| Objective:To study whether JAK-STAT signaling pathway plays an essential role in reducing H9c2 cells cardiomyocytes hypoxia/reoxygenation injury induced by S1P postconditioning and explore the potential mechanism.Methods:1. Model of H9c2 cells exposed hypoxia reoxygenation injuryH9c2 cells were placed with DMEM medium containing 10% FBS and cultured for 24 hours, then subjected to 16 hours hypoxia followed by 4 hours reoxygenation.To simulate hypoxia, the culture medium was replaced by hypoxia buffer, and then incubated for 16 h in a 95% N2 and 5% CO2 gas mixture. For reoxygenation, H9c2 cells were exposed to normal conditions for 4 hours and different drugs were appied simultaneously.2. Divide H9c2 cells into seven groups randomly(1) control(C) group: cells were placed with the only serum-free DMEM for 4h;(2) hypoxia/reoxygenation(H/R) group: cells were subjected to hypoxia for 16 h and reoxygenation for 4 h;(3) S1P group: 4 μM S1P was added before the end of hypoxia for 4 h, after which cells were treated as those of H/R group;(4)S1P+AG490 group(S1P+AG): cells were pretreated with 20 μM AG490(inhibitor of JAK2) 30 min before the end of hypoxia, then cotreated with AG490 and S1P during reoxygenation for 4 h;(5) AG group: cells were pretreated with 20 μM AG490 30 min before the end of hypoxia, then treated as those of the H/R group;(6) S1P+stattic group(S1P+ST): cells were pretreated with 1 μM stattic(inhibitor of STAT3) 30 min before the end of hypoxia, then treated with the mixture of stattic and S1P during reoxygenation for 4 h;(7) ST group: cells were pretreated with 1 μM stattic 30 min before the end of hypoxia, then treated as those of H/R group.3. Experimental assays:1) The cell viability in 7 groups was measured using the MTT assay.2) The microplate method was used to measure the activities of T-SOD and Mn-SOD in the culture medium.3) The LDH levels in cell culture medium were measured.4) Fluorescence probe JC-1 was used to assess mitochondrial transmembrane potential.5) The confocal microscope was used to measure the fluorescence intensity of intracellular calcium by Fluo-3/AM.6) Cell apoptosis was measured using FITC Annexin V Apoptosis Detection Kit I for ?ow cytometry.7) Using Bradford Protein Assay kit and Caspase 3 Activity Assay Kit to determine the caspase 3 activities in different groups.8) The phosphorylation of JAK2 and STAT3 were measured by western blot.9) The content of cytochrome C in the mitochondria and cytoplasm were measured by western blot.Results:1. Cell ViabilityH/R decreases significantly the viability of cells after hypoxia and reoxygenation compared with the C group. In the S1P group, the vaibility of cells were increased significantly.AG490 or stattic was pretreated, the viability of cells was decreased significantly than that in the S1P group.2. The Activities of SODCompared with the C group, the activities of T-SOD and Mn-SOD in H/R group were decreased significantly. As treated with S1P, the activity of T-SOD and Mn-SOD were increased. Administration of AG or ST, they were attenuated significantly.3. The LDH levelsH/R increases significantly the activity of LDH in the H/R group compared with the C group. When treated with S1P, the LDH level was decreased significantly.Administration of AG490 or stattic, caused significant enhancement of LDH activity.4. The changes of these groups in the mitochondrial transmembrane potential(?Ψm)The green/red ratio of fluorescent intensity in the group of cells exposed H/R was improved markedly compared with the C group. When treated with S1P, the ratio was decreased significantly. In the S1P+AG group or the S1P+ST group the ratio was raised again.5. The fluorescence intensity of intracellular calcium ionIn controls, the intracellular Ca2+ concentration was maintained at a basal level of 8.895±1.079. Exposure of H9c2 cells to H/R leaded to a remarkable raise in fluorescence from intracellular Ca2+. Supplementation with S1P, caused an obvious attenuated ion of the fluorescence intensity. Compared with the S1P group, incresed fluorescence intensity occurred in the S1P+AG group and in the S1P+ST group.6. The percentage of apoptotic cellsThe percentum of apoptotic cells in the C group was low; and that was markedly enhanced in the H/R model group. Meanwhile, in the presence of S1P, the rate of apoptotic cells was markedly reduced. However, when cells were cotreated with S1P and AG or S1P and ST, the percentages of apoptotic cells were markedly higher.7. The Caspase 3 activityAn increase in caspase 3 activity was observed after exposing cells to HR. When treated with S1P, the value was increased significantly. After AG or ST pretreatment,the value was significantly reduced.8. The expression of p-JAK/t-JAK and p-STAT/t-STAT1) Effect of S1P and AG on the expression of p-JAK/t-JAK, in H9c2 cells following H/RThe notable augment of the p-JAK2 expression was analysed in the cells after H/R than that in the C group. And a remarkably increase in the S1P group compared with the H/R group. AG490 could suppress the phosphorylation of JAK2.2) Effect of S1P and AG on the expression of p-STAT/t-STAT in H9c2 cells following H/RThe notable augment of the p-STAT3 expression was tested in the cells after H/R than that in the C group. And a remarkable increase in the S1P group compared with the H/R group. AG490 could significantly suppress the phosphorylation of STAT3.3) Effect of S1P and ST on the expression of p-STAT/t-STAT in H9c2 cells following H/RThe notable augment of the p-STAT3 expression was tested in the cells suffered H/R than that in the C group. And a remarkable increase in the S1P group compared with the H/R group. Stattic could significantly suppress the phosphorylation of STAT3.9. The content of cytochrome C in mitochondria and cytoplasm1) Effect of S1P, AG and stattic on the content of cytochrome C in the cytoplasm in H9c2 cells following H/RThe notable augment of the content of cytochrome C in the cytoplasm was tested after H/R than that in the C group. And a remarkable lessening in the S1P group compared with the H/R group. AG490 or stattic could suppress the effect of S1P.2) Effect of S1P, AG and stattic on the content of cytochrome C in the mitochondria in H9c2 cells following H/RThe notable reduction of the content of cytochrome C in the mitochondria was analysed in the cells suffered H/R than that in the C group. And a remarkable augement in the S1P group compared with the H/R group. AG490 or stattic could suppress the effect of S1P.Conclusion:1. The treatment of S1P could remarkably raise the cell viability, make a reduction of apoptosis, decrease the content of LDH and caspase3 activity in the culture medium, increase the activity of T-SOD and Mn-SOD, reduce the loss of mitochondrial membrane potential and mitochondrial cytochrome C as well as the concentration of intracellular calcium, which indicates that S1P protects H9c2 cells against hypoxia/reoxygenation injury.2. S1P could significantly increase the phosphorylation of JAK2 and STAT3 compared with the control H/R group. When the JAK inhibitor AG490 or the STAT inhibitor stattic were added, the effects of S1P were inhibited. Therefore S1P protects H9c2 cells against hypoxia/reoxygenation injury via JAK-STAT pathway. |
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