| Objective:To study the early effect of NADPH on human umbilical vein endothelial cells(HUVECs)and the expression of occludin and MMP9 induced by oxygen glucose deprive and reoxygenation(OGD/R).Methods: HUVECs were cultured and divided into blank control group,OGD/R group,and OGD/R+NADPH 20 umol/L group.The total experiment was devided into three parts.First,we designed to observe the change of different treated groups.The proliferation after treatment was detected by CCK8 assay.Second,we detected the oxidative stress level in different treated groups.The cytotoxicity was detected by LDH release assay.MDA?SOD were detected by commercially availabley kit.DHE was used to detecte ROS in cells.Then,The expression of occludin ?MMP9 and Bcl-2 were detected by Western Blot.Results:1.15mmol/LGSH could improve the viability of normal HUVECs lightly(1 ± 0.05 vs 1.31 ± 0.0.7,P<0.05).High concentration GSH could enhanced the viability of normal HUVECs obviously(1 ± 0.05 vs 1.41 ± 0.09,2.11 ± 028,P<0.05).Low dosages of GSH had no effect on HUVECs induced by OGD/R,while high concentration GSH could improve the viability of HUVECs induced by OGD/R obviously(0.59 ± 0.0.4 vs 1.01 ± 0.12,1.68 ± 0.19,P<0.05).2.High concentration ALA had a negative effect on normal HUVECs(0.89±0.06,vs 0.72±0.07,0.74±0.07,0.65±0.04,0.54±0.06,P<0.05).Different concentration of ALA had no effect on HUVECs induced by OGD/R groups(0.52±0.08 vs 0.46±0.04,0.48±0.06,0.47±0.04,0.49±0.06,0.51±0.04,0.56±0.05,0.58±0.08,0.60±0.03,P>0.05).3.NADPH have no effect on normal HUVECs viability(OD: 1.02±0.08 vs 1.06±0.09 1.14±0.10,1.12±0.15,1.12±0.12,1.01±0.08,P>0.05),while enhanced the cell viability induced by OGD/R in a dose-dependently way(OD:0.51±0.07 vs 0.53±0.05,0.58±0.07,0.62±0.04,0.68±0.03,0.68±0.09 P<0.05).NADPH showed a better effect than ALA and GSH.4.NADPH impacts the release of LDH in a dose-depedently way on both nomal group(inhibition of percentage:(100±11.99)% vs(95.30±5.96)%(81.94±10.22)%,(80.39±4.96)%,(72.05±7.98)%,(69.83±8.20)%,P<0.05)and model group(inhibition of percentage :(201.34±15.14)% vs(186.98±16.38)%,(187.23±18.56)%,(176.15±12.17)%,(154.78±12.97)%,(158.46±8.25)%,P.<0.05).5.The HUVECs morphology was disturbed by OGD/R.NADPH can promote the remediation of the impaired morphology.6.The vitality of SOD was damaged by OGD/R(8.29±0.71 U/mg pro vs 4.88±0.69U/mg pro,P<0.05),while it could be proved by the use of NADPH(4.88±0.69 U/mg pro vs 6.97±0.46U/mg pro,P<0.05).The content of MDA increased to twice of normal(11.11±1.22 ug/mg pro vs 26.73±2.54 ug/mg pro),the intervene of NADPH could impede this change(26.73±2.54 ug/mg pro vs 13.32±2.04 ug/mg pro).7.ROS increased apparently after reperfusion;while NADPH could restrain ROS addition.8.The down regulation of occludin and up regulation of MMP9 could be backspined partly by NADPH.9.The down regulation of Bcl-2 could also backspined by NADPH.Conclusion:1.NADPH could protect HUVECs injuryed by oxygen and glucose deprivationand reperfusion;2.NADPH showed a better effect than GSH and ALA;3.NADPH could protect HUVECs by suppressing oxidative stress;4.NADPH could protect HUVECs TJ by regulating the expression of occludin and MMP9,;5.NADPH showed a protective effect by increasing the expression of Bcl-2?... |
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