| Objective:To investigate the protective effects of sphingosine1-phosphate (SIP) postconditioning on human umbilical vein endothelial cells injured by hypoxia reoxygenation, and its mechanisms.Methods:1. Cell culture and establish the injury model of HUVECsUsing convention adherent cell culture methods, the HUVECs cells were cultured in DMEM medium with10%FBS. The HUVECs cell was conventionally cultured in an atmosphere of5%CO2at37℃. Medium was replaced and cell subculture every2-3d.HUVECs were cultured in96-well plates with DMEM containing10%FBS for24h, then HUVECs were treated under different hypoxia/reoxygenation conditions. Cell viability was tested by MTT assay and proper hypoxia/reoxygenation time was determined.2. The protective effect of SIP postconditioning on HUVECs cells during hypoxia and reoxygenation injuryHUVECs were cultured in96-well plates. Randomly divid cells into four groups: the normal (control) group; SIP low concentration group (L); S1P medium concentration group (M); SIP high concentration group (H). HUVECs cells were cultured in DMEM medium with10%FBS for24h, then Cells in normal control group were cultured in DMEM medium with10%FBS for24h. The rest of the group was treated with corresponding concentrations of the drug for24h. The final concentration was2μM,4μM,6μM, respectively. Cell viability was tested by MTT assay and observe whether S1P can affect cell proliferation.HUVECs were cultured in96-well plates. Randomly divid cells into five groups: the normal (control) group; hypoxia/reoxygenation (H/R) group; SIP low concentration group (L); S1P medium concentration group (M); S1P 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 was2,4,6μM, respectively. Cell viability was tested by MTT assay and observe whether S1P can protect HUVECs cells from H/R injury.3. The protective mechanisms of SIP postconditioning on HUVECs cells hypoxiare/oxygenation injuryChanges in cell morphology was 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 (T-SOD) and Copper/Zinc superoxide dismutase (CuZn-SOD) and manganese superoxide dismutase (Mn-SOD) activity and nitric oxide (NO) and malondialdehyde (MDA) content in cell culture medium were measured with colorimetry. Mitochondrial membrane potential in cell was observed with fluorescence microscope. Bax/Bcl-2, eNOS protein expression levels in HUVECs cells were observed with Western Blot method.Results:1. Cell culture and establish the injury model of HUVECsHUVECs cells were grows steadily and ranged as paving stones form normally. After16h of hypoxia and4h of reoxygenation, HUVECs were shrinking and distortional. Compared with control, the survival rate of cells in hypoxia/reoxygenation group decreased significantly (66.23±3.87vs100±0%, P<0.05). With moderate decrease of cell viability and good reproducibility, H16/R4was appropriate for induction of H/R model on HUVECs.2. The protective effect of SIP postconditioning onHUVECs cells during hypoxia and reoxygenation injuryThe survival rate of HUVECs cells:Compared with normal group, the cell survival rate of H/R group decreased significantly (68.89±3.56,71.62±8.42,76.68±7.69,81.88±7.2vs100±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(71.62±8.42,76.68±7.69,81.88±7.29vs68.89±3.56%, P<0.01).3. The protective mechanisms of SIP postconditioning on HUVECs cells hypoxiare/oxygenation injuryTotal SOD activity:Compared with normal group, the total SOD activity of the H/R group decreased significantly(10.38±1.07vs24.78±1.56U/mL, P<0.01); compared with the H/R group, the total SOD activity increased significantly in S1P low, medium and high concentration group(15.22±0.98,16.19±1.48,18.76±1.68vs10.38±1.07U/mL,P<0.05).CuZn-SOD activity:Compared with normal group, the CuZn-SOD activity of the H/R group decreased significantly(5.63±0.37vs11.90±0.73U/mL, P<0.01); compared with the H/R group, the CuZn-SOD activity increased significantly in SIP low, medium and high concentration group(8.36±0.93,8.86±0.45,9.83±0.57vs5.63±0.37U/mL, P<0.05).Mn-SOD activity:Compared with normal group, the Mn-SOD activity of the H/R group decreased significantly(4.76±0.96vs12.87±1.48U/mL, P<0.01); compared with the H/R group, the Mn-SOD activity increased significantly in S1P low, medium and high concentration group(6.87±0.99,7.33±1.27,8.93±1.50vs4.76±0.96U/mL, P<0.05).MDA content:Compared with normal group, the MDA content of the H/R group significantly increased (2.960±0.148vs1.480±0.099nM, P<0.01); compared with the H/R group, the MDA content decreased significantly in S1P low, medium and high concentration group(2.52±0.160,2.30±0.128,1.70±0.173vs2.96±0.132nM, P <0.05).NO content:Compared with normal group, the NO content of the H/R group decreased significantly(0.93±0.07vs24.2±1.81μM, P<0.01); compared with the H/R group, the NO content increased significantly in SIP low, medium and high concentration group(1.86±0.13,7.45±0.86,13.04±1.42vs0.93±0.07μM, P<0.05).Annexin V-FITC/PI:Cell apoptosis occurred less often in normal group. Cell apoptosis increased significantly in H/R group compared with the normal group(25.84+4.11vs8.16Π1.27%, P<0.01); Cell apoptosis rate decreased in SIP low,medium and high concentration group compared with H/R group(21.48±5.04, 17.24±3.84vs,12.45±2.09vs25.84±4.11%, P<0.01).It suggests that different concentrations of SIP can effectively inhibit HUVECs cell apoptosis caused by hypoxia/reoxygenation injury.JC-1detecting mitochondrial membrane potential:Compared with normal group, Red fluorescence declined in H/R group, green fluorescence rised in H/R group, this showed the decline of mitochondrial membrane potential; compared with the H/R group, the red fluorescence rised in all the concentrations of S1P.Expression of Bcl-2/Bax、eNOS protein:Compared with normal group, the Bcl-2/Bax level was obviously down-regulated in H/R group; compared with the H/R group, Bcl-2/Bax protein expression increased obviously in all the concentrations group of S1P. Compared with normal group, the eNOS level was obviously down-regulated in H/R group; compared with the H/R group, eNOS protein expression increased obviously in all the concentrations group of S1P.Conclusion:1. Stable H16/R4injury model of HUVECs was established.2. SIP can decrease cell apoptosis rate of HUVECs after hypoxia/reoxygenation injury, with a certain concentration dependence.3.The protection of SIP for cell apoptosis of HUVECs after hypoxia reoxygenation injury may be related to decrease the intracellular MDA content and improve intracellular SOD activity, increase mitochondrial membrane potential and enhance expression of Bcl-2/Bax anti-apoptotic protein. S1P increased the content of NO, may related to increase the expression of eNOS protein. |
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