NADPH对人脐静脉内皮细胞缺糖缺氧复灌损伤早期的保护作用

The early protective effect of NADPH on OGD/R injury of human umbilical vein endothelial cells

目的探讨还原型辅酶Ⅱ(NADPH)对人脐静脉内皮细胞(HUVECs)缺糖缺氧复灌(OGD/R)损伤早期的保护作用及其对occludin、基质金属蛋白酶(MMP)9蛋白表达的影响。方法体外培养HUVECs系,建立稳定的HUVECs缺糖缺氧复灌损伤模型,实验分3组:正常对照组、缺糖缺氧复灌组、缺糖缺氧复灌+NADPH组。细胞增殖试剂盒(CCK-8法)检测HUVECs活力;细胞毒性试剂盒检测HUVECs内乳酸脱氢酶(LDH)释放;倒置显微镜观察HUVECs形态变化;比色法检测HUVECs裂解液中超氧化物歧化酶(SOD)活力及丙二醛(MDA)含量;Western Blot法检测各组occludin、MMP9蛋白的表达。结果与缺糖缺氧复灌组比,NADPH处理可提高缺糖缺氧复灌损伤后HUVECs的存活率(P<0.05),减少损伤后HUVECs中LDH释放(P<0.05),促进HUVECs形态的恢复,减少MDA生成(P<0.05),提高SOD活力(P<0.05);缺糖缺氧复灌处理后,给予NADPH处理可抑制MMP9水平上调(P<0.05),促进occludin水平恢复(P<0.05)。结论 NADPH对HUVECs具有保护作用,其保护作用与抑制氧化应激反应,调节MMP9、occludin蛋白的表达有关。

Objective To study the early protective effect of NADPH on human umbilical vein endothelial cells （HUVECs） and the expression of occludin and MMP9 induced by oxygen glucose deprivation and reoxygenation （OGD/R）. Methods HUVECs were cultured and divided into blank control group, OGD/R group and OGD/R＋NADPH 20 μmol/L group. The proliferation of HUVECs after treatment was detected by CCK-8 assay. The cytotoxicity was detected by LDH release assay. The morphological changes of HUVECs were observed by inverted microscope. Superoxide dismutase （SOD MDA） activity and malondialdehyde （MDA） were detected by commercially available kit. The expressions of occludin and MMP9 were detected by Western blot assay. Results Compared with the OGD/R, NADPH enhanced the cell viability significantly （P〈0.05）, reduced the release of LDH （P〈0.05）, promoted the maintance of HUVECs morphology, reduced MDA generation （P〈0.05） and increased SOD activity （P〈0.05）. Following OGD/R,the treatment of NADPH can inhibit MMP9 level （P〈0.05） and promote the recovery of occludin level （P〈0.05）. Conclusion NADPH can protect HUVECs from the damage induced by OGD/R by reducing oxidative stress and regulating the expressions of MMP9 and occludin.