Proteomic Research Of Acute Rheumatic Valvulitis

Rheumatic heart disease (RHD) is an autoimmune disease with-the endoca-rdial permanent damage, which caused by the long-term and repeated attacks of rheumatic fever(RF). With the application of penicillin in preventing Group A streptococcus (GAS) infection, the incidence of RF and RHD has been declined, but it was still high in the developing countries. And we didn’t see the obvious progress about the study of its pathogenic mechanism. Although animal models of RHD have been widely applied, film forming rate is not high. And regard to the acute rheumatic heart disease, it is still in the blank. So in this study, firstly we compared and modified previous methods of animal model of RHD to find out an optimal replication method, then using the modern high-tech to explore the protein profile of acute rheumatic valvulitis(ARV). The specific research process is as follows:Part 1:A comparative study of animal model for acute and chronic rheumatic carditis in Lewis ratObjective Making comparison of the success rate of establishing the model of rheumatic heart disease about three methods to provide an optimal replication method of animal model for RHD.Methods Antigen I was a emulsifier mixed by complete freund’s adjuvant(CFA) and Group A streptococcus(GAS). Antigen II was mixed by incomplete freund’s adjuvant(IFA) and GAS. Seventy-eight female Lewis rats were randomly divided into four groups:A, B, C groups respectively had 18,18 and 24 rats, immuned with antigen I at the foot pad firstly. A week later, rats in group A and B were injected antigen I in abdominal subcutaneous 1 times a week for 4 weeks. Subsequently, rats in group A were immunized by formalin-killed and sonicated GAS, rats in group B were injected subcutaneously with antigen II. Group C was given intravenous injections of activated GAS 1 times a week for 4weeks. Rats in control group D were immunized with the same protocol outlined as treatment groups but without GAS. Respectively, at 7,12,24 weeks the rats were sacrificed 24(each group was 6). The peripheral blood rheumatoid factor (RF), anti hemolytic streptococcus antibodies (ASO), C-reactive protein (CRP) were detected. Hematoxylin-eosin (HE) and Masson staining were used to observe the pathological changes in hearts.Results 1) There were 18 survival in group C and the death rate was 25%. 2) HE staining:At 7 weeks, in group A, B, C the incidence of myocarditis was respectively 83.3%(5/6),66.7%(4/6) and 50%(3/6) and valvulitis was 100% (6/6),66.7%(4/6),100%(6/6). At 12 weeks, in group A,B,C the incidence of myocarditis was respectively 100%(6/6),50%(3/6) and 83.3(5/6) and valvulitis was 100%(6/6),66.7%(4/6),100%(6/6). At 24 weeks, in group A,B,C the incidence of myocarditis was respectively 100%(6/6),50%(3/6) and 83.3 (5/6) and valvulitis was 83.3%(5/6),50%(3/6),50%(3/6). Histopathological manifestations of group A, C was not only revealed acute damage such as cellular infiltrates as well as group B, but also the Aschoff like cells in the myocardial cells interstitial. But in group A and C there had a great degree of the inflammatory cells infiltration than group B. At 24th week:rats in group C have been detected the valve fibrosis. Masson staining of valve in RHD group at 24 weeks indicated that more collagen fibers accumulated at valves in A group than control group. None of rats in group D presented myocarditis or valvulitis.Conclusion Giving the GAS with continuous stimulation after using the mixed emulsification of CFA and GAS to immune Lewis rats for five times was a appropriate method which could provide good animal model replication for the experimental study of RHD.Part 2:Proteomic analysis of mitral valve in Lewis rat with acute rheumatic valvulitisObjective The present study was aimed to identify putative protein biomarker candidates for acute rheumatic valvulitis(ARV) and to provide the altered mitral valve protein profile for understanding the pathogenesjs of acute RHD.Methods Based on our early research we applied the method of continuous GAS stimulation on Lewis rats to duplicate the animal model of RHD. The mitral valves of rats in control group (n=10) and ARV group (n=10) were selected for proteomic analysis of ARV. The iTRAQ labeling based 2D LC-ESI-MS/MS quantitative technology was performed.Results We identified 3931 proteins in valve tissue out of which we obtained 395 differentially expressed proteins containing 176 up-regulated proteins and 119 down-regulated proteins. Changes in levels of GAPDH (6.793 times higher than the control group) and CD9 (2.63 times higher than the control group) were confirmed by Western blot or immunohistochemistry.Conclusion The differentially expressed proteins such as GAPDH, CD9 may be potential biomarkers for acute RHD. Moreover, the mitral valve protein profile may be precious data for further understanding and investigating RHD.