Study On Regulatory Effect Of Cilostazol On Maturation Of Dendritic Cells

Coronary heart disease (CHD) is the biggest threat to human health and life and has high morbidity and mortality. Atherosclerosis (AS) is the basic pathology of coronary heart disease and cerebrovascular diseases. However, its pathogenesis is still unclear. In view of its pathogenesis research has always been the difficulty and hotspot of heart medicine. In recent years it has been reported that AS is a chronic inflammation and autoimmune disease which inflammation and immunity is the intermediary and central part of AS. The core of this theory is:The activation of T lymphocytes stimulated by the atherosclerosis risk factors such as the antigen caused a series of specific cellular immunity and humoral immune responses, further activated effector cells, such as macrophages and smooth muscle cells, platelets, which resulting in dramatic increase in inflammatory products and promoting the development of atherosclerosis and plaque rupture. With the development of research, more and more evidence indicated that the inflammation and immunity run through the whole process in the occurrence and development of atherosclerosis.Dendritic cells (DCs) play an important role in a variety of inflammatory immune response. DCs originated in bone marrow CD34+pluripotent stem cells and experience three stages including progenitor cells, immature cells and mature cells. DCs are the most potent antigen-presenting cells (APCs) which responsible for acquisiting, processing, presenting antigens to T lymphocytes, thus initiating the primary and secondary immune response. DCs play a key role in the induction and regulation of immune response and its activation effect on T lymphocytes is tens to hundreds of times than other APCs such as mononuclear macrophages and B lymphocytes.In recent years, studies have found that DCs exist in normal artery wall and the numbers increased in the AS lesion which suggested it plays an important role in the development of AS. Vascular DCs are markly elevated in the normal artery that vulnerable to AS, and often gather to form clusters. DCs are clustered under larger blood eddy pressure and coexist with the activated T lymphocytes and macrophages, suggesting that DCs mediated immune mechanisms may be involved in the pathogenesis of AS in early stage. DCs are increased markly in atherosclerotic lesions particularly in the fatty streak and fibrous plaques compared with the normal arterial wall. In atherosclerotic plaques, DCs concentrated in plaque which rich in T lymphocytes and contact with T lymphocytes through its projections, rather than simply mixed together. Study has showed that atherosclerotic lesions induced by high cholesterol diet in low density lipoprotein-deficient (LDLR-/-) mice are reduced remarkably after removing CD11cDCs in arterial intimae.The maturation of DCs is the pivotal step for their function in the immune response. Activated DCs can arouse T-cell activation, further stimulating vascular inflammation and adhesion of monocytes in the atherosclerotic plaque. DCs complete their maturation process which involves the reduction of antigen processing capacity and the upregulation of adhesion molecules(CD54, CD58, CD11a/CD18, CD50), costimulatory molecules(CD40, CD80, CD86) and antigen-presenting molecules(MHCⅠ, MHCⅡ, CD1) and downregulation of endocytotic activity. Activated DCs also secret more cytokines(IL-12、IL-6、TNF-α). At this time, mature DCs can effectively present antigen to T lymphocytes and initiate the immune response. Studies have showed that alien microbes, oxidized low density lipoprotein (ox-LDL) and heat shock protein (HSP-60) can induce DCs maturation and enhance T lymphocyte response capability.Cilostazol is a selective phosphodiesterase3(PDE3) inhibitors which increase the concentration of cAMP in platelets and vascular smooth muscles through inhibiting the phosphodiseterase activity, resulting in peripheral vasodilation and inhibition of platelets aggregation. Cilostazol has been widely used to treat peripheral arterial diseases and ischemic cerebrovascular diseases. Studies have showed that cilostazol has anti-inflammatory and immunoregulation effects in addition to its anti-platelet and vasodilator effect. Cilostazol significantly decreases plaque lesion volume in low-density lipoprotein receptor-dificient mice and ApoE KO mice, reduces the inflammatory cell infiltration and inflammatory cytokines expression. In vitro experiments, cilostazol can inhibit IL-6, TNF-a and IL-1expression in lipopolysccharide (LPS) induced RAW264.7macrophage. In addition, cilostazol can inhibit the T cells proliferation and IL-17, TNF-a and IFN-y production. Cilostazol can also inhibit Th-1and Th-17differentiation and increased numbers of regulatory T cells (CD4+CD25+FoxP3+T cells, Treg cells).So cilostazol has anti-inflammatory and immunoregulation effects either in vivo or in vitro.In our experiment, we use cilostazol to intervene DCs maturation, observe the regulation effect of cilostazol on DCs maturation and investigate the mechanism preliminarily, so as to further understanding the anti-inflammatory and immunoregulation effects of cilostazol and its anti atherosclerosis mechanism, and providing certain theory basis on the clinical prevention and treatment of atherosclerosis. The paper is divided into three parts to elaborate.The first chapter:The regulation effect of cilostazol on LPS induced DC2.4 maturation.Objective:To investigate the effect of cilostazol on LPS induced DC2.4maturation.Methods:We culture DC2.4in vitro and use LPS to stimulate the maturation. We use different concentrations of cilostazol to intervene LPS induced DC2.4maturation to observe the immunophenotypic expression of CD40、CD86and MHCⅡ, endocytosis ability, secretion of cytokines(IL-6and TNF-a) and its expression of intracellular mRNA of DC2.4by means of flow cytometry、ELISA and qRT-PCR.Statistical analysis:SPSS13.00for Windows statistic software is used. All statistical analyses are expressed as mean±tandard deviation (SD) of the indicated number of experiments. Multivariate data are compared using factorial analysis and groups are compared using one-way analysis of variance or T test. Single factor data are compared using one-way analysis of variance as indicated, and are followed by a post-hoc least significant difference (LSD) test. We use Welch test and Dunnett’s T3when variances are not homogeneous. Data are evaluated at the significance level P<0.05.Results:1. The subjects are devided into four groups and experiments are repeated for three times. The variance of each groups is homogeneous(P:0.408, P:0.349and P:0.432), the results of single variance analysis are:The immunophenotypic expression of CD40、CD86and MHC Ⅱ on DC2.4have significantly different in three groups(F:129.079,83.796and80.551, P<0.001).After stimulated by LPS, the immunophenotypic expression of CD40、CD86and MHC Ⅱ on DC2.4are increased comparing with the control group (P<0.001). Cilostazol pre intervention decrease significantly the immunophenotypic expression of CD40、CD86and MHC Ⅱ on DC2.4comparing with LPS group (P<0.001) 2. The subjects are devided into four groups and experiments are repeated for three times. The result of two factorial ANOVA is:Groups with different drug treatment has a significant difference (F:10.537, P<0.001) on endocytosis function of DC2.4and different temperature has a significant difference either (F:652.762, P <0.001).There has interaction effect between the two factors (F:10.998, P<0.001). In the condition of37℃, The endocytosis ability has a significant difference in every group (F:12.437, P:0.002). After stimulated by LPS, the endocytosis ability is decreased compareing with the control group (P:0.001). Cilostazol pre intervention increase the endocytosis ability comparing with LPS group (P.0.004)3. The subjects are devided into five groups and experiments are repeated for three times. The variance of each groups is homogeneous(P:0.317and P:0.406),the results of single variance analysis are:The IL-6and TNF-α levels in cell culture supernatant are significantly different in every group(F:122.424and11.365, P<0.001and P:0.001).After stimulated by LPS, The IL-6and TNF-α levels in cell culture supernatant are increased comparing with the control group (P<0.001). We use different concentrations of cilostazol to intervene LPS induced DC2.4maturation. The concentration of10μmol/L,20μmol/L and40μmol/L cilostazol pre intervention decrease the IL-6levels (P<0.029, P<0.001and P<0.001) and concentrations of20μmol/L and40μmol/L cilostazol pre intervention decrease TNF-α levels (P:0.004and P:0.001) in cell culture supernatant comparing with LPS group, the differences are signicant. The levels of TNF-α decrease too, althrough the lOμmol/L cilostazol pre intervention doesn’t has singnificant difference comparing with LPS group (P:0.134). The IL-6and TNF-α levels decrease more obviously with the concentration of cilosatzol increasing.4. The subjects are devided into five groups and experiments are repeated for three times. The variance of each groups is homogeneous(P:0.057and P:0.213),the results of single variance analysis are:The IL-6and TNF-α intracellular mRNA expression are significantly different in every group(F:52.014and56.693, P<0.001). After stimulated by LPS, the IL-6and TNF-α intracellular mRNA expression are significantly increased comparing with the control group (P<0.001). We use different concentrations of cilostazol to intervene LPS induced DC2.4maturation. The concentration of20μmol/L and40μmol/L cilostazol pre intervention decrease significantly the IL-6and TNF-α intracellular mRNA expression comparing with LPS group, the differences are signicant (P:0.007, P<0.001and P<0.001). The levels of IL-6and TNF-α intracellular mRNA expression decrease too, althrough the10μmol/L cilostazol pre intervention doesn’t have singnificant difference comparing with LPS group (P:0.137and P:0.056). The IL-6and TNF-α intracellular mRNA expression decrease more obviously with the concentration of cilosatzol increasing.Conclusions:1. DC2.4displayed low expression of CD40、CD86and MHCⅡ and has strong endocytosis ability, it has the characteristics of immature DC cells.2. LPS can induce DC2.4maturation. After stimulated by LPS, the immunophenotypic expression of CD40、CD86and MHC Ⅱ is increased, the endocytosis ability is decreased, the IL—6and TNF—α secretion and intracellular mRNA expression is increased.3. Cilostazol can attenuate the effect of LPS on DC2.4maturation. Cilostazol pre intervention can decrease the immunophenotypic expression of CD40、CD86and MHC Ⅱ, increase the endocytosis ability, decrease the IL—6and TNF—α secretion and intracellular mRNA expression of DC2.4stimulated by LPS.The second chapter:The mechanisms of cilostazol on LPS induced DC2.4maturation.Objective:To investigate the mechanisms of cilosatzol on LPS induced DC2.4 maturation preliminarily.Methods:We add the cAMP antagonist SQ22536before cilostazol pre intervention, and determine the immunophenotypic expression of CD86and the TNF-α level in cell culture supernatant by means of flow cytometry and ELISA. We add LPS、cilostazol and IBMX (3-isobutyl-1-1-methylxanthine, a non selective PDE inhibitor) and determine the intracellular cAMP (cyclic adenosine monophosphate) concentrations in different group to observe whether the inhitory effect of cilostazol on LPS induced DC2.4maturation through the intracellular cAMP. We also use different concentrations of cilostazol to intervene LPS induced DC2.4maturation in advance and use the method of western blot to determine the activation of NF-κB (nuclear factor-kappa B). Through the experiment above, we study preliminarily the mechanism of cilostazol on LPS induced DC2.4maturation.Statistical analysis:SPSS13.00for Windows statistic software is used. All statistical analyses are expressed as mean±standard deviation (SD) of the indicated number of experiments. Data are compared using one-way analysis of variance as indicated, and are followed by a post-hoc least significant difference (LSD) test. We use Welch test and Dunnett’s T3when variance is not homogeneous. Data are evaluated at the significance level P<0.05.Results:1. The subjects are devided into four groups and experiments are repeated for three times. The variance of each groups is homogeneous(P:0.370and P:0.168), the results of single variance analysis are:The immunophenotypic expression of CD86on DC2.4and TNF-a levels in cell culture supernatant have significant difference in different groups (F:61.511and15.815, P<0.001and P:0.001). Comparing with control group, the immunophenotypic expression of CD86on DC2.4and TNF-a levels in cell culture supernatant increase after LPS stimulating (P<0.001) Cilostazol pre intervention decrease the immunophenotypic expression of CD86on DC2.4and TNF-α levels in cell culture supernatant comparing with LPS group (P<0.001and P:0.001), but the difference is not significant comparing with the SQ22536group (P:0.753and P:0.907)2. The subjects are devided into four groups and experiments are repeated for three times. The variance of each groups is homogeneous(P:0.585), the result of single variance analysis is:The intracellular cAMP concentrations have significant difference in different groups (F:6.405, P<0.001). The intracellular cAMP concentration of IBMX group increase comparing with control group (P<0.001). Comparing with control group, the intracellular cAMP concentrations of LPS group and cilostazol group have no difference (P:0.696and P:0.721)3. The subjects are devided into five groups and experiments are repeated for three times. The variances of each groups are homogeneous(P:0.238and P:0.057), the results of single variance analysis are:The expression of IκBα in cytoplasm and NF—κB P65in nucleus have significant difference in different groups (F:46.174and213.294, P<0.001).Comparing with control group, the expression of IκBα in cytoplasm decrease (P<0.001), but the expression of NF—κB P65in nucleus increase (P<0.001). Comparing with LPS group, cilostazol pre intervention with different concentrations increase the expression of IκBα in cytoplasm (P:0.012, P<0.001and P<0.001)and decrease the expression of NF—κB P65in nucleus(P:0.007, P<0.001and P<0.001). With the concentrations of cilostazol pre intervention increasing, the inhibitory effect of cilostazol on NF—κB activation is more obvious.Conclusions:1. LPS induced DC2.4maturation is through activating intracellular NF—κB.2. Cilostazol can inhibit the LPS induced activation of NF—κB in DC2.4, further inhibit LPS induced DC2.4maturation. 3. Cilostazol does not affect the cAMP levels in DC2.4and SQ22536does not affect the inhibitory effect of cilostazol on LPS induced DC2.4maturation, so the inhibitory effect of cilostazol on the activation of NF—κB in DC2.4is independent of intracellular cAMP.The third chapter:The regulation effect of cilostazol on Ox-LDL induced human peripheral blood moncyte-derived DCs maturation.Objective:To study the effect of cilostazol on Ox-LDL induced human peripheral blood moncyte-derived DCs maturation.Methods:We extract20ml perephral blood of healthy adults, through the method of Ficoll density gradient centrigugation, human peripheral blood mononuclear cells (PBMC) are isolated and are cultured with GM-CSF and IL-4to induce immature human peripheral blood mcncyte-derived DCs. We use cilostazol to intervene oxidized low—density lipoprotein (Ox-LDL)induced DCs maturation and observe the immunophenotypic expression, endocytosis ability and secretion of cytokines of DCs by means of flow cytometry and ELISA.Statistical analysis:SPSS13.00for Windows statistic software is used. All statistical analyses are expressed as mean±tandard deviation (SD) of the indicated number of experiments. Data are compared using one-way analysis of variance as indicated, and are followed by a post-hoc least significant difference (LSD) test. We use Welch test and Dunnett’s T3when variance is not homogeneous. Data are evaluated at the significance level P<0.05.Results:1. The subjects are devided into three groups and experiments are repeated for three times. The variance of each groups is homogeneous(P:0.793and P:0.612), the results of single variance analysis are:The immunophenotypic expression of CD40and CD86on DCs are significantly different in different groups (F:9.647and13.250, P:0.013and0.006). After stimulated by Ox-LDL, the immunophenotypic expression of CD40and CD86on DCs are increased comparing with the control group (P:0.005and P:0.002). Cilostazol pre intervention decrease the immunophenotypic expression of CD40and CD86on DCs comparing with Ox-LDL group (P:0.049and P:0.029)2. The subjects are devided into three groups and experiments are repeated for three times. The variance of each groups is homogeneous(P:0.733),the results of single variance analysis are:The endocytosis ability has a significant difference in different groups (F:18.941, P:0.003). After stimulated by Ox-LDL, the endocytosis ability is decreased comparing with the control group (P:0.001). Cilostazol pre intervention increase the endocytosis ability comparing with Ox-LDL group (P:0.009)3. The subjects are devided into three groups and experiments are repeated for three times. The variance of each groups is homogeneous(P:0.646and P:0.449),the results of single variance analysis are:The IL-6and TNF-a levels in cell culture supernatant are significantly different in different groups(F:39.211and25.791, P<0.001and P:0.001). After stimulated by Ox-LDL, The IL-6and TNF-α levels in cell culture supernatant are increased comparing with the control group (P<0.001) Cilostazol pre intervention decrease the IL-6and TNF-α levels in cell culture supernatant comparing with Ox-LDL group (P:0.013and P:0.046)Conclusions:1. Through the method of Ficoll density gradient centrigugation, PBMC can be isolated and successfully induced to immature DCs by culturing with GM-CSF and IL-4.The immature DCs display low expression of CD40and CD86and have strong endocytosis ability, it have the characteristics of immature DCs.2. Ox-LDL can induce DCs maturation. After stimulated by Ox-LDL, the immunophenotypic expression of CD40and CD86is increased, the endocytosis ability is decreased and the IL—6and TNF—α secretion is increased.3. Cilostazol can inhibit the maturation of DCs induced by Ox-LDL. Cilostazol pre intervention can decrease the immunophenotypic expression of CD40and CD86, increase the endocytosis ability, decrease the IL—6and TNF—α secretion of DCs stimulated by Ox-LDL.To understand comprehensively the mechanisms of cilostazol on the maturation of DCs may provide new theoretical basis for prevention and treatment of coronary heart disease.