Effects Of N_aHS Postconditioning On Hypoxia-reoxygenated Cardiomyocytes

Effects of NaHS postconditioning on hypoxia-reoxygenated cardiomyocytesObjective: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 NaHS postconditioning 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 typeâ…¡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 7 groups at random:â‘ control group,the cells of which were cultured in routine condition;â‘¡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 for 3 hours.After the hypoxia solution was replaced by reoxygenation solution,the cells were placed in the oxygenated container for 1 hour to establish the hypoxia/reoxygenation model;â‘¢NaHS preconditioning group,the cells of which were pretreated with 3 cycles for 5 minutes of 1μmol/L NaHS treatments followed by 5 minutes of hypoxia solution.Then the cells were subjected to the hypoxia/reoxygenation injury.â‘£NaHS postconditioning group,the cells of which were subjected to the hypoxia injury first,then post-treated with 3 cycles for 5 minutes of 1μmol/L NaHS treatments followed by 5 minutes of hypoxia solution,then underwent the reoxygenation process.⑤NaHS postconditioning+glibenclamide group,the cells of which were subjected to the hypoxia injury first,then post-treated with 3 cycles for 5 minutes of 1μmol/L NaHS+10μmol/L glibenclamide treatments followed by 5 minutes of hypoxia solution,then underwent the reoxygenation process.â‘¥NaHS postconditioning+5-HD group,the cells of which were subjected to the hypoxia injury first,then post-treated with 3 cycles for 5 minutes of 1μmol/L NaHS+100μmol/L 5-HD treatments followed by 5 minutes of hypoxia solution,then underwent the reoxygenation process.⑦NaHS postconditioning+atractyloside group,the cells of which were subjected to the hypoxia injury first,then post-treated with 3 cycles for 5 minutes of 1μmol/L NaHS+10μmol/L atractyloside treatments followed by 5 minutes of hypoxia solution,then underwent the reoxygenation process.The indexes for observation were as follows: the cardiocyte morphology was observed by inverted microscope;the fluorescence intensity of Ca²⁺ inside the cells was observed by laser scanning confocal microscope and the cell apoptosis was demonstrated with flow cytometry.Furthermore,the content of MDA in the myocardial cells and the activity of SOD and LDH 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 90 to 120 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 myocytes in every group:The cells in the control group grew in clusters and beat well.In the H/R group,the cells grew and beat poorly.However,most cells in the NaHS preconditioning and postconditioning groups grew well.Compared with H/R group,the beating rate was significantly increased.The condition of last three groups is more or less as that in the H/R group.3.The fluorescence intensity of Ca²⁺:The H/R group is obviously higher than the control group(595.99±33.62vsl97.17±17.97,p<0.05).The NaHS preconditioning and postconditioning groups is obviously lower compared with the H/R group(304.50±20.81vs595.99±33.62, 276.66±25.15vs595.99±33.62,both p<0.05).The last three groups are significantly higher than the NaHS postconditioning groups.(all p<0.05).4.The percentage of apoptosis cells in every group:There are lots of apoptosis cells in the H/R group compared to the control group(51.40±4.18vs6.60±0.89,P<0.05).In the NaHS preconditioning and postconditioning groups,the distribution B4 and B2 district is less than that of H/R group(24.60±2.47vs51.40±4.18,18.80±1.56vs51.40±4.18,P<0.05).Each of the last three groups is obviously higher than that of NaHS postconditioning group(p<0.05).5.The content of MDA and the activity of SOD and LDH:Compared with control group,the MDA content of the H/R group was significantly increased(p<0.05),and the SOD and the LDH activity was decreased(p<0.05).There was significant difference between the NaHS preconditioning and postconditioning groups and the H/R group(p<0.05).Each of the last three groups is significantly different from the NaHS postconditioning group(p<0.05).Conclusions:1.NaHS postconditioning can reduce the apoptosis of myocardial cells which is induced by hypoxia-reoxygenation.The protective function is similar to that of NaHS preconditioning.And this effect can be reversed by 5-HD,the specific inhibitor of mitochondrial ATP-sensitive potassium channel2.The cardioprotective mechanism of NaHS postconditioning involves inhibiting mPTP opening.3.NaHS postconditioning can reduce the lipid peroxidation induced by hypoxia-reoxygenation,and protect the SOD,LDH activity of myocardial cells.Thus,NaHS postconditioning has antioxidative effect on cardiomyocytes in the hypoxia-reoxygenation injury.