocytes was detected by Western blotting and immunofluorescence.The protein expression of TRPA1 in monocytes was higher in CHD group than that in the non-CHD group.The difference was statistically significant.4.The mRNA expression of TRPA1 in monocytes was detected by RT-PCR.The expression of TRPA1 mRNA in patients with coronary heart disease was significantly higher than that in patients of non-CHD group.The difference was statistically significant（P<0.05）.5.The level of TRPA1 mRNA expression in monocytes in Multiple vessel disease（MV）group（9.882±1.532）was significantly higher than that in Single vessel lesion（SV）group（7.005±1.135,P<0.01）.The expression of TRPA1 mRNA in Double vessel lesion（DV）group（9.790±1.1971）was significantly higher than that of SV group（7.005±1.135,P<0.01）.There was no significant difference between the MV group（9.882±1.532）and the DV group（9.790±1.1971,P=0.871）.6.The expression of TRPA1 mRNA in the Gensini score>60 points group（11.837±0.855）and the 30⁶0 points group（10.657±0.659）of monocytes were significantly higher than those in Gensini score<30 points group（8.171±1.373）,the difference was statistically significant（t=5.143,P<0.01;t=5.191,P<0.01）.The Gensini score>60 points group（11.837±0.855）was increased compared with the 30-60 points group（10.657±0.659）,and the difference was statistically significant（P<0.05）.7.The expression of TRPAl mRNA in coronary heart disease patients with DM（9.685±0.932）was significantly higher than that of non-DM group（8.982±0.756）,the difference was statistically significant（P=0.0134）.8.The expression of TRPAl mRNA in hypertensive patients with coronary heart disease（9.812±1.180）was significantly higher than that in non-hypertensive patients（8.740±1.586）,the difference was statistically significant（P=0.024）.9.The correlation between TRAP1 mRNA expression in peripheral blood monocytes and BNP in patients with coronary heart disease was analyzed by Spearman correlation analysis.The results showed a low positive correlation（r=0.342,P=0.033）.10.The correlation between the expression of TRPA1/GAPDH mRNA in peripheral blood monocytes and the Gensini score of coronary artery disease was analyzed by Spearman.The results showed a significant positive correlation between them,r=0.964,P<0.01.Conclusion1.The protein and mRNA levels of TRPA1 in monocytes in CHD patients are higher than those in non-CHD patients.With the severity of coronary artery disease,the expression of TRPA1 mRNA in monocytes increased,which can be used as a new method to predict the severity of coronary artery stenosis.2.In patients with coronary heart disease combined with risk factors such as diabetes or hypertension,the transcription level of TRPA1 mRNA in monocytes increased.3.The expression of TRPA1 mRNA in monocytes is positively correlated with BNP level in patients with coronary heart disease,which can be used as a detection index to predict the long-term prognosis of patients with coronary heart disease.