| It is well known that both ischemic pre-conditioning(IPC) and post-conditioning(IPost), which describe the cardioprotection obtained from applying transient episodes of myocardial ischemia and reperfusion either before or after the index ischemic event respectively, appear to recruit a common mechanistic pathway and have been respected as effective cardioprotective strategy against myocardial ischemia and reperfusion(MI/R) injury. However, harnessing the cardioprotective effect of IPC necessitates an intervention which can be applied before the index ischemic period, thereby restricting its clinical application to situations in which the ischemic event can be anticipated, such as in the case of patients undergoing cardiac bypass surgery or in patients. Recent years, pharmacological IPC and IPost have been identified as novel strategies for attenuating MI/R injury by imitating the cardioprotective effects of IPC and IPost, and pharmacological IPost has been an important direction for the cure of MI/R injury because of its controllability and easy operation.Hydrogen sulfide (H2S), which plays an important role in regulation of cardiovascular system, is the third gasotransmitters after Nitric Oxide (NO) and Carbon Monoxide (CO) were identified as important endogenous signaling gas. Emerging evidence showed that treatment with H2S before ischemia in MI/R rat exerted protective effects on cardiomyocytes, but whether administration with H2S during the reperfusion period can exhibit cardioprotective effects against MI/R injury is rarely reported. Therefore, in the present study, we observed the phenomenon that whether treatment with H2S during reperfusion period can exhibit exact cardioprotective effects against myocardial regional MI/R injury at heart level in vivo and in vitro and cellular level. Our results demonstrated that H2S administration during the reperfusion period showed protective effects against regional MI/R in heart and hypoxia-reoxygenation (H/R) injury in cultured H9c2(2-1) myocytes respectively, and the corresponding optimal doses of H2S were 50μmol/kg and 50μmol/L respectively. Meanwhile, the similar protective effects of exogenous administration with H2S were also testified in hyperlipoidemia and aged rats subjected to regional MI/R.Basing on the protective effects of H2S administered during reperfusion period against MI/R and the optimal dose of H2S, the effects of I/R and treatment with H2S on H2S/CSE system, the role of exogenous H2S in preconditioning, and the effects of PAG on postconditioning intervention were observed by detection of H2S content, the activity and mRNA level of CSE; the protective effects of H2S and its possible anti-apoptotic role were determined by TUNEL, Caspase-3 activity assay, AO/EB and Rhodamine-123 staining at the animal and cellular level; whole-cell patch clamp technique was applied to detect the protective role of H2S on KATP channels in cell membrane and mitochondrion, and combinating with the antagonists, the contribution of KATP channels to cardioprotective effects of H2S was also analyzed; the relationship between NO,PKA,PKC,IP3K and the protective role of H2S was also observed at the animal and cellular level by using the antagonists. The study is divided into two parts as below.PART 1 Cardioprotective effects of administration with H2S during reperfusion period against MI/R injuryAim: To examine whether the administration of H2S during reperfusion period exhibits cardioprotective effects against MI/R injury and the corresponding optimal dose of H2S.Material and Methods:1. Materials:Male Sprague-Dawley adult rats (4-5 months) weighing 260-280 g; Male Sprague-Dawley adult rats (4-5 weeks) weighing 110-130 g; Male Sprague-Dawley adult rats (22-24 months) weighing 490-510 g; H9c2 (2-1) cell: Strain: BD1X, Organ: heart, Tissue: myocardium2. Method:(1) Establishment of experimental model and sample collection:(1)The establishment of rat regional MI/R model in vivo and tissue collection;(2)The establishment of H9c2(2-1) H/R model and cell collection;(3)The establishment of rat regional MI/R model in vitro and tissue collection;(4)The establishment of rat regional MI/R induced-arrhythmia model in vitro and tissuecollection;(5)The establishment of hyperlipoidemia rat regional MI/R model in vivo and tissuecollection:(6)The establishment of aged rat regional MI/R model in vivo and tissue collection.(2) Index determination(1) Determination of myocardial infarct size;(2) Determination of biochemical index:i . Measurement of Creatine Kinase(CK) content; ii. Measurement of Maleic Dialdehyde(MDA) in serum and culture medium supernatantiii. Detection of Lactate Dehydrogenase(LDH) content in perfusion solution and culture medium supernatant;iv. Measurement of Triglyceride(TG), Total Cholesterol(TC), Low Density Lipoprotein Cholesterol(LDLC) in serum of rat on an empty stomach.(3) Histomorphology Observation:Myocardium tissue HE Staining; Myocardium microstructure observation.(4) Measurement of cardiac function(5) Measurement of reperfusion arrhythmiaResults:1. The effects of H2S administered with different dose during reperfusion period on rat I/R heart:Animals were randomized to receive one of the following treatments (6 groups):(1) Sham group: LAD without occlusion, total time course is 210 min。(2) I/R group: LAD with reversible occlusion, 30 min ischemia followed by 180 min reperfusion; administer normal sodium 0.5 ml by intraperitoneal injection at 5 min before reperfusion.(3) H2S(6.25μmol/kg) group: 30 min ischemia followed by 180 min reperfusion; administer NaHS (6.25μmol/kg) dissolved in normal sodium 0.5 ml by intraperitoneal injection at 5 min before reperfusion.(4) H2S(12.5μmol/kg) group: the procedure is the same as group (3) except the concentration of NaHS is 12.5μmol/kg.(5) H2S(25μmol/kg) group: the procedure is the same as group (3) except the concentration of NaHS is 25μmol/kg.(6) H2S(50μmol/kg) group: the procedure is the same as group (3) except the concentration of NaHS is 50μmol/kg.1.1 Effects of H2S administered with different concentration during reperfusion period on myocardial infarct size of regional MI/R rats:1.1.1 The percentage of the area at risk to left ventricle area (AAR/LV): there is no significant difference among the groups, which indicates that the ischemic area is roughly identical among each group of animal models and then these groups are comparable.1.1.2 The percentage of myocardial infarction area to area at risk (AN/AAR): the ratios of AN/AAR in different does H2S(6.25μmol/kg, 12.5μmol/kg, 25μmol/kg, 50μmol/kg) treatment group are 37.01%±2.74 P,31.89%±2.45%,26.61%±5.51% and 18.86%±2.57% respectively, which decreased obviously compared to I/R group; Compared with I/R group (46.26 %±3.55 %), the myocardial infarct size in different does H2S groups significantly reduced in a does-dependent manner. This indicates that myocardial protection generated by administration of H2S during the reperfusion period does exist in MI/R rats, and this effect was increased in a does-dependent manner from 6.25μmol/kg to 50μmol/kg of H2S. 1.2 Effects of H2S administered with different dose during reperfusion period on the CK and MDA level in regional MI/R rats in vivoThe decrease of the CK and MDA parallels with that of myocardial infarct area. Compared with I/R group (1.74±0.09 U/ml; 5.93±0.81 nmol/ml), the concentration of CK and MDA in 6.25μmol/kg H2S group(1.59±0.08 U/ml; 5.40±0.81 nmol/ml), 12.5μmol/kg H2S group(1.49±0.11 U/ml; 4.73±0.38 nmol/ml), 25μmol/kg H2S group(1.22±0.13 U/ml; 3.70±0.37 nmol/ml), 50μmol/kg H2S group( 1.11±0.09 U/ml; 3.07±0.45 nmol/ml) significantly decreased(P<0.05).2. Effects of different concentration of H2S administered during the reoxygenation period on cultured H9c2(2-1) cells subjected to H/R injury.Animals were randomized to receive one of the following treatments (8 groups):(1) Normoxia group: cultured in an atmosphere of 5% CO2/95% air for 5 h;(2) H/R group: hyposia with 95% N2 and 5% CO2 for 3 h followed by 2 h reoxygenation in 5% CO2 and 95% air;(3) 12.5μmol/L H2S group: add 10μl H2S solution into the cultured medium before reoxygenation (final concentration 12.5μmol/L);(4) 25μmol/L H2S group: the procedure was the same as the above process in group (3) except that the final concentration of H2S is 25μmol/L;(5) 50μmol/L H2S group: the procedure was the same as the above process in group (3) except that the final concentration of H2S is 50μmol/L;(6) 75μmol/L H2S group: the procedure was the same as the above process in group (3) except that the final concentration of H2S is 75μmol/L;(7) 100μmol/L H2S group: the procedure was the same as the above process in group (3) except that the final concentration of H2S is 100μmol/L;(8) 200μLmol/L H2S group: the procedure was the same as the above process in group (3) except that the final concentration of H2S is 200μmol/L.Effects of different concentration of H2S administered during the reoxygenation period on LDH release and MDA content in supernatant of cultured H9c2(2-1) cells subjected to H/R injury.Compared with the H/R group(800±29.81 U/L; 5.97±0.90 nmol/ml), both the LDH release and MDA contents decreased significantly in H2S groups; compared with H/R group, the LDH release and MDA contents significantly reduced(P<0.05) in H2S groups at 12.5μmol/L(466.67±42.16 U/L; 2.56±0.25 nmol/ml), 25μmol/L(161.11±44.31 U/L; 1.40±0.29 nmol/ml), 50μmol/L (61.11±25.09 U/L; 0.70±0.39 nmol/ml) in a dose-dependent manner; however, the LDH release and MDA contents increased in H2S groups at 75μmol/L(244.44±58.37 U/L; 3.80±1.04 nmol/ml), 100μmol/L(622.22±80.74 U/L; 4.88±0.99 nmol/ml), 200μmol/L (650±104.88 U/L; 5.58±0.66 nmol/ml) a dose-dependent manner, but there is no statistical significant in LDH release between 100μmol/L and 200μmol/L H2S groups, and in MDA contents among 75μmol/L,100μmol/L,200μmol/L H2S groups(P>0.05). These results indicated that (1) H2S administration in the reperfusion period showed cardioprotection on cultured H9c2(2-1) cells subjected to H/R; (2) the optimal dose is 50μmol/L at which H2S could offer its maximal effects.3. Protective effects of H2S administered during reperfusion period on Morphology of rat MI/R injuryAnimals were randomized to receive one of the following treatments (3 groups):(1) Sham group: LAD without occlusion, total time course is 210 min;(2) I/R group: LAD with reversible occlusion, ischemia 30 min followed by 180 min reperfusion; administer normal sodium 0.5 ml by intraperitoneal injection at 5 min before reperfusion;(3) H2S group: ischemia 30 min followed by 180 min reperfusion; administer NaHS(50μmol/kg) dissolved in normal sodium 0.5 ml by intraperitoneal injection at 5 min before reperfusion.3.1 Protective effects of H2S administered during reperfusion period on pathomorphological changes in rat myocardium after MI/R injuryBy the optical microscopy, the myocardial tissue showed extensive necrosis and severe muscle fiber fracture, myocardial cells dissolution and disappearance, neutrophils exudation in I/R group. Rats receiving H2S displayed a reduced degree of myocardial neutrophilic infiltrate, necrosis, hemorrhage, and spindle-shaped interstitial cells as compared with rats receiving sham operation.3.2 Protective effects of H2S administered during reperfusion period on ultrastructural changes in rat myocardium after MI/R injuryAfter ischemia and reperfusion myocardial samples were qualitatively assessed by transmission electron microscopy for structural changes. Under the TEM, it showed myofibrils disarrangement, large areas of myofibrils fracture, amalgamation, disappearance, sarcomere structure unclear, myocardial nuclear swelling, the nuclear membrane rupture, nuclear chromatin asymmetry, condensation, margination, mitochondrial morphological abnormalities and swelling, the ridge disarrangement, fracture, disappearance, forming cavity, glycogen granules between the mitochondrial vanishment, some mitochondria parceled by double membrane, and completely desquamated from the mother cells, forming apoptotic bodies in I/R group. In H2S groups, myocardial ultrastructural changes were discriminating.4. Cardioprotective effects of H2S administered during reperfusion period on regional MI/R rat heart in vitroIsolated hearts were randomized to receive one of the following treatments (3 groups):(1) Sham group: LAD without occlusion, total time course is 210 min;(2) I/R group: LAD with reversible occlusion, ischemia 30 min followed by 180 min reperfusion; administer Tyrode's solution by the flow rate of 8 ml/min at 5 min before reperfusion;(3) H2S group: ischemia 30 min followed by 180 min reperfusion; administer NaHS (50μmol/L) by the flow rate of 8 ml/min at 5 min before reperfusion.4.1 The time-course curve of LDH release in perfusate from isolated rat regional MI/R model in vitroThe results indicated that the release of LDH began to gradually increase after reperfusion, and reached the peak at 30min after reperfusion, then gradually decreased.4.2 Cardioprotective effects of H2S administered during reperfusion period on isolated rat heart subjected to MI/R in vitro4.2.1 Cardioprotective effects of H2S administered during reperfusion period on the LDH release in the perfusate from isolated rat heart subjected to MI/R in vitroCompared with sham group, the LDH release in the perfusate significantly increased in I/R group(2526.32±254.66 U/L vs. 131.58±38.57 U/L, P<0.01), indicating that regional I/R rat heart model was successfully established; compared with I/R group, LDH release in H2S group significantly decreased(501.75±176.35 U/L, P<0.01).4.2.2 Effects of H2S administered during reperfusion period on cardiac function recovery of isolated rat heart subjected regional MI/RCompared with sham group, + dp/dtmax(%), -dp/dtmax(%), LVSP-LVDP(%) are significantly lower(P<0.01), however, the HR(%) was enhanced in I/R group(P<0.01); compared with I/R group, every index referred above was significantly improved(P<0.01 or P<0.05).4.2.3 Effects of H2S administered during reperfusion period on reperfusion arrhythmiaBoth the frequency of premature ventricular beat (PVB) in I/R group was significantly increased as compared with that in sham group(76.83±6.85 vs. 14.43±4.13, P<0.01), indicating that the reperfusion arrhythmia model was successfully established; compared with I/R group, the frequency of PVB was significantly reduced in the H2S group(22.33±9.29, P<0.01).5. Cardioprotective effects of H2S administered during reperfusion on hyperlipoidemia rat heart subjected to MI/R injuryThere are 2 group including normal adult rat group and hyperlipoidemia rat group in this study, and each group is divided into 3 subgroups as below:(1) Sham group: LAD without occlusion, total time course is 210 min;(2) I/R group: LAD with reversible occlusion, 30 min ischemia followed by 180 min reperfusion; administer normal sodium 0.5 ml by intraperitoneal injection at 5 min before reperfusion;(3) H2S group: 30 min ischemia followed by 180 min reperfusion; administer NaHS (50μmol/kg) dissolved in normal sodium 0.5 ml by intraperitoneal injection at 5 min before reperfusion.5.1 Establishment of hyperlipoidemia rat modelHyperlipoidemia rat group was fed with high fat diet for 8 weeks, and the serum TG, TC, LDLC level in hyperlipoidemia rat group is significantly higher than that in normal diet group(P<0.05), indicating that the hyperlipoidemia rat models were successfully established.5.2 Effects of H2S administered during reperfusion period on myocardial infarct size of hyperlipoidemia ratsThere is no difference in AAR/LV between groups (P>0.05). The ratio of AN/AAR in H2S + hyperlipoidemia group was significantly increased than that in sham+hyperlipoidemia group (22.90 %±1.54 % vs. 56.98 %±2.47 %, P<0.01), while was significantly higher than that in H2S +normal diet group (46.25 %±3.55 % vs. 56.98 %±2.47 %, P<0.05); compared with sham+ hyperlipoidemia group, the ratio of AN/AAR was significantly increased in sham+normal diet group(15.89 %±1.92 % vs. 8.86 %±2.95 %, P<0.05).5.3 Effects of H2S administered during reperfusion period on cardioprotective rate of hyperlipoidemia ratsCompared with H2S+normal diet group, the cardioprotective rate in H2S+ hyperlipoidemia group was significantly increased(91.67±1.83 vs. 88.15±2.91, P<0.05), indicating that H2S played a more effective protective role in hyperlipoidemia group.6. Cardioprotective effects of H2S administered during reperfusion period on aged rat heart subjected to MI/R injuryThere are 2 group including normal adult rat group and aged rat group in this study, and each group is divided into 3 subgroups as that in hyperlipoidemia rats.6.1 Effects of H2S administered during the reperfusion period on myocardial infarct size of aged rats There is no difference in AAR/LV between groups (P>0.05). The ratio of AN/AAR in H2S + aged group was significantly decreased than that in sham+ aged group (22.52 %±1.69 % vs. 54.65 %±1.72 %, P<0.01), while was significantly higher than that in H2S+normal adult group (46.25 %±3.55 % vs. 54.65 %±1.72 %, P<0.05); compared with sham+ aged group, the ratio of AN/AAR was significantly increased in sham+normal adult group(15.51 %±1.28 % vs. 8.86 %±2.95 %, P<0.05).6.2 Effects of H2S administered during reperfusion period on cardioprotective rate of aged ratsCompared with H2S+normal adult group, the cardioprotective rate in H2S+ aged group was significantly increased(91.70±2.00 vs. 88.15±2.91, P<0.05), indicating that H2S played a more effective protective role in aged rats.PART 2 Mechanisms of the cardioprotective effects of administrationwith H2S during reperfusion period on rat MI/RAim: To explore the potential mechanism involving cardioprotective effects of H2Sadministered during reperfusion period on MI/R heart in ratsMaterials and Methods:1. Materials:Male Sprague-Dawley adult rats (4-5 months) weighing 260-280 g; H9c2 (2-1) cell: Strain: BD1X, Organ: heart, Tissue: myocardium2. Methods:(1) Establishment of experimental model and sample collection:①The establishment of rat regional MI/R model in vivo and tissue collection;②The establishment of H9c2(2-1) H/R model and cell collection;③Cell isolation procedureThe rats were decapitated and the hearts were rapidly excised and mounted onto a Langendorff perfusion apparatus, and were immediately perfused with Ca2+-free Tyrode solution equilibrated with O2 until spontaneous contractions ceased. Ventricles were separated and minced in Krebs solution before being filtered through a nylon mesh. The viable cells were subsequently separated by sedimentation for 10 min twice. The ventricular myocytes were then re-suspended in the Krebs solution, and Ca2+ was slowly added to the cell suspension till it reached final concentration of 1.8 mmol/L.④establishment of other animal models and sample collection are the same as that in Part1.(2) Index determination①Determination of biochemical index: i. Measurement of LDH, MDA and SOD content (the same as Part 1);ii. Measurement of H2S content and Cystathionineγ-synthase(CSE) activity by sensitive sulphur electrode assay②Detection of mRNA expression level of CSE real-time quantitive PCR③Cell apoptosis detectioni. Myocardial apoptosis is detected by TdT-mediated dUTP nick end labeling (TUNEL)ii. Caspase-3 relative activity assayiii. AO/EB stainingiv. Mitochondrial transmembrane potential detection④patch-clamp whole-cell recording techniqueConventional patch-clamp whole-cell recording method was applied in this study.Result:1. Effects of H2S administered during reperfusion period on H2S/CSE systemAnimals were randomized to receive one of the following treatments (3 groups):(1) Sham group; (2) I/R group; (3) H2S group(50μmol/kg).1.1 Effects of H2S treatment on H2S level and CSE activity in serum and myocardium from the area at riskCompared with sham group, H2S level in serum, tissue and CSE activity in myocardium of area at risk were significantly decreased in I/R group(38.05±1.95μmol/L vs. 47.22±3.02μmol/L, P<0.01; 37.69±1.63μmol/g prot vs. 48.00±3.51μmol/g prot,P<0.01; 10.57±1.45μmol/L/min·g prot vs. 18.97±2.00 nmol/L/min·g prot, P<0.01); and the above indexes in H2S group(43.28±3.24μmol/L; 43.77±3.05μmol/g prot; 14.01±1.612 nmol/L/min·g prot) were enhanced as comparing with I/R group(P<0.01).1.2 Effects of H2S treatment on expression of CSE in myocardium from the area at risk.The mRNA expression of CSE in I/R group was significantly lower by 13 % than that in sham group (P<0.01); and in H2S group, the mRNA expression of CSE in myocardium from the area at risk was significantly higher by 6 % than that in I/R group(P<0.01).2. Role of endogenous H2S in postcondioning--Effects of PAG on ischemiapostconditioning(IPost)'s protection2.1 Effects of PAG on serum and tissue enzyme indexes of myocardium IPost in vivo Animals were randomized to receive one of the following treatments (4 groups):(1) Sham group;(2) I/R group: LAD with reversible occlusion, ischemia 30 min followed by 180 min reperfusion; administer normal sodium 0.5 ml by intraperitoneal injection at 5 min before reperfusion; (3) POST group: LAD with reversible occlusion, ischemia 30 min followed by 180 min reperfusion; hearts were administered with normal sodium 0.5 ml by intraperitoneal and subjected to three alternate episodes of 10 sec LAD occlusion and 10 sec reperfusion at 15min before reperfusion;(4) PAG group: hearts were administered with PAG (DL-propargylglycine, a specific inhibitor of CSE, 50mg/kg) 0.5ml by intraperitoneal at 15min before reperfusion, other processes are the same as that in group (3).The results demonstrated that in POST group, the CK in serum and MDA in heart were significantly decreased(1.23±0.14 U/ml vs. 1.74±0.09 U/ml, P<0.01; 9.36±0.51 nmol/mg prot vs. 16.02±2.02 nmol/mg prot, P<0.01) respectively, while SOD level in heart was significantly reduced as comparing with that in I/R group(39.97±2.16 U/mg prot vs. 26.33±2.21 U/mg prot, P<0.01); in PAG group, the CK level was significantly higher(1.48±0.12 U/ml vs. 1.23±0.14 U/ml, P<0.01 ) and the SOD level was lower(31.15±2.33 U/mg prot vs. 39.97±2.16 U/mg prot) than that in POST group, the MDA showed an increasing trend though with no any statistical significance. All above results indicated that PAG partly blocked the protection induced by postconditioning, in which endogenous H2S involved.2.2 Effects of PAG on enzyme indexes in medium supernatant of cultured H9c2 (2-1) cell subjected to hypoxia postconditioningCultured H9C2 myocytes were randomly assigned to 4 groups:(1) Normoxia group;(2) H/R group: add 5μl culture medium pretreated with hypoxia at 0.5h before reoxygenation(3) POST group: add 5μl culture medium pretreated with hypoxia at 0.5h before reoxygenation(4) PAG group(5 mmol/L): add 5ul PAG dissolved in culture medium pretreated with hypoxia at 0.5h before reoxygenation( final concentration of PAG is 5 mmol/L).Above results indicated that in POST group, both LDH and MDA in medium supernatant significantly decreased as comparing with that in H/R group(172.41±48.77 U/L vs. 752.87±50.76 U/L, P<0.01; 3.40±0.46 nmol/ml vs. 6.93±0.41 nmol/ml, P<0.01); the LDH and MDA in PAG group were significantly higher than that in HPost group(540.23±35.61 U/L vs. 172.41±48.77 U/L, P<0.01; 4.80±0.33 nmol/ml vs. 3.40±0.46 nmol/ml, P<0.01), indicating that PAG attenuated the protection induced by hypoxia postconditioning, in which endogenous H2S involved.3. Effects of KATP channel activation on protection induced by H2S administration during reperfusion period 3.1 Effects H2S on the activation of KATP channel in cardiomyocytesThe effect of H2S on Katp channel in cardiomyocytes was observed by patch-clamp whole-cell recording technique. The results demonstrated that H2S can activate glibenclamide sensitive homeostatic membrane outside currents. As glibenclamide is specific plasma membrane KATP channel inhibitor, the results indicated that the Katp channel in cardiomyocytes can be activated by direct effects of H2S.3.2 Effects of Katp channel blocker on cardioprotection produced by H2S3.2.1 Effects of Katp channel blocker on cardioprotection induced by H2S administered during the heart reperfusion periodAnimals were randomized to receive one of the following treatments (5 groups):(1) Sham group;(2) I/R group;(3) H2S group(50μmol/kg);(4) Gli group(0.3 mg/kg): administer with glibenclamide (0.3 mg/kg) 0.5 ml by intraperitoneal injection at 5 min before reperfusion;(5) 5-HD group(5mg/kg): administer with 5-HD(5-hydroxydecanoic acid, a selective mitochondrial KATP channel antagnist, 5mg/kg) 0.5 ml by intraperitoneal injection at 5 min before reperfusion.The results demonstrated that in H2S group, serum CK and myocardial MDA level significantly decreased( 1.11±0.09 U/ml vs. 1.74±0.09 U/ml, P<0.01; 8.77±0.73 nmol/mg prot vs.16.02±2.02 nmol/mg prot, P<0.01), while the myocardial SOD content significantly increased as comparing with that in I/R group(42.59±4.72 U/mg prot vs. 26.33±2.21 U/mg prot, P<0.01); compared with H2S group, serum CK(1.48±0.12 U/ml) and myocardial MDA level (13.78±2.75 nmol/mg prot) in Gli group were significantly higher(P<0.01 and P<0.05 respectively), and the myocardial SOD level(31.15±2.33 U/mg prot) were significantly lower, and the indexes showed statistical significances with I/R group except the SOD level; the indexes except MDA content showed statistical differences between H2S group and 5-HD group, indicating that the KATP channel in both plasma membrane and mitochondrion involved in the protection by H2S.3.2.2 Effects of KATP channel blocker on cardioprotection induced by H2S treatment in vitro The groups are same as 3.2.1.3.2.2.1 Effects of KATP channel blocker on LDH release in perfusate from isolated heart subjected to I/RIn H2S group, LDH release level in perfusate significantly decreased as comparing with that in I/R group(501.75±l76.35 U/L vs. 2526.32±454.66 U/L, P<0.01); compared with H2S group, LDH release in Gli group significantly increased(2456.14±225.83 U/L vs. 501.75±176.35 U/L, PO.01), but did not show any statistical difference with I/R group(P>0.05); the LDH release in 5-HD group(1778.95±287.97 U/L) increased as comparing with H2S group, but did not show any statistical difference with I/R group.3.2.2.2 Effects of Katp channel blocker on cardiac function recovery following regional I/R in vitroCompared with I/R group, the indexes of cardiac function significantly recovered in H2S group(P<0.01); the degrees of recovery in Gli group(P<0.05) and 5-HD group(P<0.01) were attenuated at different levels as compared with H2S group (P<0.01), indicating that the KATP channel in plasma membrane and mitochondrion involved in the protection produced by H2S intervention.3.2.2.3 Effects of KATP channel blocker on reperfusion arrhythmiaCompared with I/R group, the PVB frequency significantly decreased in H2S group (22.33±9.29 vs. 76.83±6.85, P<0.01); the PVB frequency in Gli group (70.50±14.110) and 5-HD group(43.00±7.54) significantly increased as compared with H2S group (P<0.01), indicating that the KATP channel in plasma membrane and mitochondrion involved in the protection produced by H2S intervention.3.3 Effects of KATP channel blocker on cardiomyocyte protection induced by H2S treatment during the reoxygenation periodCells were randomized to receive one of the following treatments (5 groups):(1) Normoxia group;(2) H/R group;(3) H2S group(50μmol/L);(4) Gli group(2μmol/L);(5) 5-HD group(100μmol/L).Compared with H/R group, LDH and MDA level in perfusate significantly decreased in H2S group (94.44±53.40 U/L vs. 772.22±32.77 U/L, P<0.01; 3.30±0.28 nmol/ml vs. 6.97±0.40 nmol/ml, P<0.01). In Gli group, perfusate LDH and MDA level significantly increased as comparing with that in H2S group(750.00±27.89 U/L vs. 94.44±53.40 U/L, PO.01; 6.27±0.72 nmol/ml vs. 3.30±0.28 nmol/ml, P<0.01), but with no any statistical difference with H/R group. There are statistical differences in the above indexes between 5-HD group and H2S group, H/R group, respectively. The results demonstrated that both the KATP channel in plasma membrane and mitochondria involved in the protection induced by H2S.4. Effects of H2S treatment during reperfusion on myocardial I/R induced apoptosis4.1 Effects of H2S treatment during reperfusion on apoptosis induced by myocardial I/R in vivo Animals were randomized to receive one of the following treatments (3 groups):(1) Sham group: LAD without occlusion, total time course is 210 min;(2) I/R group: LAD with reversible occlusion, ischemia 30 min followed by 180 min reperfusion; administer normal sodium 0.5 ml by intraperitoneal injection at 5 min before reperfusion;(3) H2S group: administer with NaHS(50μmol/kg) 0.5 ml by intraperitoneal injection at 5 min before reperfusion;4.1.1 myocardium apoptosis detection by TUNELMyocardial apoptosis was significantly higher in I/R group than that in sham group as evidenced by increased apoptotic index(1.44%±0.14% vs. 19.07%±1.02%, P<0.01); Compared with I/R group, the apoptotic index significantly decreased in H2S group(9.63%±1.02% vs. 19.07%±1.02%, P<0.01), indicating that administration of H2S during the reperfusion period can significantly inhibit myocardial apoptosis induced by I/R injury,.4.1.2 Caspase-3 relative activity assayThe caspase-3 relative activity in I/R group was significantly enhanced as comparing with sham group (3.12±0.61 vs. 1.00±0.08, P<0.01); compared with I/R group, the caspase-3 relative activity in H2S group significantly decreased (1.55±0.29 vs. 1.00±0.08, P<0.01). It is consistent with the result detected by TUNEL, indicating that administration of H2S during the reperfusion period can significantly inhibit myocardial apoptosis induced by I/R injury.4.2 Effects of H2S treatment during reoxygenation on H/R induced cell apoptosisCells were randomized to receive one of the following treatments (3 groups):(1) Normoxia group; (2) H/R group; (3) H2S group(50μmol/L);4.2.1 Cell apoptosis detection by AO/EB stainingCompared with normoxia group, the apoptotic index in H/R group significantly increased(43.07%±4.34% vs. 3.47%±0.78%, P<0.01 ); In H2S group, the apoptotic index significantly decreased as comparing with that in H/R group(26.30%±2.93% vs. 43.07%±4.34%, P<0.01); The results demonstrated that administration of H2S during the reoxygenation period can significantly relieve myocardial apoptosis induced by H/R injury.4.2.2 Mitochondria membrane potential detection by Rhodamine123 stainingCompared with normoxia group, the relative fluorescence intensity in H/R group significantly increased (208.77±15.42 vs. 100±9.79, P<0.01); in H2S group, the relative fluorescence intensity significantly decreased as comparing with that in H/R group (162.57±15.00 vs. 208.77±15.42,P<0.01); the results indicated that administration of H2S during the reoxygenation period may play a role in relieving decreased mitochondria membrane potential induced by H/R injury. 4.2.3 Caspase-3 relative activity assayCompared with normoxia group, the Caspase-3 relative activity in H/R group significantly increased (1.74±0.29 vs. 1.00±0.08, P<0.01); in H2S group, the Caspase-3 relative activity significantly decreased as comparing with that in H/R group(1.26±0.13 vs. 1.74±0.29, P<0.01); the results were consistent with that detected by AO/EB staining, and demonstrated that administra... |
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