| Objective:To investigate the protective effects of sphingosine1-phosphate (SIP) postconditioning on rat myocardial cells injured by hypoxia reoxygenation and to explore the mechanism of protective effects involve in PI3K/Akt and ERK1/2signaling pathway.Methods:1. Hypoxia reoxygenation was administered to establish the injury model of H9c2cellsH9c2cells were cultured for24hours with DMEM medium containing10%FBS. Then replace normal culture medium by simulated hypoxia fluid. The culture medium was pre-saturated by95%N2and5%CO2. Then put it in hypoxic incubator (95%N2and5%CO2) for Several hours in37℃. For reoxygenation, cardiomyocytes cells exposed to hypoxia were put in DMEM medium without containing FBS. Culture it under normal conditions for several hours. The viability of H9c2cells was detected using MTT method. The appropriate time was choosen to establish the hypoxia/reoxygenation injury model.2. The protective effect of SIP postconditioning on hypoxia/reoxygenation-indued injury involving PI3K/Akt signaling pathway in H9c2cells.The cultured rat H9c2cells were randomly divided into five groups:(1) control group;(2) hypoxia/reoxygenation (H/R) group;(3) S1P group;(4) S1P+LY group;(5) LY group; Cells in control group were cultured without FBS.Cells in H/R group receive hypoxic for16h and reoxygenation for4h; Cells in SIP group was treated with4μM S1P though reoxygenation for4h; Cells in S1P+LY group pretreat10μM LY2940002(inhibitor of PI3K/Akt) for30min. then add4μM SIP though reoxygenation for3.5h; Cells in LY group was treated with10μM LY2940002though reoxygenation for4h. The viability of H9c2cells were detected using MTT method. The content of MDA in the culture medium and the activity of T-SOD and Mn-SOD, were measured with colorimetry. The concentration of intracellular free calcium ion were labeled by Fluo-3AM, then using confocal microscopy to detect the fluorescence intensity. Using flow cytometric analysis, the rate of cell apoptosis were determined. Western blot was performed to exam expression of p-Akt and t-Akt in H9c2cells respectively.3. The protective effect of S1P postconditioning on hypoxia/reoxygenation-indued injury involving ERK1/2signaling pathway in H9c2cells.The cultured rat H9c2cells were randomly divided into five groups:ditto for (1)～(3);(4) S1P+PD group;(5) PD group; Cells in S1P+PD group pretreat50μM PD98059(inhibitor of ERK1/2) for30min, then add4μM S1P though reoxygenation for3.5h; Cells in PD group was treated with50μM PD98059though reoxygenation for4h. The viability of H9c2cells were detected using MTT method. The content of MDA in the culture medium and the activity of T-SOD and Mn-SOD, were measured with colorimetry. The concentration of intracellular free calcium ion were labeled by Fluo-3AM, then using confocal microscopy to detect the fluorescence intensity. Using flow cytometric analysis, the rate of cell apoptosis were determined. Western blot was performed to exam expression of p-Akt and t-Akt in H9c2cells respectively.Results:1. Hypoxia reoxygenation was administered to establish the injury model of H9c2cells After hypoxia/reoxygenation injury, cells were shrinking and rupturing. Compared with control group (100%±0), the vaibility of cells in hypoxia/reoxygenation group (67.2%±5.62%) decreased significantly after being in hypoxia for16h and reoxygenation for4h. The model was used in the following experiments due to good reproducibility of experimental results.2. The protective effect of S1P postconditioning on hypoxia/reoxygenation-indued injury involving PI3K/Akt signaling pathway in H9c2cells.Compare with the H/R group, in S1P group, the vaibility of cells were increased significantly (73.13%±1.21%vs60.44±3.19%.P<0.01); the content of MDA in the culture medium was decreased significantly (2.94±0.072nmol/L vs1.57±0.050nmol/L, P<0.01); the activity of T-SOD (19.46±1.31U/ml vs10.67±1.44U/ml, P<0.01) and Mn-SOD (9.24±0.77U/ml vs6.60±0.97U/ml, P<0.01) were increased significantly; the fluorescence intensity of intracellular calcium was decreased significantly (0.75±0.043vs2.77±0.11, P<0.01); the rate of apoptosis was decreased significantly (16.80%±0.57%vs28.83%±1.23%, P<0.01).Compare with the SIP group, in group pretreated with LY294002, LY+SIP group, the vaibility of cells were decreased significantly (63.41%±2.74%vs73.13%±1.21%, P<0.01); the content of MDA in the culture medium was increased significantly (3.04±0.097nmol/L vs1.57±0.050nmol/L, P<0.01); the activity of T-SOD (13.60±1.46U/ml vs19.46±1.31U/ml, P<0.01) and Mn-SOD (5.01±0.61U/ml vs9.24±0.77U/ml, P<0.01) were decreased significantly; the fluorescence intensity of intracellular calcium was increased significantly (1.27±0.038vs0.75±0.043, P<0.01); the rate of apoptosis was increased significantly (23.46%±0.91%vs16.80%±0.57%, P<0.01).Compared with control group, the expression of p-Akt protein in H/R group was increased obviously (P<0.01); compared with the H/R group, p-Akt protein expression was increased obviously in S1P group (P<0.01); when added LY294002, the p-Akt protein expression was decreased significantly (P<0.01).3. The protective effect of S1P postconditioning on hypoxia/reoxygenation-indued injury involving ERK1/2signaling pathway in H9c2cells.Compare with the S1P group, in group pretreated with PD98059, PD+S1P group, the vaibility of cells were decreased significantly (62.64%±4.54%vs73.13%±1.21%, P<0.01); the content of MDA in the culture medium was increased significantly (2.94±0.072nmol/L vs1.57±0.050nmol/L, P<0.01); the activity of T-SOD (13.54±2.53U/ml vs19.46±1.31U/ml, P<0.01) and Mn-SOD (5.11±0.64U/ml vs9.24±0.77U/ml, P<0.01) were decreased significantly; the fluorescence intensity of intracellular calcium was increased significantly (1.27±0.032vs0.75±0.043, P<0.01); the rate of apoptosis was increased significantly (27.6%±0.83%vs16.80%±0.57%, P<0.01).Compared with control group, the expression of p-ERK1/2protein in H/R group was increased obviously (P<0.01); compared with the H/R group, p-ERK1/2protein expression was increased obviously in SIP group (P<0.01); when added PD98059, the p-ERK1/2protein expression was decreased significantly (P<0.01). Conclusion:1. On the condition of hypoxia for4h and reoxygenation for16h, the injury model of H9c2cells induced by the hypoxia/reoxygenation was established successfully with good reproducibility.2.4μM SIP could increase the vaibility and the activities of antioxidant enzyme of H9c2cells; decrease the concentration of intracellular calcium ion; and reduce the peroxidation injury and apoptosis; then it protects H9c2cells against hypoxia/reoxygenation injury.3. It is the first discovery that SIP promotes the expression of p-Akt in H9c2cells injuried by H/R. The protection of SIP was inhibited by LY294002, the inhibitor of PI3K/Akt. SIP protects H9c2cells against hypoxia/reoxygenation injury through activating PI3K/Akt signaling pathway. SIP could also promote the expression of p-ERK1/2in H9c2cells injuried by H/R. The protection of SIP was inhibited by PD98059, the inhibitor of ERK1/2. SIP also activates ERK1/2signaling pathway to protect H9c2cells against hypoxia/reoxygenation injury. |
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