Study On The Effect Of Interleukin-8on Atherosclerosis Stability And The Intervention Of Xiongshao Capsule

Coronary atherosclerotic heart disease refers to coronary stenosis or obstruction and occlusion of the lumen caused by severe coronary atherosclerosis or convulsion and thrombosis, which results in myocardial ischemia or myocardial infarction. While, inflammation goes through the whole process of coronary heart disease as the basic pathology.Recent findings showed that inflammation play an important role in the early atherosclerosis since its initial stage. Infiltration of neutrophils and monotypes caused by large numbers of inflammation factors, which can promotes the lipid deposition of endarterium and causes early pathologic of atherosclerosis. Moreover, inflammation is one of the most notability characters of vulnerable plaque, occurring in fibrous cap and adventitia. Macrophage activated in plaque cap can result in Plaque Rupture through MMPs, cell factors and other medium involved in inflammation, initiate and expand ACS. Consequently, chronic inflammation related to cardiovascular disease, as the symbol of atherosclerosis, plays a key role in the developing process of AS.Coronary heart disease (CHD) is the leading cause of death worldwide. Genetic factors and environmental factors are responsible for the development of CHD. 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), protein kinase C beta1(PKCβ1), HLA-DQB1, FOLR3, PTGDS were involved in the development of CHD with BSS. Most of them played the key role in the regulation of CHD with BSS. In this study, we observe the effects of IL-8on the development of CHD with BSS in vitro and vivo.This study is divided into two parts:literature review and experimental study.1. Review:p38MAPK signal channel and the cell activation.2. Experimental Study:Including the following four parts. Study Ⅰ:Objective:To observe the injury of ox-LDL on human umbilical vein endothelial cells (HUVECs).Method:HUVEC were incubated for different times with the same dosage ox-LDL:0h (NOR group),6h,12h,24h and48h.Results:①Morphological results showed that all groups neither except NOR group can induce HUVECs injury obviously. The cell survival rates and cell viability in these groups decreased significantly neither compared with NOR group (P<0.05, P<0.01).②Biochemical indicator shows:Compared with NOR group, the volume of ET-1emissions increased obviously and it is time-dependent from0h-12h, peak at12h, then the emissions decreased gradually. Compared with NOR group, the volume of NO emissions decreased and the lowest at24h.Compared with NOR group, the volume of IL-8、IL-1β、E-selection、ICAM-1emissions increased obviously. For IL-8, it existed in a significant time-response relationship from Oh to12h, decreased gradually after12h.Conclusions:①From0to24h, HUVECs injury by ox-LDL was increased gradually.②The intervention in short-term by ox-LDL would releases a mass of inflammation factors, and the expression level of IL-8would reach high peak in12h.Study Ⅱ:Objective:Study on the effects of IL-8on the adherence of neutrophils from human peripheral blood to human umbilical vein endothelial cells and the intervention of Xiongshao capsule.Methods:We cultivate and identify the human umbilical vein endothelial cells and separate the neutrophils from human peripheral blood, make VEC1×108/L vaccinate in96-well plates, add the appropriate subject of drugs or solvents respectively by the following:1,Blank control2,IL-8(100ng/ml)3,IL-8+high dosage of Xiongshao Capsule (XSC) group (XSCH)4, IL-8+low dosage of XSC group (XSCL)5, IL-8+Simvastatin, each set of six complex,3times’ independent experiments. Add10%corresponding serum in each group. Results:Neutrophils were identified by their characteristic morphology in microscope using Wright-Gimsa staining. HUVECs were identified to be more than99%endothelial cells by their characteristic cobblestone morphology in an inverted microscope and by immunoctyochemical demonstration of factor Ⅷ staining.Compared with blank control, the adhesive efficiency of neutrophils to HUVECs of adding the stimulating factor IL-8was increased considerably (P<0.01).Compared with model group, the adhesive efficiency of XSCL group, XSCH group and Sm group were lower (P<0.05, P<0.01), showing dose-dependent manner.Conclusion:IL-8could induce the adherence of neutrophils from human peripheral blood to human umbilical vein endothelial cells, and the Xiongshao capsule could reduce this adherence, showing dose-dependent manner.Study Ⅲ:Objective:Influence of IL-8on HUVECs and p38MAPK pathway and the intervention of Xiongshao capsule.Methods:We cultivate and identify the human umbilical vein endothelial cells and culture HUVECs in media containing different concentrations:1,Normal control group(nonnal group, NOR):cultivating HUVECs without any stimulus factors;2,IL-8(100ng/ml);3,IL-8+SB203580(SB group):5μmol/L SB203580pretreatment for30min;4,IL-8+Xiongshao capsule (XSC group). Added10%medicinal serum to Xiongshao capsule group and the same normal rats serum to blank control, model group and inhibitors group.Results:①Compared with the normal control group, IL-8model group’s cell activity reduced considerably (P<0.01); Compared with model group, IL-8antagonists (SB203580) group and Xiongshao capsule (XSC) group’s cell activity increased considerably (P<0.01).②p-p38MAPK and iNOS protein detection show:Compared with the normal control group, IL-8model group endothelial cell p-p38MAPK protein expression considerably increased (P<0.01).And IL-8model group comparison, SB group and XSC group p-p38MAPK protein expression considerably reduced (P<0.01); compared with the normal control group, IL-8model group endothelial cells iNOS protein expression considerably increased (P<0.01);with IL-8model group comparison, SB group and Xiongshao iNOS protein expression considerably reduced (P<0.01).Conclusion:IL-8can induce HUVECs dysfunction and injury obviously, the phosphorylation levels of p-p38MAPK was increased significantly. And Xiongshao capsules can reduce the phosphorylation level and protect HUVECs.Study Ⅳ:Objective:Study on the effect of IL-8on atherosclerosis stability and the intervention of Xiongshao Capsule in ApoE-/-mice.Methods:ninty8-week-old male ApoE-/-mice were randomly divided into the model group, ApoE-/-+simvastatin group (Sm group)、ApoE-/-+IL-8antagonist (reparixin group)、ApoE-/-+high dosage of Xiongshao Capsule (XSC) group (XSCH) and ApoE-/-+low dosage of XSC group (XSCL), eighteen mice in each group. And eighteen8-week-old male C57BL/6J mice were selected as the control group. Mice in the control group were put on a normal diet, and others fed with a high-fat diet for12weeks.①Deteciton of aortic histopathology. Aortic root tissues were dyed with HE to evaluate atherosclerosis plaques change.②Deteciton of hematologic indices. Levels of TC, TG, LDL-C and HDL-C were determined using standard enzymatic methods. The protein level of membrane CD11b/CD18on neutrophils in ApoE-/-mice was analyzed by flow cytometry and levels of IL-8, IL-1and sE-selectin in sera were examined by ELISA.③Detection of protein level of p38MAPK, p-p38MAPK, MMP-9and iNOS in aorta: The protein level of p38MAPK, p-p38MAPK, MMP-9and iNOS in aorta was determined by Western blot.Results:①Aortic atherosclerotic plaque in ApoE-/-mice contains a lot of lipid, macrophages and VSMCs, whereas collagen composition was reduced. Similar to Simvastatin, IL-8antagonists may ameliorate athersoclerosis by decreasing lipid level and macrophages contents and inhibiting VSMCs proliferation. ②High level of TC, TG, and LDL-C, and increased protein level of membrane CD11b/CD18on neutrophils were found in ApoE-/-mice, along with increased levels of IL-8, IL-1and sE-selectin in sera. Additionally, similar to Simvastatin, IL-8antagonists pretreatment inhibited this response in ApoE-/-mice. After XSC treatment for12weeks, levels of TC, TG, and LDL-C were decreased compared with ApoE-/-mice, along with the decreased levels of both protein level of membrane CD11b/CD18on neutrophils and inflammatory factors such as IL-8, IL-1and sE-selectin in sera. Similar to Simvastatin, XSCH inhibited this inflammation response in ApoE-/-mice.③Increased protein level of p-p38MAPK, MMP-9and iNOS in aotra were found in ApoE-/-mice compared with C57mice. Additionally, similar to Simvastatin, IL-8antagonists pretreatment inhibited protein level of MMP-9in ApoE-/-mice. Similar to Simvastatin, XSC inhibited protein level of p-p38MAPK, MMP-9and iNOS in ApoE-/-mice aotra.Conclusion:①IL-8may be involved in the development of aortic atherosclerotic plaque destabilization.②Xiongshao capsule may alleviate inflammatory reaction, stabilize atherosclerotic plaque and improve atheroslcerotic formation. The role of Xiongshao capsule in preventing atherosclerosis was related to IL-8.