Endogenous H₂S-induced Postconditioning Protects Hypoxia/Reoxgenated Cardiomyocytes And Its Relative Mechanism

Endogenous H₂S-induced postconditioning protects Hypoxia/reoxgenated cardiomyocytes and its relative mechanismObjective:The myocardial cells in neonatal SD rats were cultured to establish the hypoxia-reoxygenation model,the pathophysiologic changes of which are similar to those of the myocardial ischemia-reperfusion injury in vivo.The purpose of the study was to investigate the effect of H₂S on hypoxia/reoxygenation injury in cultured cardiomyocytes and do further research on its protective mechanism.Methods:The myocardial cells were isolated from the neonatal Sprague-Dawley rats by collagenase, and purified by treatment with the differential adhesion technique and the chemical inhibition method,then cultured in the Dulbecco's modified eagle culture medium consisting of 20%fetal bovine serum.The condition of cell growth and spontaneous beating were observed under the inverted microscope.The myocardial sarcomeric actin and cardiac troponin T were identified by immunofluorescent technique.Primary cultured myocardial cells of rats were divided into 5 groups at random:(Control group),the cells of which were cultured in routine condition;hypoxia/reoxygenation group(H/R group),the cells of which were treated as follows:the normal cultured medium was replaced by hypoxia solution.Then the cells were cultured hermetically for3 hours.After the hypoxia solution was replaced by reoxygenation solution,the cells were placed in the oxygenated container for 1hour to establish the hypoxia/reoxygenation model;Hypoxic postcondiontioning group(HPC group),the cells of which were treated as follows:the normal cultured medium was replaced by hypoxia solution.Then the cells were cultured hermetically for 3 hours,After the hypoxia solution was replaced by reoxygenation solution,the cells were placed in the oxygenated container for 5 minutes,then in hypoxied container for 5 minutes,repeat 3 times,in total 30 minutes;at last the cells were placed in the oxygenated container for 1 hour to establish the hypoxic postconditioning model;hypoxia/reoxygenation +L-propargylglycine(H₂S metabolic enzyme inhibitor) group(H/R+L-PAG group),the cells were treated as HPC group,But add 10μmol/L L-PAG in reoxygenated culture solution during the 3 circles;hypoxia/reoxygenation +Chelerythrine(PKC inhibitor) group(H/R+Che group),the cells were treated as HPC group,But add 3μmol/L Chelerythrine in reoxygenated culture solution during the 3 circles.The indexes for observation were as follows:the content of LDH was detected by chemistry chromatometry; the cardiomyocytes morphology and beating rate were observed by inverted microscope,the cell apoptosis was demonstrated with flow cytometry and the intracellular calcium fluorescence intensity were detected by Laser scanning confocal microscope.Furthermore,the content of MDA in the myocardial cells and the activity of SOD in the supernatant of the culture medium were detected.Results:1.Identification of myocardial cells:The microscope showed that the cells grew into clusters and beat regularly,the frequency of which were between 80 to 100 beats per minute. When the primary antibodies of myocardial sarcomeric actin and troponin T were used, the cells emitted green fluorescence under the fluorescence microscope.2.The morphology of cardiac myocytes in every group:In the H/R group,the cells grew poor.The beating rate was significantly slow in the H/R group(18.26±3.50beat/min) when compared with H/R+L-PAG group(38.50±2.38 beat/min) and H/R+Che group(41.52±2.65beat/min)(p<0.05).In addition,the cells in HPC group beat faster than those two groups,and the difference was obvious(p<0.05)3.The percentage of apoptosis cells in every group:Bl,B2,B3 and B4 represented the late phase of apoptosis,death cells,normal cells and the first phase of apoptosis.The percentage of apoptosis in H/R group was obviously higher than in HPC group(43.72±4.72%vs8.93±1.04%,p<0.05);the apoptosis rate in H/R+L-PAG group and H/R +Che group decreased obviously when compared with that of H/R group(17.41±#2.56%and 19.16±1.47%vs 43.72±4.72%,p<0.05).4.The intracellular calcium fluorescence intensity in H/R group was obviously higher than normal group(567.43±45.28 vs 112.37±13.67 p<0.05);The intracellular calcium fluorescence intensity in H/R+L-PAG group,H/R +Che group and HPC group decreased when compared with that of H/R group(245.12±19.03,350.28±35.22 and 170.62±22.07 vs 567.43±45.28,p<0.05).5.The content of MDA and the activity of SOD:compared with normal group,the MDA content of the H/R group was significantly increased(p<0.05),and the SOD activity was decreased(p<0.05);The MDA content of the H/R+L-PAG group and the H/R +Che group was obviously lower than that of the H/R group(p<0.05),and the SOD activity were higher(p<0.05).however,above two groups compared with HPC group,The MDA content increased obviously(p<0.05),The SOD activity content decreased significantly (p<0.05).6.The LDH concentration in H/R group was obviously higher than in normal group (59.64±2.71 vs 19.31±1.58 U/L,p<0.05);The LDH in H/R+L-PAG group,H/R +Che group and HPC group decreased when compared with that of H/R group(45.66±1.49, 43.27±1.35,37.18±1.63 vs 59.64±2.71 U/L,p<0.05);The LDH in H/R+L-PAG group,H/R +Che group and HPC group increased when compared with that of HPC group(p< 0.05).Conclusions:1.Endogenous hydrogen sulfide may participate in the protective of hypoxic postconditioning in cardiac myocytes.2.Endogenous hydrogen sulfide can reduce the lipid peroxidation induced by hypoxia/reoxygenation,and reduce the injury of the cardiac myocytes,Thus,endogenous hydrogen sulfide has antioxidative effect on myocardial cells in the hypoxia/reoxygenation injury.3.Endogenous hydrogen sulfide participates the protective of hypoxic postconditioning by activating PKC in cardiac myocytes,It may release the calcium overload by regulating intracellular handling by PKC.