Effect Of Serum Amyloid P Component On Inflammation And Oxidative Stress And Its Mechanism In Macrophage

Part Ⅰ. Effect of Serum Amyloid P Component on inflammation and oxidative stress and its mechanism in macrophageBackgroundAtherosclerotic cardiovascular disease (ASCVD) is one of the diseases with the highest morbidity and mortality in the world. The basic pathological physiology of ASCVD is atherosclerosis (AS), whose genesis and development have not been clearly elucidated. The most recognized theories currently include theory of thrombosis, lipid-infiltration theory, injury-reaction theory and inflammatory theory. No matter lipid deposition or inflammation, oxidative stress was recognized as the major pathological characteristics of the formation of atherosclerotic plaque. And the lipid deposition, inflammation and oxidative stress mentioned in theories are all characteristics and promoters thorough development and progression of AS. Abnormity of blood lipid metabolic is one of the risk factors. The increase of oxidative stress would generate abundant reactive oxygen and reactive nitrogen species. These reactive species would promote the oxidative modification of low-density lipoprotein cholesterol (LDL-C). Those generated oxdized low-density lipoprotein (Ox-LDL) would be more likely to damage endothelial function, promote deposition of cholesterol under endothelium, activate migration and phagocytosis of macrophage and finally lead to result in generation of foam cells and accelerate AS.Multiple studies have demonstrated that inflammation and immune response are involved in every stage of formation and development of AS. Many inflammatory factors including interleukins (IL),interferons (IFN), tumor necrosis factor (TNF) are expressed in atheromatous plaques. And almost all cells participated in AS could secrete cytokines and were provoked to immune response by related cytokines. This is one of major causes of influence of inflammatory immune reponse on AS and an important target preventing AS.Serum amyloid P (SAP), C reactive protein (CRP) and pentraxin 3 (PTX3) belonged to pentraxin family and connected closely to inflammatory immune reponse. At first SAP was regarded as an amyloid and functioned in the development of systemic amyloidosis. As researches continued, SAP was found to be involved in innate immunity, inflammation and amyloidosis. And its effects on cardiovascular disease had been illuminated in recent years. Studies have revealed that the concentration of SAP in patients with coronary heart disease, especially with unstable angina, was significantly higher than in normal patients. However, its pathophysiological role in cardiovascular disease was not explicit.ObjectiveIt is aimed to investigate the effect of different concentration of serum amyloid P component (SAP) on inflammation and oxidative stress and explore it’s probably mechanism through observering the effect of SAP on proteins related to inflammation in RAW264.7 cells.Methods1. Cell culture and groups of ExperimentCells were cultured with DMEM added 10%fetal bovine serum (FBS) and 100U/ml penicillin-streptomycin. Before each experiment, cells were divided in four groups and recultured in six well plates with 1*106 cells each plate. After 24 hours FBS-free culture, different SAP groups were added different concentrations of SAP, while the control group was added nothing more.24 hours later, we collected samples for test.Cells were divided into four groups:1) the normal control group,2) 1.25ug/ml SAP group,3) 2.5ug/ml SAP group, and 4) 5ug/ml SAP group.2. Effect of different concentrations of SAP on inflammation and oxidative stress ofRAW264.7cell.After RAW264.7 cells were treated with different concentrations of SAP for 24 hours, cell supernatants harvested to measure IL-1β, TNF-a and nitric oxide (NO) by enzyme-linked immunosorbent assay (ELISA). Expression of intercellular adhesion molecule 1 (ICAM-1) and Fc gamma receptor (FcyR) were measured by flow cytometry. RT-PCR was used to re-measure expression of mRNA of IL-1β,TNF-a and ICAM-1.3. Effect of different concentrations of SAP on proteins related to inflammation.After RAW264.7 cells were treated with different concentrations of SAP for 24 hours, cells were treated with RIPA to extract proteins. Expression of Proteins (Spleen tyrosine kinase, Syk, extracellular signal-regulated kinase, ERK 1/2 and pERK 1/2) that related to information were detected by Western Blot.4. Statistical analysisAll data are presented as the mean ± SD. The statistical analysis was carried out by Student’s t test or one-way ANOVA coupled with Bonferroni-Dunn post hoc test which are performed using of the SPSS for windows software. P<0.05 was considered to be statistical significant.Result1. Effect of different concentration of SAP on secretions of IL-1β, TNF-α, ICAM-1 and NO in cell supernatants.SAP could concentration-dependently increase secretions of inflammatory factors, IL-1β and TNF-α, ICAM-1 and NO. Compared with control group, secretions of IL-1β in three SAP groups were all significantly increased with statistical difference. Expressions of TNF-α in SAP 2.5ug/ml group and SAP 5ug/ml group were more than both the control group and SAP 1.25ug/ml group. Expressions of ICAM-1 and NO in SAP 5ug/ml group were increased compared with both the control group and SAP 1.25ug/ml group. While secretions of inflammatory factors in other groups had no statistically significant difference, ascending trends still existed.2. Effect of different concentration of SAP on expression of mRNA of IL-1β, TNF-α and ICAM-1Compared with control group, expressions of mRNA of inflammatory factors, IL-1β, TNF-α and ICAM-1 were significantly concentration-dependently increased after adding different concentrations of SAP with statistical difference. In SAP 5ug/ml group, expressions of mRNA of these three inflammatory factors were much more than other three groups. Expressions of mRNA of IL-1β and TNF-α in SAP 2.5ug/ml group were increased compared both control and SAP 1.25ug/ml group. While expressions of mRNA of inflammatory factors in other groups had no statistically significant difference, ascending trends still existed.3. Effect of different concentrations of SAP on expressions of proteins related to inflammation.Syk was significantly increased after treated with SAP. Compared with the control group, expression of Syk was increased in all SAP groups with statistical difference. Moreover expression of Syk in SAP 2.5ug/ml and SAP 5ug/ml group was much more than SAP 1.25ug/ml group. Howerver treated with SAP, expressions of FcγR and pERK were not statistical increased or decreased compared the control group.ConclusionThis reseach observed effects of different concentrations of SAP on inflammation and oxidative stress. We found that after combined with FcγR, SAP could increase expression of Syk without impact on the expression of FcγR, and then increase secretions of inflammatory factors, IL-1β, TNF-a, ICAM-1 and NO. Thus, we suspected that SAP may promote inflammation and oxidative stress in the atherosclerosis process. However, it was still too early to make conclusions. Concrete mechanisms still needed more experiments to explore.Part II. Effects of Probucol on Restenosis after Percutaneous Coronary Intervention:A Systematic Review and Meta-AnalysisBackgroundCoronary heart disease (CHD) is the leading cause of death and disability worldwide. As the leading killer of human, prevention and treatment of CHD seems to be most important issues for us. In resent years, percutaneous coronary intervention (PCI) with stent placement gradually become the standard nonsurgical treatment for CHD, especially for patients who were suffering from severe coronary stenosis. No matter percutaneous transluminal coronary angioplasty (PTCA) or stent-implantation could significantly dredge narrow vessels, restore blood flow to the myocardium and finally reduce myocardial necrosis within a short time. This treatment benefit symptoms and prognosis of patients with CHD a lot. However, there remained a serious shortcoming that will influence long-term effect of PCI. That is restenosis after PCI. Restenosis had high incidence in patients who have undergone either PTCA (30%-50%) or stent-implantation (15%-20%). How to reduce restenosis rate after PCI is an important clinical problem that urgently need to be addressed. Probucol is a lipid-lowering drug which shared a long history. It can efficiently reduce total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C). Moreover probucol was also effective for degrading xanthoma in patients suffering familial hypercholesterolemia (FH). At the very first time, probucol was found as an industrial antioxidant which had a powerful antioxidant effect. Owing to this antioxidant effect, probucol could protect normal cells from oxidative stress which was one of leading causes of restenosis after PCI. There were many researches that investigate effect of probucol on restenosis after PCI, however, they did not come to an agreement. Thus, we conduct this meta-analysis to assess whether treatment with probucol reduced diameter restenosis in patients after PCI and explore its probably mechanism.MethodTwo researchers independently searched Electronic databases, PubMed, EMBASE, ScienceDirect and the Cochrane Library, and hand-searched conference articles, books and the reference lists of studies. Read abstracts, we screened articles that were randomized controlled trials related to probucol, CHD and restenosis after PCI. Then we read full text of these articles carefully to determine which article should be included. Our meta-analysis had two groups:1) treatment group was CHD patients who underwent PCI and regularly took probucol without other lipid-lowering drugs, and 2) control group was CHD patients undergone PCI and took only CHD routine medications without any lipid-lowering drugs. All the patients were followed for at least 3 months and conducted follow-up coronary arteriography 6 months later to evaluate lumen diameter of PCI segments. Combined two angiography findings, we assessed restenosis rate, minimal luminal diameter (MLD), late loss and major adverse cardiac events (MACE). Sensitivity analysis, subgroup analysis, funnel plot,, et al were used to verify consistency of results and evaluate selection bias. All analyses were performed with RevMan 5.2 software.Result1) Our search strategy generated 327 results and 37 articles were examined in detail. After read full text,10 studies were included in the analyses which examined a total of 859 patients.2) Probucol reduced the risk ratio of restenosis among both lesions (RR:0.59 [0.43,0.80], P= 0.0007) and among patients (RR:0.52 [0.40,0.68], P＜0.00001). An increase in MLD was observed with probucol compared with the control group (SMD= 0.45 [0.30,0.61], P＜0.00001), whereas a significant reduction in luminal late loss was observed in probucol group (SMD=-0.41 [-0.60,-0.22], P<0.0001). For the most concerned indicator, Treatment of probucol reduced incidence of MACE by 31 percent compared with standard treatment after PCI (RR= 0.69 [0.51,0.93], P= 0.01).3) We found that heterogeneity of MDL was high. Subgroup analyses found that the effect of probucol on MLD among patients who underwent PTCA appeared to be more significant upon follow-up at 6 months than the effect among patients who instead underwent stent implantation. This difference probably was the source of heterogeneity of MDL.4) No evidence of publication bias was identified based on funnel plots. According to the sensitivity analysis, results of the meta-analysis were verified consistency.ConclusionProbucol significantly reduced the restenosis and late luminal loss, increased MLD, reduced the incidence of MACE and probably increased the long-term survival rate after PCI.