| Background In the study of diabetes,the combination of advanced glycation end products(AGEs)and receptor of advanced glycation end-products on the cell surface(RAGE)plays an role of inhibiting vascular proliferation.The same results were obtained in animal experiments.However,the circulating soluble RAGE(s RAGE)can combines with AGEs by competing against RAGE on the cell surface,and blocks RAGE binding with ligand.so their interactions can promote angiogenesis and promote coronary collateral circulation(CCC)formation.At present,the research of AGEs and s RAGE is mainly limited to diabetes mellitus and diabetic animal model,no studies have been done on people and animals with non diabetes.In this study,the reason for choosing AGEs and s RAGE is the animal model used in our previous experimental animal protein chip detection is the formation of collateral circulation non diabetic animal model,and protein chip results have shown that the high expression of RAGE in plasma.It is possible that the interaction between AGEs and s RAGE is not only present in patients with diabetes mellitus,but also may be widely found in non-diabetic patients.Objective To investigate the expression and significance of AGEs and s RAGE in patients with coronary heart disease(CHD)and the formation of collateral circulation.The aim of this study is to screen out new and stronger molecular biomarkers that can promote the formation of collateral circulation,and to provide a new method and theoretical basis for the diagnosis and treatment of coronary heart disease.Methods1.Preliminary animal experiment: The rat model of myocardial ischemia was established by ligating the left anterior descending branch.Protein chip was used to detect the difference of the plasma protein expression in the collateral circulation group.To screen out the protein factors that may be related to the formation of collateral circulation.This study is mainly a verification test by expanding the sample size in clinical patients.2.Clinical specimen collection and data collection:Coronary angiography(CAG)was performed routinely.After the success of the vascular puncture,blood samples were collected and added into the EDTA-K2 anticoagulant tube.Then we selected 2ml whole blood sample,which was divided into 2 tubes,and preservate them on the low temperature-80 degree.The remaining blood samples were centrifuged After centrifugation,we chose the upper plasma,divided them into 2 tubes,and preservate them on the low temperature-80 degree.We selected the patients whose large branch of coronary artery(such as left anterior descending branch,left circumflex artery or righe coronary artery)stenosis degree is greater than 90% at least by the coronary angiography.According to the coronary collateral circulation,the patients were divided into two groups,which one was good,the other was not good.,there were 30 cases in the two groups.At the same time,coronary angiography showed that 30 cases without coronary atherosclerosis were used as control group.The general clinical data of all subjects,such as age,sex,hypertension,diabetes and medication were recorded.3.ELISA analysis:The contents of AGEs and s RAGE in plasma were detected quantitatively by double antibody sandwich enzyme-linked immunosorbent assay.,the operation strictly according to the kit instructions.4.Western Blotting detection:AGEs and s RAGE levels were detected by Western Blotting in plasma samples from the other tube.(1)Protein concentration determination:Bradford colorimetric method was used to detect the content of protein in the absorbance value at A595.(2)Protein separation:In this study,12%SDS polyacrylamide gel electrophoresis was used to separate proteins.(3)Electric transfer:This study uses water bath type electric transfer.4.Statistical analysis: SPSS 21 statistical software package was used to count the data,analysis the expression level and difference of AGE and s RAGE in the group which the collateral circulation was well formed,collateral formation was not good and in the control group.,and analyzed the influencing factors.Results1.In the animal model of myocardial ischemia induced by non diabetic collateral circulation,the high expression of RAGE was detected by protein chip assay.2.WB and ELISA verification test results showed that the expression of AGEs and s RAGE in the CCC group of good and the CCC group of bad were significantly higher than those in the control group in diabetic and non diabetic patients.For patients with diabetes,compared with the control group,The expression levels were significantly increased in the CCC group of good(AGEs:0.312±0.018 vs 0.192±0.055,s RAGE:0.244±0.103 vs0.161±0.028)and the CCC group of bad(AGEs:0.370±0.090 vs 0.192±0.055,s RAGE:0.272±0.061 vs 0.161±0.028).Similarly,for non-diabetic patients,compared with the control group,The expression levels were also significantly increased(P < 0.05)in the CCC group of good(AGEs:0.361±0.032 vs 0.167±0.021,s RAGE:0.235±0.061 vs0.133±0.013)and the CCC group of bad(AGEs:0.332±0.138 vs 0.167±0.021,s RAGE:0.223±0.055 vs 0.133±0.013).While the CCC group of good and CCC group of bad comparison,the expression of AGEs and s RAGE in diabetic and non-diabetic patients were different.In diabetic patients,the expression of AGEs and s RAGE in CCC group of good was lower than that in the CCC group of bad(AGEs:0.312±0.018 vs 0.370±0.090,s RAGE:0.244±0.103 vs 0.272±0.061),but the difference was not statistically significant(P>0.05).However,in the non diabetic patients,the expression of AGEs and s RAGE in the CCC group of good was higher than that in the CCC group of bad(AGEs:0.361±0.032 vs 0.332±0.138,s RAGE:0.235±0.061 vs 0.223±0.055),the difference was not statistically significant too(P>0.05).3.In univariate analysis,male patients,patients with hypertension,the higher the hs-CRP level,the intima-media thickness of the carotid plaque thickness,size,degree of coronary artery stenosis in comparison between the three groups,the difference was statistically significant.Conclusion1.In non-diabetic animal experiments,The protein chip showed the expression of s RAGE in the CCC group of good increased.2.The data showed that : for diabetic and non-diabetic patients,the exprseeion of AGEs and s RAGE increased significantly in the collateral circulation formation which was good or bad.It is suggested that AGEs and s RAGE may be related to the formation of collateral circulation.Subgroup analysis showed that: for the diabetic patients,the expression of AGEs and s RAGE in the plasma was negatively correlated with CCC grading.Then for the non diabetic patients,the expression of AGEs and s RAGE in the plasma was positively correlated with CCC grading.But the difference was not statistically significant.The mechanism is unknown,whether it is related to the smaller sample size,we can study in the future.3.Male patients,patients with hypertension,the higher the hs-CRP level,the intima-media thickness of the thicker,can promote the formation of CCC.Currently.they are not considered the independent factors to promote the formation of CCC,carotid artery plaque and the severity of coronary artery stenosis are the independent factors affecting the formation of CCC.4.The formation of CCC is related to the degree of coronary artery stenosis,which shows that CCC is more common in patients with severe coronary artery disease,the more severe vascular stenosis,the more likely to form CCC. |
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