| Background Atherosclerosis is an ancient disease which is the main reason inducing cardiovascular diseases like coronary disease and miocardial infarction etc.Cardiovascular disease has currently been a leading cause of death worldwide. Stains are the preventative of coronary disease, hypertension and cerebrovascular disease,among which atorvastatin would have bioactivity under no metabolism entering into human body, and atorvastatin has the advantages of fast-effect, strong lipid-decreasing and long duration, which can effectively decrease the incidence and mortality of cardiovascular disease. Therefore, whether atorvastatin can be able to activate PPARγ to drive the differentiation from monocyte to M2 macrophage could be determined through the changes of M2 in human monocytes before and after patients’ receiving of atorvastatin treatment; moreover, its mechanism was exploited as well aiming to lay foundation for learning the curing mechanism of atorvastatin.The pathological response such as the injury of artery endothelial cells and the Lipid infiltration, etc. can have influences on the incidence and the development of atherosclerotic plaques. Besides, the related aspects of the incidence and the development of atherosclerotic plaques were the changes of oxidative stress,inflammation, blood coagulation and the immunity level whose changing could be reflected by 11 inflammatory factors: cystatin C(Cys C), homocysteine(Hcy),D-Dimer(D-D), High sensitive C-reactive protein(hs-CRP), uric acid(UC),malonyldialdehyde(MDA), interleukin 6(IL 6), fibrinogen(FIB), soluble CD40ligand(Scd40L), lipoprotein(a)(LP(a)) and Serum amyloid A(SAA). Therefore,these 11 inflammatory factorss were selected to study aiming at providing a sound theoretical basis for finding atherosclerotic plaques early and its development.Over the years, the data mining of bioinformatics has been more and more popular including data base, statistics, machine learning and artificial intelligence, etc.Therefore, after the determination of inflammatory factors related to atherosclerosis,these inflammatory factors were screened further based on Logistic regression analysis; and then their applicable value in the diagnosis of atherosclerosis were evaluated. What’s more, for the purpose of being able to assistant the doctors to diagnose and to improve their diagnosis rate, the inflammatory factor diagnostic model for early diagnosis of atherosclerosis was established based on support vector machine(SVM), BP Neural Network, Logistic regression analysis and receiver operating characteristic curve(ROC).Objective The purpose of the study is to evaluate the changes of M2 marker in human monocytes before and after patients receiving atorvastatin treatment and to determine whether atorvastatin is able to activate PPARγ to drive the differentiation from monocyte to M2 macrophage. And the deep exploitation of its mechanism is to learn atorvastatin curing mechanism well. Furthermore, the determination of inflammatory factors is aiming to provide a sound theoretical basis for finding atherosclerotic plaques early and its development, and the establishment of the inflammatory factor diagnostic model is to assistant the doctors to diagnose and to improve their diagnosis rate.Materaila and methods1. Twenty patients with coronary artery disease but without diabetes from the First Affiliated Hospital of Zhengzhou University were selected to carry out atorvastatin treatment. Collecting 10 ml peripheral vein blood on the day before patients started to receive atorvastatin treatment and two months later after they received the treatment for study. Peripheral blood mononuclear cells were collected through density gradient centrifugation on Ficoll-Hypaque and the real-time fluorescence quantitative PCR was employed to detect the level of PPARγ, CD206 and CD163 m RNA, and ELISA was adopted to determine the content of TNF-α, MCP-1,PPARγ, ERK, p38 MAPK, phospho-ERK and phospho- p38 MAPK. Finally,Graoh Pad Prim 5.0 software was used to analyze the data in the study.2. Two hundred patients with atherosclerosis and a hundred healthy people were included in the study as subjects and then their clinical data were collected and recorded detailedly. Besides, blood serum, Cys C, Hcy, hs-CRP, UA, TG, TCLDLC and HDL-C were collected and then they were determined by automatic analyzer;D-D and LP(a) were detected by immunoturbidimetry; IL-6, SAA, and Scd40 L were detected by ELISA; MDA was detected by chromatometry. Furthermore, single ROC curves of the inflammatory factors were made to evaluate their values in the diagnosis of congenital heart disease.3. Inflammatory factors were screened out to be used in the modeling based on Logistic regression analysis; then early diagnostic model for atherosclerosis was established based on ROC, SVM, and BP Neural Network.Research findings1. The investigation on the effect of atorvastatin on peripheral blood PPARγof patients suffered with atherosclerosis The expression level of M2 markers as CD206, IL-10 and CCL18 can be dramatically raised by atorvastatin while that of CD163 was just prompted slightly and showed no significant difference. Meantime, that of PPARγ on monocytes was obviously augmented after the atorvastatin treatment.As time goes on, the expression of m RNA of PPARγ increased after treatment of10 μM atorvastatin, which had reached a steady state at 12 hours to 24 hours later.The larger dose atorvastatin is, the more expression of m RNA of PPARγ are, and the differences between various doses are significant. What’s more, the level of a P2 m RNA share a same trend with that of PPARγ. 24 hours later after treatment of 10μM atorvastatin, all the level of the m RNA expression of PPARγ, the activity of PPARγ and the a P2 m RNA expression can be affected by PPARγ agonist and antagonist.As the result of in vitro test showed, atorvastatin can enhance the effect of the decreased expression of CD163 which was caused by IL-4 in M2 macrophage, and cultivate M1 macrophage with supernate cultivated M2 macrophage could restrain the expression of TNF-α and MCP-1.Atorvastatin can obviously promoted the phosphorylation of p38 MAPK while had no noticeable effect on ERK1/2. The activation and expression of PPARγ induced by atorvastatin can be obviously dose-dependent suppressed by SB203580, the p38 MAPK specific inhibitor, while there is no significant effect on suppression when MAPK/ERK specific inhibitor was added.2 The detection of atherosclerosis related inflammatory factors There’s no significant difference between the subjects’ data of two group; the level of 11 kinds of inflammatory factors in atherosclerosis group were all higher than that of control group; as the ROC curves showed, every single inflammatory factor has a certain value for atherosclerosis diagnosis, among which the highest sensitivity UA is 98, the lowest FIB is 55.5, and the highest specific UA is 99, the lowest FIB is 78,and the highest value for area under the curve(AUC) UA and SAA are 0.995, the lowest FIB is 0.721.3. The establishment of atherosclerosis diagnosis model with related inflammatory factors Six independent variables which were selected into Logistic Regression equation are as follow: Hcy, Hs-CRP, IL-6, D-D, Cys C and MDA. What the classification showed is that the forecast accuracy of the sixth step is 99%.The combination detection was performed with 6 inflammatory factors which were incorporated in Logistic Regression equation, and result showed its sensitivity is57%, specificity is 97%, AUC is 0.821; after the D-D model removed, its sensitivity is 64%, specificity is 90%, AUC is 0.828, which means all that indices were superior to that of the combination detection. Then another two combination of Hcy, Hs-CRP,MDA and IL-6, D-D, Cys C were conducted to analysis of ROC curves, and the results indicated that the sensitivity of union of Hcy, Hs-CRP, MDA is 67%,specificity is 94% and AUC is 0.869, which were worse than the result of the combination of IL-6, D-D, Cys C with 87% sensitivity, 92% specificity and 0.936 AUC.The accuracy of atherosclerosis diagnosis model based on SVM is 82.5%.The accuracy of atherosclerosis diagnosis model on the basis of BP neural network is 77.5%Conclusion1. It’s the first time to find that there’s a high expression of M2 marker in human peripheral blood circulating mononuclear cells after patients receiving atorvastatin treatment. And both in vivo tests and in vitro experiments conformed that atorvastatin can induce the expression and activation of PPAR( in human mononuclear cells, making mononuclear cells differentiate into M2 macrophage.Besides, the mediate of that differentiation of atorvastatin was p38 MAPK but not ERK1/2. Moreover, this study laid solid biological foundation for deep study on the function mechanism of atorvastatin.2. Every selected inflammatory factor had some certain diagnostic values for atherosclerosis through their ROC curves.3. The established early diagnostic models for atherosclerosis based on Logistic regression analysis, ROC combining detection, SVM, and BP Neural Network,respectively were all have certain diagnostic value, and could provide certain references for the clinical diagnosis of physicians. |
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