Effect Of Nasal Tolerance To Heat Shock Protein60on Atherosclerosis And Potential Mechanism

Part Ⅰ. Effect of nasal heat shock protein60on the development and progress of atherosclerosis in ApoE-/-miceObjective:To investigate the effect of nasal HSP60on the development and progress of atherosclerosis in ApoE-/-mice, and provide basis for clinical application of antigen-induced tolerance in chronic inflammatory diseases.Methods:Six-week-old male ApoE-/-mice were randomly divided into HSP60-administered group mice and PBS-administered group as control. HSP60-and PBS-administered mice then were randomly divided into7-week-old-administered group (early group) and16-week-old-administered group (late group), respectively. All mice were nasally administered and killed at16or24weeks of age. Aortic roots from mice were immediately snap-frozen, sliced and then stained with hematoxylin and eosin (HE) and with oil red O. Finally, the plaque areas in aortic roots were measured by using software.Results:A significant decrease by up to53.7%was shown in plaque areas of aortic roots at16weeks of age when compared between HSP60-and PBS-administered group from7-week-old-administered mice (226432±29676μm2vs489250±31339μm2; P<0.01). However, for10-week-old-administered mice, no evident change in plaque areas when compared with control mice was observed at24weeks of age (845917±61180μm2vs800685±39654μm2; P>0.05).Conclusions:Nasal administration of HSP60can decrease the development of atherosclerosis in ApoE-/-mice, but not inhibit the progress of atherosclerosis. Moreover, administration of a lower dose HSP60only for a few days resulted in a prolonged atheroprotective effect for at least10weeks. Part II. Study on the potential mechanism of the atheroprotective effect of nasal heat shock protein60in ApoE-/-miceObjective:To investigate the potential mechanism by which nasal administratin of HSP60can inhibit the development of atherosclerosis in ApoE-/-mice and the effect of Treg cells and cytokine TGF-β in HSP60-induced immune tolerance.Methods:After1week high-cholesterol diet,7-week-old male ApoE-/-mice were nasally administered HSP60or PBS as control. At4th and14th day after the last nasal administration, cell proliferation assay was used to measure the number of CD4+LAP+and CD4+CD25+Foxp3+Treg cells and their propotion in CD4+cells in the spleens and CLNs from ApoE-/-mice. ELISA was applied to the detection of the levels of cytokines TGF-P, INF-y and so on in CLNS from ApoE-/-mice at4th day,14th day, and8th week after the last nasal administration. After8weeks from the last administration, the levels of biomarkers in the plaques, such as TGF-β, INF-y, CD25, Foxp3and MCP-1, were assessed by real-time PCR.Results:No marked difference was found in the proportion of CD4+CD25-LAP+T cells in CD4+cells in spleens and CLNs from ApoE-/-mice between HSP60-and PBS-administered group at4th day after the last nasal administration, but a significant increase in the proportion both in spleens and CLNs from HSP60group when compared to PBS group at14th day after administration (P<0.01). At4th day,14th day and8th week three time points, the levels of TGF-P were all higher in nasal HSP60group by comparison with PBS control group (P<.01). INF-y level was significantly decreased in HSP60-treated group as compared with PBS group at8weeks after administration (P<0.05). For real-time PCR, it was shown that the relative mRNA expression of MCP-1and IFN-γ were remarkably reduced (P<0.05) and TGF-β was observably increased (P<0.05) in plaques from HSP60group as compared to PBS control group.Conclusions:The atheroprotective effect of nasal tolerance to HSP60may be associated with the induction of Treg cells, especially CD4+LAP+T cells, which function as a regulatory role mainly depending on TGF-β secretion.