Effect And Mechanism Of Hydrogen Sulfide Postconditioning On MIRI In Rat Hearts

ObjectiveTo investigate the effect of hydrogen sulfide （H₂S） postconditioning onischemia-reperfusion （IR） myocardium and explore its possible mechanism.MethodsTwenty-four healthy male Sprague-Dawley （SD） rats were randomlydivided into sham-operation （Sham） group, IR group, H₂S postconditioning（H₂S） group （n=8）. Sodium hydrosulfide （NaHS） was used as an exogenousdonor of H₂S. Hearts in Sham group were observed for4.5h continuouslywithout left anterior descending coronary artery （LAD） ligation. Hearts inIR group underwent surgery for LAD ligation for0.5h and followed by4hof reperfusion.5min before reperfusion,1ml saline was injected intoabdominal cavity. Hearts in H₂S group were processed in the same way asIR group, except that1ml NaHS （160μmol/kg, diluted with saline） wereadministered to rats by intraperitoneal injection. The left ventricularsamples of each group were collected after4h of reperfusion forexamination. Myocardial apoptosis indexes （AI） were detected with TUNEL. Hypoxia-inducible factor-1alpha （HIF-1α） and vascularendothelial growth factor （VEGF） mRNA were assessed by RT-PCR. Inaddition, the expressions of HIF-1α and VEGF protein were measured byWestern blot.Cardiomyocytes of neonatal SD rats in primary culture were randomlydivided into five groups: control （CON） group, hypoxia-reoxygenation（HR）group, dimethyl sulfoxide （DMSO） group, H₂S postconditioning （H₂S）group，and LY294002+H₂S postconditioning （LY+H₂S） group. In CONgroup, cardiomyocytes were grown in an incubator at37℃and aeratedwith95%air and5%CO₂and incubated for4h. In HR group,cardiomyocytes were incubated with hypoxic solution in an incubator at37℃under condition of hypoxia （95%N2+5%CO₂）, then incubatedwith reoxygenation solution in an atmosphere of5%CO₂for2h. InDMSO group,2h after hypoxia intervention, cardiomyocytes werecultured in an atmosphere of5%CO₂and95%N2with0.02%DMSOreoxygenation solution for30min, and then cultured with DMSO-freereoxygenation solution for1.5h. In H₂S group,2h after hypoxiaintervention, cardiomyocytes were incubated with NaHS reoxygenationsolution （final concentration of40μmol/L） for30min, and then incubatedwith NaHS-free solution for1.5h. In LY+H₂S group,2h after hypoxiaintervention, cardiomyocytes were cultured with LY294002（finalconcentration of15μmol/L） and NaHS （final concentration of40μmol/L） reoxygenation solution for30min, then cultured with LY294002-free,NaHS-free reoxygenation solution for1.5h. Cultured medium andcardiomyocytes were collected before hypoxia,2h after hypoxia, and2hafter reoxygenation, respectively. Survival rates of cardiomyocytes weredetected by MTT and activities of CK and LDH in cultured medium wereassayed. Apoptosis rates of the cardiomyocytes were detected by flowcytometry and the expressions of HIF-1α, p-Akt and total Akt protein wereexamined by Western blot.Results4h after reperfusion, myocardial AI in Sham group, IR group and H₂Sgroup were （5.04±0.54）%,（36.39±0.69）%and （17.52±1.02）%by TUNEL.Compared with IR group, AI of H₂S group significantly decreased（P<0.01）. RT-PCR results showed that mRNA expressions of HIF-1α andVEGF in H₂S group markedly increased, compared with IR group[（99.67±1.53）%,（100.67±3.51）%, all P＜0.01]. Western blot analysesrevealed that protein expressions of HIF-1α and VEGF in H₂S groupevidently increased in comparison with IR group [（78.00±17.35）%,（137.67±15.50）%, P＜0.05and P＜0.01, respectively].2h after reoxygenation, survival rates of cardiomyocytes in CONgroup, HR group, DMSO group, H₂S group and LY+H₂S group were（95.24±1.32）%,（62.03±1.29）%,（60.86±2.12）%,（71.55±1.46）%and（63.65±2.01）%by MTT. Survival rate of H₂S group was evidently higher in comparison with HR group and LY+H₂S group （all P＜0.01）. Flowcytometry results indicated that apoptosis rates of cardiomyocytes in CONgroup, HR group, DMSO group, H₂S group and LY+H₂S group were（6.98±1.33）%,（17.38±2.04）%,（17.48±2.47）%,（10.37±1.51）%and（15.17±1.56）%. Apoptosis rate of H₂S group was markedly lower,compared with HR group and LY+H₂S group （all P＜0.01）. Meanwhile,compared with HR group and LY+H₂S group, activities of CK and LDH inH₂S group significantly decreased [（244.99±21.38）U/L,（371.64±18.62）U/L,all P＜0.01]. Western blot analyses revealed that expressions of HIF-1αand p-Akt protein in H₂S group remarkably increased in comparison withHR group and LY+H₂S group [（63.93±4.64）%,（62.59±2.11）%, P＜0.05and P＜0.01, respectively].ConclusionH₂S postconditioning can protect rat myocardium against IR injury.The possible mechanism for this protective effect is that H₂Spostconditioning activates PI3K/Akt signaling pathway, promotes proteinexpression of HIF-1α and inhibits myocardial apoptosis, thus alleviatesMIRI.