The Action Of Differential Gene Interleukin-8in Coronary Heart Disease Patients With Blood Stasis Syndrome

Coronary heart disease (CHD) is the leading cause of death worldwide. Genetic factors and environmental factors are responsible for the development of CHD. The underlying mechanisms of CHD have not yet been elucidated. Previous studies have investigated the differential gene expression profiles in peripheral leukocytes from CHD patients with blood stasis syndrome (BSS) by oligonucleotide microarray technique. We have shown that the immune inflammatory response is correlated to the development of CHD and interleukin-8(IL-8), Fc receptor III A of immunoglobulin G (FcyRIIIA, also named CD16), protein kinase C beta1(PKCβ1), HLA-DQB1, FOLR3, PTGDS were involved in the development of CHD with BSS. Of them, IL-8, FcyRIIIA and PKCβ1played the key role in the regulation of CHD with BSS. In previous studies, It was shown that FcyRIIIA mediated the adhesion of monocytes to endothelial cells and affected the stability of atheromatous plaque in the way of inflammation factors. IL-8induced the activation of platelet by only or synergism with arachidonic acid and Adenosine diphosphate. FcyRIIIA and IL-8may participate in the progress of CHD with BSS in these ways.We conducted further studies to confirm the correlation of chemokine IL-8to CHD with BSS and its action in the development of CHD with BSS. We investigated the protein expression of IL-8in serum in CHD patients with BSS, CHD patients with non-BSS and the healthy control by enzyme linked immunosorbent assay. There was a significant increase of IL-8at the protein level in CHD patients with BSS. We also investigated the platelet leukocyte aggregate in whole blood in CHD patients with BSS, CHD patients with non-BSS and the healthy control by flow cytometry. There was a remarkable increase of platelet monocyte aggregation and platelet neutrophil aggregation in CHD patients with BSS. Previous study showed that IL-8induced the activation of adhesion molecules MAC-1at the membrane surface of leukocyte and MAC-1plays a role in the platelet leukocyte aggregation. We come up with hypotheses that IL-8could induce the platelet leukocyte aggregation by the activation of adhesion molecules MAC-1in the progress of CHD with BSS.In our study, we observed the level of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation by the model of IL-8induced monocytes, neutrophils and platelet in whole blood of CHD patients with BSS, CHD patients with non-BSS and the healthy control. There were marked elevations in activation of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation after inducing of IL-8in the healthy control. The level of activation of adhesion molecules MAC-1, platelet monocyte aggregation and platelet neutrophil aggregation in CHD patients with BSS was much higher than those of CHD patients with non-BSS. Meanwhile, there were abundant expression of the tissue factor on the surface of platelet-bound monocytes and platelet-bound neutrophils. Neutrophils also secreted Cathepsin G、NE and PAF, which induce the activation of platelet and have the features of coagulation activity. In addition, IL-8induced the activation of platelet directly after binding with the receptor CXCR1which expressed on the surface of platelet. Moreover, we observed inhibiting effect of activating blood circulation herbs on the above mentioned biological effect of IL-8.This study is divided into two parts:literature review and experimental research.1Literature review:Including the following two reviews:Advances in CHD with BSS in Genomics and Proteomics Research and the advances of IL-8in CHD.2Experimental research:Including the following four parts.Study I:The determination of serum IL-8and platelet leukocyte aggregation in CHD with BSS.Objective:To discuss the correlation of serum level of IL-8, the platelet monocyte aggregation and platelet neutrophil aggregation with the CHD with BSS.Methods:All cases were divided into CHD patients with BSS (53), CHD patients with non-BSS (49) and the healthy control (45). CHD patients were diagnosed according to the1999ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the2002ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least50%was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. Patients with BSS were diagnosed according to guidelines for the diagnosis of BSS. The IL-8in serum was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by enzyme linked immunosorbent assay. The platelet monocyte aggregation, platelet neutrophil aggregation and the expression of GPⅡb/Ⅲa at the surface of platelet were determined in whole blood by flow cytometry.Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, medical history and herbal therapy beween CHD patients with BSS and CHD patients with non-BSS.①There was a significant increase of IL-8in serum in CHD, compared to the healthy control (47.91±2.93). Additionally, the level of IL-8in serum in CHD patients with BSS (72.58±4.12) was higher than that in CHD patients with non-BSS (58.72±3.90)②The expression of GP Ⅱb/Ⅲa at surface of platelet was much increased in CHD patients with BSS (19.68±0.98) than that in CHD patients with non-BSS (9.97±0.85). However, the expression of GP Ⅱb/Ⅲa at surface of platelet had raising trend in CHD patients with non-BSS, compared to the healthy control.③There were significant increases of platelet monocyte aggregation and platelet neutrophil aggregation in CHD that those in the healthy control. Additionally, the platelet monocyte aggregation and platelet neutrophil aggregation increased remarkably in CHD patients with BSS(46.91±2.66) than those in CHD patients with non-BSS(31.56±3.17).Conclusion:The serum level of IL-8, the platelet monocyte aggregation and platelet neutrophil aggregation were markedly correlated to the CHD with BSS.Study II:The effect of IL-8on the expression of adhesion molecules MAC-1on monocytes and neutrophils, and the expression of tissue factor at the surface on platelet-bound monocytes and platelet-bound neutrophils.Objective:To study the active mechanism of IL-8induced platelet leukocyte aggregation and the activation of IL-8in the development of CHD with BSS.Methods:CHD patients were diagnosed according to the1999ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the2002ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least50%was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. All cases were divided into CHD patients with BSS, CHD patients with non-BSSand the healthy control,15in each group. Taken6ml blood and divived into four groups:CON,35μl pure water, IL-8group,100ng/ml IL-8, Reparixin group,100ng/ml IL-8+1μM Reparixin, fMLP group. Then, these were incubating at37℃for20min and added0.5%paraformaldehyde. After30min standing, the adhesion molecules MAC-1, platelet monocyte aggregation, platelet neutrophil aggregation and the expression of tissue factor at its surface.Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, medical history and herbal therapy beween CHD patients with BSS and CHD patients with non-BSS.①The expression of adhesion molecules MAC-1at monocytes (8.26±2.23) and neutrophils(5.93±2.83(induced by chemokine IL-8markedly increased than that in CON group in the healthy control. After addition of Reparixin which is a non-selective antagonist to IL-8receptors, the expression of adhesion molecules MAC-1at monocytes (3.08±2.08) and neutrophils (4.9±1.79) significantly decreased in Reparixin group in the healthy control.②The expression of adhesion molecules MAC-1at monocytes and neutrophils in IL-8group in CHD remarkably increased than those in the healthy control. Additionally, the expression of adhesion molecules MAC-1at monocytes and neutrophils in IL-8group in CHD patients with BSS significantly increased than those in CHD patients with non-BSS (P<0.05).③The platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group increased remarkably than those in CON group in the healthy control. After addition of Reparixin, the platelet monocyte aggregation and the platelet neutrophil aggregation decreased markedly than those in Reparixin group in the healthy control.④The platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group in CHD remarkably increased than those in the healthy control. Additionally, the platelet monocyte aggregation and the platelet neutrophil aggregation in IL-8group in CHD patients with BSS significantly increased than those in CHD patients with non-BSS (P<0.05).⑤The expression of tissue factor at the surface of platelet-bound monocytes and platelet-bound neutrophils increased significantly in IL-8group in CHD patients with BSS than those at surface of platelet (P<0.01). Additionally, the expression of tissue factor at the surface of platelet-bound monocytes and platelet-bound neutrophils in IL-8group in CHD patients with BSS increased markedly than those in CHD patients with non-BSS (P<0.05)Conclusion:The chemokine IL-8may participate in the development of CHD with BSS in the way of inducing the activation of adhesion molecules MAC-1at the surface monocyte and neutrophil, and the formation of platelet monocyte aggregation and platelet neutrophil aggregation.Study III:The study on expression of IL-8receptor CXCR1at the membrane of platelet and the role of CXCR1in the activation of platelet.Objective:To study the effect of IL-8on the activation of platelet.Methods:Human blood platelet from5healthy volunteers was isolated by gradient centrifugation and was purified by the filter which can deplete the leukocytes. The mRNA expression of chemokine IL-8was detected in the way of Real-time RT-PCR. Human blood from15healthy volunteers was prepared for platelet rich plasma (PRP) and platelet poor plasma(PPP). The platelet aggregation was determined by turbidimetry and the expression of membrane protein GPⅡb/Ⅲa at the platelet by the flow cytometry.Results:①The relative expression of CXCR1mRNA was between1.22-2.37. The purification of RNA was in accord with experiment requirement of Real-Time RT-PCR. ②IL-8induced the activation of platelet in different concentrations in dose-dependent manner. Reparixin decreased the platelet aggregation and the expression of membrane protein GP II b/IIIa at the platelet, which reverse the effect of CXCRl in the activation of platelet.Conclusion:①One of chemokine IL-8receptor CXCR1expressed moderately at the platelet.②IL-8induced the activation of platelet directly by binding to the CXCR1at the the platelet.Study IV:Effects of IL-8on the secretion of peripheral blood neutrophils and the intervention effect of active blood herbs on their secretions.Objective:To study the effects of IL-8on the secretion of peripheral blood neutrophils and the intervention effect of active blood herbs on their secretions.Methods:Human blood neutrophils was isolated by gradient centrifugation and was cultured in RPMI1640medium for2hours, according to groups as follows:CON group:35μl RPMI1640medium, IL-8group:100ng/ml IL-8, Reparixin group:100ng/ml IL-8+25μl Reparixin, fMLP group:1μMfMLP, Ligustrazine group:100ng/ml IL-8+20μl ligustrazine, Paeoniflorin group:100ng/ml IL-8+20μ plaeoniflorin. After the samples were treated as mentioned above, the human blood neutrophils were cultured for30min and collected the cell culture supernatant. The concentrations of Cathepsin G, NE and PAF were detected by enzyme linked immunosorbent assay.Results:①The secretion of Cathepsin G and NE from human blood neutrophils induced by recombinant human cytokines IL-8increased remarkably than those in CON group(P<0.01).②The secretion of PAF from human blood neutrophils induced by recombinant human cytokines IL-8increased significantly than that in CON group and decreased markedly in Reparixin group than that in IL-8group.③The secretion of Cathepsin G and NE from human blood neutrophils in Ligustrazine group decreased significantly than those in IL-8group (P<0.05). The secretion of PAF from human blood neutrophils in Paeoniflorin group decreased remarkably than that in IL-8group (P<0.05)Conclusion:①IL-8induced the secretion of Cathepsin G, NE and PAF from human blood neutrophils, which induce the activation of platelet and have procoagulant activity.②The active blood herbs reverse the effects of IL-8on the secretion of Cathepsin G, NE and PAF from human blood neutrophils, which was similar to activation of receptor antagonist.