| Background and aimViral myocarditis is an inflammatory disease, in which the heart was infected by virus , but it's mechanism was not clearly understood, and lack of dependable therapeutical methods, finding drugs from Chinese traditional medicine is new way for viral myocarditis therapy.The anti-virus activity of Momordica charantia L (MCL)has been found by many researchs. Momordicin is a 28kd protein (MD28)purified from the pulp of MCL, Our prevenient primary researchs showed that: MD28 not only has a direct anti-virus activity in vitro,but also shows therapeutical effect on BALB/C CVB3 myocarditis in vivo.Aim: To find a large-scale purification method of purifying MD28 from MCL, and go deep into therapeutic effects of MD28 on CVB3 myocarditis, and to understand the mechanism from three aspects including NF-kB, inflammatory factros, apotosis.MethodsPreparing MD28 from the plup of MCL, the quality of MD28 was monitored by high performance liquid chromatography, the protect effects on cultured CVB3 inffected myocardiocyte of MD28 was measured, including PBC (percents of beat cells,PBC) , CPE(cytopathic effect,CPE), myocardiocyte related enzymes and TCID50.Precautionary and therapeutic experiment were taken on CVB3 inffected BALB/C. in the precautionary experiment, MD28 was injected 3 days before the virus injection, and terminated as the virus inoculation begining. In the therapeutic experiment of MD28, dose-effects and time-effects (MD28 was injected 30 minutes and 3 days after virus'sinfection respetively) were studied. There are 6 groups animal in the dose-effect experiment:Control of Normal (C), Control of Disease(d), Control of Momordicin(Y), High dose group(H,25mg/kg.d), Medium dose group(M, 8.3mg/kg.d), Low dose group(L, 1.79mg/kg.d). survival rate, pathologic score , Troponin-cTn, TCID50 and CVB3 mRNA were measured.The activity of NF-kB was measured by EMSA(Electrophoretic Mobility Shift Assays), Impacts of MD28 on NF-kB, TNF-alpha and iNOS in CVB3 myocarditis were measured by situ-hybridize, PCR, western blot in need.Using flowmetro ,The myocardiocyte of rat was used in measure of apoptosis at the cells level in vitro, and using microscope and TUNEL to measure apoptosis at the level of tissue ,The activity of caspase3 was measured by ELISA. Impacts on bax, bcl-2 and caspase3 of MD28 in CVB3 myocarditis were measured by situ-hybridize, PCR, western blot in need.ResultsResults at the level of cellsA pure object component was purified from MCL indicated by SDS-PAGE and reverse-high-performance-liquid-chromatography. The toxicity of MD28 is feebleness on myocariocyte(the IC50=7.86mg/ml), a distinct protective effect of MD28 on CVB3 inffected myocardiocyte was also found(ED50=0.045mg/ml ) .at the concentration of 80 , 40, 20, 10 g/ml, MD28 has distinct effects of enhancing PBC, reduction of CPE(cytopathic effect), replication of CVB3 , and at the concentration of 80, 40 g/ml, MD28 reduced LDH and AST remarkably.Results at the level of animal model(1) There were distinct dose-effect relationship(r=0.995) in therapeutic experiment, the ED50 is 0. 295mg/kg.d, and the survival rate were enhanced remarkably(d:36.67%, H:100%, M:90%, L:70%)in the H and M groups, and the TCID50, inflammatory areas , necrotic areas, were reduced markly in three therapeutic groups(H, M, L);(2)No precautionary effects were found of MD28 on CVB3 inffected BALB/C, and a time-effect relationship was found in the therapeutic experiment, the survival rate is 100% in the 30 minutes group(d=36.67%), and 53.33% in the 3days group(d=33.33%).(3)Troponin-cTn and CVB3 mRNA were remarkably reduced in the three therapeutic groups. Results of the mechanism researchsOnly a little NF-kB was activated in the C, and remarkably activated in the dat3,7,14,21d respectively(compared to C), and it's activity was remarkably inhibited in both H and L, and the disassemble of IkB can also be inhibited. iNOS and TNF-alpha were up-graded in the d, and MD28 can't inhibit iNOS, but can reduce TNF-... |
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