The Protective Effect And Mechanism Research Of Hydrogen Sulfide Postconditioning On Adult Rat Cardiomyocytes Underwent Hypoxia/Reoxygenation

Objective:To explore the effects of hydrogen sulfide(H2S) postconditioning on adult rat cardiomyocytes underwent ischemia/reoxygenation model and to found the protective mechanisms of H2 S postconditioning.Methods: 1. Adult rat cardiomyocytes were isolated and the same hatch cells were divided into 4 groups. The cells in normal group(group N) were cultured in normal condition for 120 min. The cells of ischemcia/reoxygenation group(group IR) were put in hypoxic chamber for 60 min followed by reoxygenation for 60 min.In ischemia postconditioning group(group IPTC) were executed that was consisted of 3 cycles of 3 min reoxygenation /3 min hypoxia before reoxygenation.In H2 S postconditioning group(group S), 10μmol/L Na HS were administrated in the culture medium for 2 min followed by reoxygenation. At the end of reoxygenation, F-actin/G-actin was detected with laser scanning confocal microscopy and the level of p-p38 MAPK was detected with Western-blot. 2. The same hatch cells were divided into 8 groups. Group N, group IR, group IPTC and group S were treated samely with the part 1. Before hypoxia, cytochalasin D(1μg/m L) was added in the medium for 60 min in all 4 groups. At the end of treatment, intracellular calcium ion concentration(Ca2+) and p H value were detected with laser scanning confocal microscopy.Results: 1. The fluorescence intensity of F-actin/G-actin: the value of group N were lower than other groups(P<0.05); the value of group IPTC and S were higher than group IR(P<0.05); the value of group S was higher when compared with groupIPTC(P<0.05).The level of p-p38MAPK: the level of p-p38 MAPK in group IR was significantly higher than other groups(P<0.05); there were no difference among groups N,IPTC and S(P>0.05) 2. The fluorescence intensity of intracellular Ca2+and p H value when using Cytochalasin D(Cy D) The fluorescence intensity of of Ca2+:(1) the intensity of group IR was higher than groups N, group IPTC and S(P<0.05);there were no difference among groups N,IPTC and S(P>0.05);(2) The intensity of Cy D groups were higher than respective corresponding groups and group N(P<0.05);(3) The difference between Cy D groups and the mean value of group N were represented with N-N, IR-N,IPTC-N and S-N respectively. IR-N, IPTC-N and S-N was higher than N-N(P<0.05); IR-N was higher than IPTC-N and S-N(P<0.05); IPTC-N is higher than S-N(P<0.05). The fluorescence intensity of p Hi:(1) p Hi of group IR was lower when compared with other groups(P<0.05), there were no significant difference among group N, IPTC and S(P>0.05).(2) p Hi of Cyd in group N and IR were higher when compared with corresponding groups and group N(P<0.05), however the value of Cy D groups of IPTC and S were lower than their corresponding groups and group N(P<0.05).Conclusion 1. Hydrogen sulfide postconditioning could promote F-actin to remodel to maintain the normal structure of cytoskeleton. 2. Hypoxia/reoxygenation promotes the phosphorylation level of p38 MAPK in adult rat cardiomyocytes which could be suppressed the phosphorylation level of p38 MAPK by hydrogen sulfide postconditioning to exert cardioprotection. 3. Hydrogen sulfide postconditioning could alleviate calcium overload and stabilize the p H value of cardiomyocytes underwent ischemia/reperfusion injury. The protective mechanism is related with the ability of postconditioning stabilizing cytoskeleton and suppressing depolymerization of microfilament.