Study On The Relation Between The Effection Of Astragalus Polysaccharids On The Gene Expression Of Dendritic Cells And The Generation Of Atheroslerosis

Coronary heart disease(CHD)has been the main threaten to human health since the arrival of 21th century.The effective prevention and treatment of CHD has being the emphasis and challenge of the whole society of human beings.However,the pathogenesis of CHD has not been clearly identified so it is a great hindrance to the clinical treatment of CHD.Atherosclerosis(AS)is the pathologic basis of cardiac cerebro vascular diseases.In the past,there were mainly 3 theories,including lipidose deposition,injury repair and thrombogenesis,to explain the forming of AS.On basis of these theories,factors such as aging,hyperlipemia,hypertension,hyperglycaemia, hypercysteinemia and high blood uric acid are termed as high risks of CHD because they may induce blood vessel endothelium injury and lipidose deposition.Some progresses have been made in prevention of the genesis and advancement of AS by getting rid of or controlling of these high risks.However,people are beginning to question the effects of the preventions and treatments on these high risks for that the morbility age of CHD are younger than ever before,and the progress of AS speeds up. With the terming of ACS on acute progress stages of CHD,People re-recognized the pathologic and pathophysiologic characteristics of CHD in their developmental stages. Studies on AS found that unstable plaques are the pathological basis of ACS.The interventions of the inflammatory cells and immunocells to unstable plaques are key factors that leading to the instability and easily rupture of AS plaques.It is considered that systemic activation of the inflammatory factors and inflammatory reactions exist in ACS cases.What's more,people found that immunocells involve in all the AS developmental stages.They had proposed a new point of view that CHD is a process of chronic inflammation and a disease of autoimmunity.It is considered that inflammatory reactions and immune reactions induced by endogenous and exogenous risks are the pathological basis of AS.Dendritic cells(DCs)are the most powerful specific antigen presenting cells that play a role of presenting antigens in the immunologic process.Minor DCs may intensively activate T cells,which may initiate the specific cellular immunity reaction. Their capacities of T cells activation are as 100-1000 folds as that of macrophages. They play an important role in the induction and regulation of immunologic response. It is confirmed by recent studies that arterial wall may offer a good microenviroment to the immunological reactions.A Vascular-associated lymphoid tissue(VALT)that similar to mucous membrane-associated lymphoid tissue in respiratory tract and gastrointestinal tract,exist in the normal endarterium.Some cell masses which are comprised of immunological competent cells and antigen presenting cells distribute diffusedly in these tissues.They may monitor and screen the potential endogeneous or exogenous harmful antigens in the vascular tissues.Vascular dendritic cells(VDCs) can be found in VALT.Normally speaking,only a very small number of VDCs which in an immature form,can be found in the vessel tunica intima and tunica adventitia. Aggregation of VDCs in the affected regions increase and,they always aggregate with T cells and macrophages in the inflammatory infilitrated regions.People presume that DCs may play a role in initiating inflammatory and immunological reactions in the genesis and development of AS for that DCs co-emerge with T cells in the weak affected regions.Recent studies showed that oxidated LDL and nicotine may promote the advancement of AS by activating DCs-mediated acquired immunological reactions.This suggest that DCs may play a role in the pathogenesis of AS and that they may play an important role in the regulation of the triggering and amplification of inflammatory and immunological reactions in AS.Astragalus polysaccharids,one of the most important natural active ingredients in Astragalus,have the function of bi-directional regulation of blood sugar,promoting immunity,enhancing macrophage activity and promoting the transformation of lymphocyte,activating T cells,B cells and promoting the formation of antibodies in order to fight against various viral and bacterial diseases.In addition,the study found that APS play an important role in various diseases,for instance,APS have anti-tumor effect,but APS can not inhibit effectively the growth of tumor cells in vitro, suggesting that its anti-tumor effects of APS may be associated with immune effects. APS can reduce the incidence of type 1 diabetes of the non-obese mice significantly, and protect the the ultrastructure of pancreatic islets,restore the balance between Th1 cells and Th2 cells.APS can reduce NF-kB mRNA expression levels in the renal cortex of SD rats,enhance the expression of IkB mRNA,and can control the progress of diabetic nephropathy effectively.APS can inhibit metal-induced oxidative stress and renal ischemia-reperfusion injury,stimulate the immune regulation,inhibit glomerular mesangial cell proliferation and matrix synthesis,as well as reduced the expression ofβ1 integrin in mesangial cell.In relation with the research of cardiovascular disease,APS can reduce total cholesterol,apolipoprotein H, low-density lipoprotein level of healthy people effectively,as well as triglycerides levels in more than 50-year-old middle-aged healthy population.On the study of the hamster model of diabetic cardiomyopathy,APS exert the effect of anti-diabetic cardiomyopathy by inhibiting the chymotrypsin-Angâ…¡system.Besides,more studies have shown that astragalus polysaccharide increase the content of cAMP in platelets and inhibit platelet aggregation and thrombosis through the inhibition of platelet calmodulin and phosphodiesterase activity.Moreover,Astragalus Polysaccharides are one of the major components in Astragalus injection,and in the treatment of angina pectoris,Astragalus injection can reduce the number of angina attacks,extend the attack time interval,improve the clinical efficacy significantly. Above all,Astragalus injection intervening in the development of CHD may be related to the immune regulation function of APS.Considering that DCs are key in natural defense and acquired immune and closely related to the occurrence and development of CHD,so in the treatment of CHD,whether APS play a role in regulating immune function though exerting on DCs?After treatment with APS,how the DCs functional status in antigen-presenting, and whether affect secretion of the functional proteins and immunomodulatory molecules,and the gene expression of these proteins in DCs or not.Study on genetic changes of DCs is the key in resolving these problems.Our studies can mainly be divided into 3 portions.Peripheral blood mononuclear cell(PBMC)and serum abstracted from clinical cases are the objectives of our study.The gene expressions and functional changes of PBMC derived DCs after treatment with APS is the starting point in our study.Through immunofluorescence,flow cytometry,DNA microarray,RT-PCR and ELISA,we have focused on the relationship between the occurrence and development of AS and mature status,immune function and other gene expression differences of PBMC peripheral blood-derived DCs after APS treatment,and discussed deeply the status and the role of antigen-presenting cell function after treatment with APS in the treatment of AS.Aiming to provide an experimental evidence and target of immune therapy for the immune-mediated hypothesis in the development of AS,and review new theoretical platform and technical mean for the prevention and treatment of cardio-cerebrovascular disease,this study have the long-term economic value and important social significance.The results are as follows:1.DCs derived from peripheral blood in a mature state after APS treatment,have the ability to induce T cell proliferation.1.1 Culture and identification of DCsIn cultured 2 day,cells adhesion to the bottom of polystyrene bottle in the control group,part cells are suspended growth in medium in the experimental group, smaller,and no significant difference shape.In cultured 6 day,cells adhesion to the culture bottle bottom with round or oval in shape in control group;cells,showing suspended growth in medium,part of them aggregation in clusters,dendritic-like protrusionsin on cell surface in different quatity and shape,have the best growth status in LPS group,100mg/L group.Large number of vacuoles-like structure in the majority of the cytoplasm cell,collapsed cells,and a large number of atypical cell debris can be seen in 200mg/L APS group.In APS 100mg/L group,the phenotype of human peripheral blood PBMC derived DCs are CD1α⁺,CD80⁺,CD83^low,CD86⁺, HLA-DR⁺by flow cytometry analysis.1.2 Detection cytokine IL-12 in cultured supernatant.Because of a large number of apoptotic cells in 200mg/L,the control group,LPS group,50mg/L APS group,100mg/L APS have been chosed to detect the cytokine IL-12 in cell supernatant.In culture 6 days,the content of IL-12 have significantly statistical difference in 4 groups of cells cultured supernatant(P=0.000);compared the content of IL-12 in cells cultured supernatant of LPS group,l00mg/L APS with the control group,50mg/L APS group,there have significantly statistical difference (P=0.000),and the content of IL-12 in cells cultured supernatant in the fomer two groups significantly higher than in the latter two groups.1.3 The phenotype of peripheral blood PBMC derived DCs varied in each group.Because of a large number of apoptotic cells in 200mg/L,the control group,LPS group,50mg/L APS group,l00mg/L APS have been chosed to detect the phenotype of peripheral blood PBMC derived DCs varied in each group.There have statistical difference in CD1α,CD86 of DCs phenotype between l00mg/L APS group,LPS group and control group,50mg/L APS group(P＜0.05),and the CD 1α,CD86 of DCs phenotype in l00mg/L APS group,LPS group Showed up-regulated expression;there have statistical difference in HLA-DR of DCs phenotype between LPS group and 50mg/L APS group(P＜0.05),and the HLA-DR of DCs phenotype in 50mg/L APS group Showed up-regulated expression.1.4 Allogenic mixed lymphocytic reactions in PBMC derived DCs in each group. There have a significant difference ³H-TDR incorporation rate in 4 groups(P＜0.01).At the proportion of 1:50,1:20 of DCs mixed with T lymphocytes,there have significantly statistical difference between l00mg/L APS group,LPS group and other two groups in the rate of H-TDR incorporation(P＜0.01),and the rate of H-TDR incorporation in l00mg/L APS group,LPS group significantly higher than the other two groups;but there have no statistical difference between the control group and 50mg/L in the ³H-TDR incorporation rate(P＞0.05)2 Detection different gene expression of the peripheral blood PBMC derived DCs by gene chipThe result of gene chip detection show that,the expression gene of functional protein after APS treatment in peripheral blood DCs have seven genes increased markedly,they are CD36,IGSF6,IL27,INHA,LIPA,MAP4K3 and SOD2,and belong to antigen recognition receptors,cytokine,cell enzymes and cell signal transduction system.And 26 genes showed decreased expression,they are ADAM19, ADAR,BASP1,BTG1,CCR7,CD40LG,CST7,CXCL9,CXCR4,DCTN2,EBB, ISG15,ICAM1,IFI16,IL15,ISG20,CD207,LTA,MARCKS,MARCKSL1, NFKB1,NFKB2,PNRC1,RAC1,RELB and TNFRSF11B,and belong to cell enzymes,antigen recognition receptors,cytokine receptor,cytokine and cellular signal transduction systems.The peripheral blood PBMC after APS treatment are reduced in the expression of cell adhesion and chemotactic migration of DCs receptors and related proteins such as AD AM19,CCR7,CD40LG,CST7,CXCL9 and CXCR4;the gene expression with related to the induction of cell proliferation and activation signal transduction pathway and functional protein are also decreased, such as ADAR,BASP1,BTG1,DCTN2,ISG15,ISG20,MARCKS,MARCKSL1, NFKB1,NFKB2,PNRC1,RAC1,RELB and TNFRSF11B;and the gene expression with related to antigen-presenting and immune response related protein are decreased, such as EBI3,IFI16,IL15,CD207,and LTA.In upregulated expression genes,there have antigen-binding protein gene such as CD36,digestion and degradation of antigen related cell and lysosome protein gene such as LIPA and SOD2,as well as inhibition inflammation and immune responses protein gene expression such as IL27 and INHA.3 Detection the results of gene chip by fluorescence quantitative PCR showed that gene chip have better credibility in detection DCs gene.Application fluorescence quantitative PCR in detection of microarray gene expression of CD36 and IL-27,as well as down-regulated expression gene FIF16 showed that,compared with control group,CD36 and IL-27 gene expression in DCs group after APS treatment increased width,FIF16 gene expression decreased width is similar with gene chip detection.Through above three-part experiment,we can draw the conclusions as follows:â‘ The phenotype expression in peripheral blood DCs after APS treatment are in a mature state; â‘¡The levels IL-12 secreted in peripheral blood PBMC derived DCs after APS treatment,are significantly higher than the control group,and have the ability to induce allogenic lymphocytic proliferation;â‘¢The expression gene of functional protein after APS treatment in peripheral blood DCs have seven genes increased markedly,they are CD36,IGSF6,IL27,INHA, LIPA,MAP4K3 and SOD2,and belong to antigen recognition receptors,cytokine, cell enzymes and cell signal transduction system.And 26 genes showed significant decreased expression,they are ADAM19,ADAR,BASP1,BTG1,CCR7,CD40LG, CST7,CXCL9,CXCR4,DCTN2,EBI3,ISG15,ICAM1,IFI16,IL15,ISG20,CD207, LTA,MARCKS,MARCKSL1,NFKB1,NFKB2,PNRC1,RAC1,RELB and TNFRSF11B,and belong to cell enzymes,antigen recognition receptors,cytokine receptor,cytokine and cellular signal transduction systems.â‘£Detection the CD36 and IL-27 gene which upregulated expression,and IFI16 gene which downregulated expression in microarray by fluorescence quantitative PCR,verified the results of the gene microarray.