Protective Effects Of Sphingosine-1-Phosphate Postconditioning On Hypoxia/Reoxygenation Injury In Cultured Rat H9c2Cardiocytes

Objective:To investigate the protective effects of sphingosine1-phosphate (SIP) postconditioning on rat myocardial cells injured by hypoxia reoxygenation, and its mechanisms.Methods:1. Cell culture and establishment of cell hypoxia reoxygenation injury modelUsing convention adherent cell culture methods,the rat H9c2cells were cultured in DMEM medium with10%South American fetal bovine serum. The H9c2cell strain in rats was conventionally cultured in a37℃,5%CO2incubator. Medium was replaced and cell subculture every2-3days.Replace normal culture medium by simulated hypoxia fluid. The culture medium was pre-saturated for10minutes by95%N2and5%CO2. Then put it in hypoxic incubator (95%N2and5%CO2)for16h in37℃; For reoxygenation, cardiomyocytes cells exposed to hypoxia were put in drug-containing low glucose DMEM medium with95%air and5%CO2. Culture it under normal conditions for4h and establish hypoxia/reoxygenation injury model.2. The protective effect of SIP postconditioning on rat cardiomyocytes during hypoxia and reoxygenation injuryThe cultured rat H9c2cells were randomly divided into five groups:(1) the normal (control) group;(2) hypoxia/reoxygenation (H/R) group;(3) SIP low concentration group (L);(4) SIP medium concentration group (M);(5) SIP high concentration group (H). Cells in normal control group were cultured without FBS.Cells in H/R group receive hypoxic for16h and reoxygenation for4h; The rest of the group was treated with corresponding concentrations of the drug though reoxygenation for4h. The final concentration was2mM,4mM,6mM, respectively.3. The protective mechanisms of S1P postconditioning on rat H9c2cells hypoxia reoxygenation injuryChanges in cell morphology were observed with optical microscope. MTT method was used to measure cell survival. Using flow cytometric analysis, the rate of cell apoptosis were determined. The activity of total superoxide dismutase (Total Super Oxide Dismutase, T-SOD) and Copper/Zinc Super Oxide Dismutase(Copper/Zinc Super Oxide Dismutase,CuZn-SOD) and manganese superoxide dismutase (Manganese Super Oxide dismutase, Mn-SOD) activity and malondialdehyde (Malondialdehyde, MDA) content in cell culture medium were measured with colorimetry. The concentration of intracellular calcium ion in cell were observed with fluorescence microscope. HSP70protein expression levels in H9c2cells were observed with Western Blot method.Results:1. H9c2cells were spindle with good refractive index. After hypoxia/reoxygenation injury, cells were shrinking and rupturing. Compared with control group, the survival rate of cells in hypoxia/reoxygenation group decreased significantly after being in hypoxia for16h and reoxygenation for4h, with stable and moderate reduced degree. The model was used in the following experiments due to good reproducibility of experimental results.2. The survival rate of H9c2cells:Compared with normal group, the cell survival rate of H/R group decreased significantly (68.72%±6.02%vs100%±0%, P <0.01); Compared with H/R group, the cell survival rate of S1P low, medium and high concentration group increased significantly, with obviously reduced injury (73.90%±10.77%vs68.72%±6.02%,80.15%±9.78%vs68.72%±6.02%,80.85%±9.50%vs68.72%±6.02%, P<0.01, P<0.01, P<0.01)3. MDA content in the cell culture medium:Compared with normal group, the MDA content of the H/R group significantly increased (2.55nmol/L±0.132nmol/L vs1.37nmol/L±0.097nmol/L, P<0.01); Compared with H/R group, MDA content decreased significantly in SIP low, medium and high concentration group (1.98nmol/L±0.125nmol/L vs2.55nmol/L±0.132nmol/L,1.71nmol/L±0.095nmol/L vs2.55nmol/L±0.132nmol/L,1.67nmol/L±0.193nmol/L vs2.55nmol/L±0.132nmol/L, P<0.01, P<0.01, P<0.01)。4. Total SOD activity in cell culture medium:compared with normal group, the total SOD activity of the H/R group decreased significantly (11.18U/ml±1.15U/ml vs21.14U/ml±1.33U/ml, P<0O01); compared with the H/R group, the total SOD activity increased significantly in S1P low, medium and high concentration group (14.00U/ml±0.89U/ml vs11.18U/ml±1.15U/ml,17.77U/ml±1.60U/ml vs11.18U/ml±1.15U/ml,18.45U/ml±1.52U/ml vs11.18U/ml±1.15U/ml, P<0.05,P<0.01,P<0.01)。CuZn-SOD activity in cell culture medium:compared with normal group, the CuZn-SOD activity of the H/R group decreased significantly (5.61U/ml±0.25U/ml vs11.87U/ml±0.61U/ml, P<0.01); compared with the H/R group, the CuZn-SOD activity increased significantly in SIP low, medium and high concentration group (6.95U/ml±0.98U/ml vs5.56U/ml±1.12U/ml,9.11U/ml±1.58U/ml vs5.56U/ml±1.12U/ml,9.46U/ml±1.59U/ml vs5.56U/ml±1.12U/ml,P<0.01, P<0.01, P<0.01)。Mn-SOD activity in cell culture medium:compared with normal group, the Mn-SOD activity of the H/R group decreased significantly (5.56U/ml±1.12U/ml vs9.27U/ml±1.07U/ml, P<0.01); compared with the H/R group, the Mn-SOD activity increased significantly in SIP low, medium and high concentration group (6.95U/ml±0.98U/ml vs5.56U/ml±1.12U/ml,9.11U/ml±1.58U/ml vs5.56U/ml±1.12U/ml,9.46U/ml±1.59U/ml vs5.56U/ml±1.12U/ml, P<0.01, P<0.01, P<0.01)。5. Determination of intracellular free calcium fluorescence intensity:the fluorescence intensity of the normal group maintained at62.04±1.61level; the fluorescence intensity of the H/R group increased significantly after hypoxia/reoxygenation injury, and with significant difference compared with the normal group (82.65±2.21vs62.04±1.61,P<0.01). After S1P intervention, the cell fluorescence intensity of S1P low, medium and high concentration group decreased in some degree (77.76±0.45vs82.65±2.21,72.97±0.97vs82.65±2.21,69.96±2.02vs82.65±2.21, P<0.01, P<0.01, P<0.01).6. Flow cytometric analysis of the determination of the rate of apoptosis; Cell apoptosis occurred less often in normal group. Cell apoptosis increased significantly in H/R group compared with the normal group (20.2%±9.37%vs5.6%±3.27%, P<0.01); Cell apoptosis rate decreased in S1P low, medium and high concentration group compared with H/R group(13.7%±11.78%vs20.2%±9.37%,10.5%±8.66% vs20.2%±9.37%,10.1%±7.39%vs20.2%±9.37%, P<0.05, P<0.01, P<0.01).It suggests that different concentrations of SIP can effectively inhibit H9c2cell apoptosis caused by hypoxia/reoxygenation injury.7. Expression of HSP70protein:compared with normal group, the expression of HSP70protein in H/R group obviously increased; compared with the H/R group, HSP70protein expression increased obviously in all the concentrations group of S1P.Conclusion:1. Hypoxia/reoxygenation injury has a strong inhibited effect on H9c2cell proliferation. And a certain concentration and time dependence is presented. Injury is moderate and the results are stable through hypoxia for4h and reoxygenation for16h, with good reproducibility. It apply to the injury model.2. SIP can decrease cell apoptosis rate of H9c2after hypoxia/reoxygenation injury, with a certain concentration dependence.3. The protection of SIP for cell apoptosis of H9c2after hypoxia/reoxygenation injury may be related to decrease the intracellular MDA content and intracellular calcium ion concentration, improve intracellular SOD activity and enhance expression of HSP70anti-apoptotic protein.