| Objectives:To find novel compound which is useful to treat ischemic heart diseases,we compared the protective effects of 7 cysteine-containing compounds on neonatal rat cardiomyocytes(NRCs) injury induced by hypoxia/reoxygenation(H/R), including S-allyl-L-cysteine(SAC),S-ethyl-L-cysteine(SEC),S-propyl-L-cysteine (SPC),S-allylmercapto-L-cysteine(SAMC) and their analogues S-butyl-L-cysteine (SBC),S-pentyl-L-cysteine(SPEC),and S-propargyl-L-cysteine(SPRC).Furthmore, to elucidate the role of SPRC,SAC and SPC on ischemic heart of rats and to explore the involved pathways,we evaluated the pharmacological effects of three cysteine containing compounds on H2S production,apoptosis and antioxidant defences in an acute myocardial infarction(MI) rat model.Besides,we determined the therapeutical role of SPRC on chronic heart failure after myocardial infarction.This study will provide novel evidence for treatment of ischemic heart diseases.Methods:1.Methylthiazol tetrazoium(MTT) assay for cell viability,lactate dehydrogenase(LDH) release as an index of cytotoxicity and malondialdehyde(MDA) content indicating lipid peroxidation were measured.To study the mechanism, intracellular glutathione(GSH) level in NRCs,OH radical scavenging activity and activities of antioxidant enzymes including superoxide dismutase(SOD),catalase (CAT)were determined.Hoechst 33342 staining was performed to observe apoptosis. Reverse transcriptional PCR(RT-PCR) were used to detect mRNA expression.2.SD rats were subjected to left coronary artery occlusion.Infarct size and mortality were determined.Plasma H2S level and cystathionase-γ-lyase(CSE) activity in the ventricular tissues were determined spectrophotometrically.The enzymatic activities of SOD and glutathione peroxidase(Gpx),glutathione redox status and MDA content were also determined.3.The infarct size was measured by Evan's blue and TTC double staining and myocardial function reflected by blood pressure.The myocardial tissues in peri-infarct and infarct areas were used for gene and protein expression studies as well as myocardial metabolites analysis.4.Hemodynamic indexes including LVSP,LVEDP and±dp/dtmax were eassayed in each group after operation 6 weeks.The hearts and lungs were harvested to test heart weight index or lung weight index.HE staining was used to evaluate the pathological changes of heart following ischemia.Results:Part 1:Intervention with seven cysteine-containing compounds significantly protected the cardiomyocytes against H/R injury.All of the 7 compounds increased GSH level,and CAT activities.SAC restored both SODs activities and mRNA expression.SPC,SBC,SPEC and SAMC significantly preserved CuZn-SOD activity.And SEC and SPRC increased Mn-SOD activity and gene expression.SAC, SEC,SPC,SAMC and SPRC inhibit caspase-3 activity and apoptosis.These agents, except SEC,also significantly increased CSE mRNA expression and H2S concentration in culture media.Part 2:SPC,SAC and SPRC treated animals demonstrated reduced mortality and infarct size as compared to the MI vehicle control group(P＜0.05);this correlating with an increase in plasma H2S concentrations.In addition,all three compounds were found to preserve SOD and GPx activities and tissue GSH content whilst reducing the formation of the lipid peroxidation product MDA in ventricular tissues.Propargylglycine,a selective inhibitor of CSE,abolished the protective effects of each compound in the current model.Part 3:The protective effects of SPRC were confirmed by significant reduction in infarct size,improvement in cardiac function and well preserved myocardial ATP level.Interestingly,SPRC was shown to increase Bcl-2 protein expression by 1.8- fold in peri-infarct area and 3.0-fold in infarct area and decrease Bax protein expression by 0.88-fold in peri-infarct and 0.65-fold in infarct region when compared to MI rats.The mRNA expressions of Bcl-2 and Bax showed similar patterns as the protein expressions. SPRC induced CSE in both areas,especially in infarct area compared with MI rats. Moreover,Plasma H2S concentration was significantly higher in SPRC-pretreated group.These effects of SPRC were abolished by PACt Part 4:Heart weight index of SPRC-treated rats was decreased,but had no significant difference when compare with CHF vehicle rats(P＜0.05).But the lung weight index was increased when compared to Sham group,and SPRC sharply decreased the lung weight index when compare with CHF vehicle group(P＜0.05).Experimental results showed the increase of LVSP and±LVdp/dtmax in SPRC treated group.Cardiac function improved in SPRC-treated rats.SPRC preserved SOD activity and decreased MDA level with an increase in plasma H2S concentrations in the plasma.There was no significant difference of Bcl-2 protein and mRNA expression in all treated groups.But SPRC decreased Bax,Caspase-9 and Caspase-3 mRNA expression in peri-infarct and remote area.And SPRC could inhibit the expression of TGF-β1 and Collagen I,there are obvious statistical differences compared with CHF vehicle group(P＜0.05)Conclusion:1.The present study demonstrated that the cysteine-containing compounds have protective effects on NRCs H/R injury via the antioxidant and anti-apoptotic pathway,modulated by endogenous H2S.The cysteine-containing compounds may be considered as valuable agents for ischemia heart disease, especially the three carbon chain compounds,such as SPC,SAC and SPRC.2.SPC, SAC and SPRC have cardioprotective effects in MI by reducing the deleterious effects of oxidative stress and by modulating the endogenous levels of H2S and preserving the activities of antioxidant defencive enzymes like SOD.3.SPRC is efficacious in response to MI by inhibiting apoptosis.The properties are associated with the modulation of H2S signaling pathways.4.The results indicated that SPRC have the cardioprotective effects on chronic heart failure after myocardial infarction, which was showed by improved heart function,decreased apoptosis in remote and peri-infarct area,and inhibit ventricular remodeling.Endogenous H2S have partly involved in the cardioprotective effects of SPRC. |
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