Study On The Anti-atherosclerotic Mechanisms Of Immune Tolerance Induced By Hsp60

Heat shock protein 60(HSP60) is a protein which the stimulated and deadendothelial cells ofen secrete and release. Susceptible to external factors such as stress,hypertension, infection and other stimuli, inarterial endothelial cells can cause theexpression of HSP60,which identified by the dendritic cells and lymphocytes,thereby triggering the autoimmune response. In recent years the relationship betweenHSP60 andatherosclerosis has drawn increasing attention from scholars athome andabroad. Many studies have evidence that HSP60 play an important role in ASpathogenesis. HSP60 as an autoantigen can trigger the immune response ofthevascular wall, start the formation of AS and to promote its further development.This topic from a new angle to study the mechanism of oral HSP60-induced immunetolerance and anti-AS, contrast the similarities and differences between preventiongroup(oral administrating HSP60 before modeling AS) and treatment group (oraladministrating HSP60 after modeling AS) in the induction of immune tolerance,anti-atherosclerosis and improvement of the myocardial ischemic injury, and explorethe timing of administration of oral HSP60,whether it have the double effect ofprevention and treatment, provide a new strategy for the prevention and treatment ofAS and ischemic heart disease.1. Explore the specific mechanism of immune tolerance induced by oral HSP60mechanism.2. Explore the anti-atherosclerotic mechanisms of oral HSP60 1. 40 male SD rats (body weight 100±10g) were randomly divided into four groups,healthy control group (C group), atherosclerosis group (AS group), H1 group (oraladministrating HSP60 before modeling AS) and H2 group(oral administrating HSP60after modeling AS),each group have 10 rats.2. In the first part of the experiment, the content of CD4+ and CD8 + IL-10, TGF-βand regulatory T cells (Treg) in the aorta, spleen,lymph nodes and peripheral blood ofabove four groups rats were detection by immunohistochemistry, flow cytometry,ELISA to evaluate the effect of immune tolerance induced by oral administrationof HSP60.3. In the second part of the experiment, the blood lipid of rats in four groups weredetected by enzymatic colorimetric method, the aortic and myocardial pathologicalchangeS were observed by HE staining, transmission electron microscopy andimage analysis system, the intimal (CID) and vessel diameter (CD) ratios werecalculated by the image analysis system, the expression of CD36, SR-A, NF-κBand HSP60 in the the aortic root, myocardial VEGF , HIF-1αgene and proteinexpression were observed by IHC, RT-PCR and Western blot analysis.the contentof HSP60, MMP-9, hs-CRP in the serum were detected by ELISA.4. Above indicators and their correlation studies were analyzed by the statisticalmethods. The immune tolerance mechanism induced by oral HSP60 foranti-atherosclerosis were studied from the tissue morphology, protein science,genetics, serology, immunology and ultrastructure.1. The results of the first part of the experiment show that oral HSP60 can induceimmune tolerance in H1 and H2 groups, the levels of CD8suppressor T cells, CD4+CD8+Foxp3+Treg cells in peripheral blood and spleen lymphoid tissue increased, the content of TGF-β,which secreted by Treg cells and have the function of suppressingthe immune and inducing immune tolerance,and IL-10,which is able to suppress thespecific effector T cells, and other cytokines in the peripheral blood also increased.On the contrary, the above indicators of atherosclerosis group were decreased.There are positive correlation between above indicators. However, the levels ofCD4+ T cells in the H1,H2 groups are decreased,and those in AS groups areincreased. There are negative correlation between CD4 + T cells and otherindicators. In addition,the experimental results also showed that immune toleranceeffect induced in H1 group was better than that in H2 group. It suggested that theearlier taking HSP60 orally , the better immune tolerance induced by it. (P <0.05).2. The results of the second part of the experiment show that oral administrationof HSP60 does not lower blood lipids, but it can reduce the degree of arterial intimalthickening. And the sooner taking HSP60 orally, the better reducing the intimalthickening. Under the light microscope, the aortic root intima of AS group wassignificantly thicker than group C (control group), In the AS group, it was can beseen that smooth muscle cell proliferation, derangement,inflammatory cells, foamcells infiltration, muscle fiber fracture, atherosclerotic plaques, myocardial tissuealsotraumatic changes, structural disorder of cardiac muscle fibers,the muscle cell gapwidened ,interstitial edema. In the picture collected by the electron microscope, it anbe seen that myocardial mitochondrial swelling, structural of mitochondria disorder,fracture, vascular and myocardial lesions ofthe oral HSP60 group is lighter than theAS group, the group H1(prevention group) lesions lighter than the H2 group(treatment group). It suggested that the earlier the oral HSP60, the betteranti-atherosclerosis effect (P <0.05).3. From the detected results by Immunohistochemistry, RT-PCR, Western blot andELISA, it can be seen that the indicators'expression, such as the gene and proteinexpression of HSP60,SR-A,CD36,NF-κB in aorta , VEGF, HIF-1αin myocardium,and the content of HSP60 ,MMP-9 and hs-CRP in serum of AS group, were significantly higher than that in the control group. Oral HSP60 group can significantlyreduce the level of expression of each of these indicators, and the level ofexpression in the H1 group was lower than that in the H2 group. It also prompted thesooner the oralHSP60, the better anti-atherosclerosis effect.4. The correlation analysis showed that, there are positive correlation between theintimal thickness(IT)/ inner diameter (ID) ratio of aorta and the serum lipids withineach group, IT/ID ratio of aorta and the levels of HSP60 in serum and aortic, theexpression of HSP60 in aortic and its SR-A, CD36, NF-κB expression, the IT/IDratio of aorta and its SR-A, CD36, NF-κB expression, serum lipids and the HSP60 inaortic and serum. VEGF and HIF-1αexpression in the myocardium and the contentof HSP60 in aorta and serum, the levels of MMP-9, hs-CRP and the contentof HSP60 in serum, the protein expression level of aortic HSP60 and the geneexpression levels of SR-A,CD36, NF-κB, myocardial HIF-1α,and VEGF.(P <0.05).1. Through the first part of the experiment, we have come to the conclusion of the oralHSP60 can induce immune tolerance, and its mechanism may be as follows: HSP60as oral toleragen contact with gastrointestinal mucosa, acting on the gut-associatedlymphoid tissue (GALT),and stimulate CD4 +CD25+ Foxp3 +Treg cells to secreteinhibitory cytokines IL-10 and TGF-beta,produce the HSP60 antigen-specificimmune tolerance (noresponse or low response), and thus can reduce the localimmuneand inflammatory response. And more early oral administration of HSP60, the moreeasy to induce immune tolerance.2. By the second part of the experiment, we have come to the conclusion of oralHSP60 resistance to atherosclerosis, the mechanism are:the oral HSP60-inducedimmune tolerance, inhibition of the arteries immune and inflammatory response(NF-κB, hs-CRP decreased) to reduce the intimal injury and HSP60 antigen exposure(aortic HSP60 expression, serum HSP60 content decreased), reduce the degree of autoimmune response, thereby reducing the degree of intimal thickening(IT/ID ratiodecrease), inhibition of smooth muscle hyperplasia (smooth muscle scavengerreceptorSR-A gene and protein expression declined), to reduce foam cell macrophage CD36gene and protein expression decreased (with light microscopy and electronmicroscopy,it can be observed that plaques formation significantly reduced ),improving myocardial blood supply (myocardial vascular endothelial growth factor(VEGF) and hypoxia inducible factor HIF-1αgene and protein expressiondecrease,myocardial lesions alleviate), to maintain the stability of the fibrous plaques(serum matrix metalloproteinases MMP-9 content reduced, the ability ofdegradation plaque fibrous cap and arterial endometrial matrix and destruction ofplaque integrity and stability decreased).3. The study found that oral administration of HSP60 has double effect ofprevention and treatment to AS. before and after modeling AS, oral HSP60 canreduced the intimal smooth muscle proliferation and improved the stenosis. To someextent, the effect of oral HSP60 before modeling is better than that of oral HSP60after modeling, that is, the sooner the oral administration of HSP60, the better.anti-atherosclerosis effect.4.The experiments also revealed that it can not completely prevent andcureatherosclerosis if we rely solely on oral HSP60,there still exists the phenomenonof intimal and smooth muscle proliferation in oral HSP60 group,which may be just forthe HSP60-specific immune tolerance induced by oral HSP60 only inhibit immuneinflammation triggered by HSP60, and it is not very good for the immuneinflammation triggered by high blood lipids, oxidized LDL (ox-LDL).Therefore, itis suggested that the joint use of a variety of mainly toleragens (such as HSP60, ox-LDL, etc.)to induce the immune tolerance,and combined with lipid-lowering drugs(such as statin drugs) to eradicate AS pathogens , namely high blood cholesterol, inorder to achieve better effect of prevention and treatment to AS .There may evenreach the effect of radical cure for AS. I also ongoing experimental research, and look forward to for the majority of arterial atherosclerosis and resulting cardiovascularandcerebrovascular disease patients at an early date to bring the Gospel.