| Background Rheumatic heart disease(RHD)still retains high morbidity and mortality,the basic research on RHD is still important in the treatment of penicillin in the treatment of streptococcal infection.In the previous study,we compared the construction methods of various RHD animal models to obtain a more stable RHD modeling method and studied the proteomics of acute rheumatic heart valve,and created the corresponding differential protein expression profile.OBJECTIVE(1)To further improve the original modeling method,to get the human RHD pathology similar to the animal model.(2)To study the proteomicsof mitochondria in chronic RHD rats,to construct the differential protein expression profiles,and to find out the similarities and differences between the differential proteins in acute and chronic phase.To elucidate the mechanism of RHD development and find out New interventions provide the basis.METHODS 47 Lewis rats were randomized into 25 patients with chronic RHD(CRHD)and 22 normal controls(Control group).At week 1,the CRHD group injected subcutaneously with antigen I(inactivated GAS solution +complete Freund's adjuvant emulsifier)at the foot pad.The interval was 7 days and the antigen I was injected subcutaneously at the 2nd week,3 weeks,4 weeks,and 5 weeks.Control group at the same time point,the same dose,the same part of the injection of saline + complete Freund's adjuvant emulsifier.At week 6,the CRHD group was injected subcutaneously with 0.5 ml of antigen II(inactivated GAS bacteria)for 7 days until the 28 th week,29 th,30th and 31 st weeks to stop antigen stimulation.Week mold end.Control group at the same time point,the same dose,the same parts of the injection of saline.At the end of the model,rats were sacrificed and serum ASO and CRP were measured.Rat myocardium and mitral valve were stained with HE and Masson,and the incidence of myocarditis and valvular disease was calculated and the collagen fiber deposition was quantified.10 cases were randomly selected as iTRAQ quantitative subjects,and the remaining 6 cases were used as protein validation test subjects.Mitral valve tissue protein in chronic RHD rats was extracted and 10 cases of mitral valve tissue protein were mixed and analyzed by iTRAQ quantitative 2DLC-ESI-MS /MS.The expression of EMT / EndMT related proteins ?-SMA,COL18A1 and periostin were verified by Western Blot protein quantification.RESULTS(1)During the modeling,3 died in the CRHD group and 1 died in the Control group.(2)There was no significant difference in ASO and CRPbetween the CRHD group and the control group at the end of the 32 nd week(p= 0.054;p = 0.156).(3)HE staining results showed that there were 5 rats in the CRHD group with myocardium inflammatory cell infiltration.16 rats with mitral valve tissue there is chronic lymphocytes,plasma cell infiltration,collagen hyperplasia,calcium deposition and angiogenesis and so on.The proportion of myocarditis accounted for 22.7%(5/22)and the proportion of valvular disease was 72.7%(16/22).In the control group,one of the myocardium had diffuse inflammatory cell infiltration,and no inflammatory cell infiltration was found in the mitral valve Control group rats were not found in myocardial tissue or mitral valve tissue inflammatory cells.Control group myocarditis accounted for 4.8%(1/21),the proportion of valve disease accounted for 0%(0/21).(4)Masson staining showed that the staining of the control group showed pale blue and the collagen fibers had no obvious hyperplasia.The staining of CRHD group was deepened and the proliferation of collagen fibers was obvious.(5)The optical density of collagen in CRHD group was significantly higher than that in control group(p <0.01)by collagen deposition.The number of proteins was 2554,among which 167 were different proteins(77 were up-regulated and 90 were down-regulated).(6)By the pathway enrichment analysis of differential proteins,it was found that there were multiple protein uptake in the TGF-?signaling pathway,Notch signaling pathway,Wnt signaling pathway and VEGF signaling pathway related to EMT /EndMT.(7)Western blot analysis of EMT / EndMT-related proteins ?-SMA,COL18A1 and periostin,found that compared with the Control group,CRHD group the three protein expression increased,the difference was statistically significant(p <0.05).This result is consistent with the trend of iTRAQ binding to 2D LC-ESI-MS / MS quantitative proteomics analysis.CONCLUSIONS By prolonging the time of antigen stimulation and the time of non-antigen stimulation before leaving the white specimen,we can obtain the chronic RHD rat model which is similar to the pathological manifestation of human chronic RHD;The expressions of alpha-SMA,COL18A1 and periostin in EMT / EndMT-related proteins were increased in mitral flap of rats with chronic rheumatic heart disease.EMT / EndMT may be involved in the development of valvular heart disease,blocking EMT / EndMT is expected to become a new treatment of rheumatic heart disease. |
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