抗衰老Klotho蛋白对过氧化氢诱导的人脐静脉内皮细胞氧化损伤的保护作用及其机制

Protective effect of anti-aging Klotho protein on hydrogen peroxide induced oxidative damage in human umbilical vein endothelial cells and its mechanism

目的研究抗衰老Klotho蛋白对过氧化氢(H2O2)氧化损伤的人脐静脉内皮细胞(HUVEC)的影响及其作用机制。方法用H2O2诱导HUVEC构建氧化损伤模型。将HUVEC设置对照组、H2O2损伤组、不同浓度(1、10、100μg/L)Klotho蛋白组、丝氨酸-苏氨酸蛋白激酶(AKT)作用组。噻唑蓝法检测细胞存活率;特定试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)含量;酶联免疫吸附法检测一氧化氮(NO)、内皮素1(ET-1)、细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)、核因子κB(NF-κB)的含量;SA-β-半乳糖苷酶染色检测细胞衰老情况;流式细胞术检测活性氧(ROS)和细胞凋亡;Western blot检测Bcl-2、Bax、NF-κB p65及p-AKT的表达变化。结果与对照组相比,H2O2损伤组细胞存活率显著下降,LDH、MDA含量显著增加,SOD、GSH活性显著下降,NO含量下降,ET-1、ICAM-1、VCAM-1、NF-κB、细胞衰老率、ROS含量和细胞凋亡率显著增加,Bax蛋白表达上升而Bcl-2蛋白表达显著降低,NF-κB p65磷酸化水平显著升高而p-AKT/AKT水平下降(均P<0.05)。在不同浓度Klotho蛋白组,细胞存活率逐渐上升,LDH、MDA含量逐渐下降,SOD、GSH活性随之上升,NO含量上升,ET-1、ICAM-1、VCAM-1、NF-κB、细胞衰老率、ROS含量和细胞凋亡率逐渐下降,Bax蛋白、NF-κB p65磷酸化水平逐渐下降,Bcl-2蛋白、p-AKT/AKT水平逐渐上升(均P<0.05)。AKT作用组上述指标的检测结果与Klotho蛋白组相反。结论抗衰老Klotho蛋白能提升H2O2氧化损伤后HUVEC的存活率,增强细胞功能和抗氧化作用,减少细胞炎性反应、衰老和凋亡,其通过抑制Bax、NF-κB p65及促进Bcl-2、p-AKT/AKT而发挥作用。

Aim To study the effect of anti-aging Klotho protein on oxidative damage induced by hydrogen peroxide （H202 ） in human umbilical vein endothelial cell （HUVEC） and its mechanism. Methods The oxidative damage model was built by HUVEC treated by H202. HUVECs were individed into control group, H202 damage group, different concentrations （ 1, 10, 100 μg/L） Klotho protein groups and serine-threonine protein kinase （AKT） action group. Cell survival rate was detected by methyl thiazolyl tetrazolium assay. Lactate dehydrogenase （LDH）, malondialdehyde （MDA）, superoxide dismutase （SOD） and reduced glutathione （GSH） content were detected by specific Kit. The content of nitric oxide （ NO）, endothelin-1 （ ET-1 ）, intercellular cell adhesion molecule-1 （ ICAM-1 ）, vascular cell adhesion molecule-1 （VCAM-1） and nuclear factor-kappa B （NF-κB） were detected by enzyme-linked immunosorbent assay. SAbeta galactosidase staining was used to detect cell senescence. Reactive oxygen species （ROS） and cell apoptosis were determined by flow cytometry. Expression of Bcl-2, Bax, NF-KB p65 and p-AKT were detected by Western blot. Resuits Compared with the control group, the survival rate of HUVEC was significantly decreased, the contents of LDH and MDA were significantly increased, the activities of SOD and GSH were significantly decreased, the ET-1, ICAM-1, VCAM-1, NF-KB, ROS, cell senescence rate and apoptosis rate were significantly increased, the expression of Bax protein and phosphorylation level of NF-KB p65 were significantly increased but Bcl-2 protein and p-AKT/AKT decreased, in H2O2 damage group （all P〈0.05）. In different concentrations of Klotho protein groups, the cell survival rate was gradually increased, the contents of LDH and MDA were gradually decreased, the activities of SOD and GSH were increased, NO content was increased, the ET-1, ICAM-1, VCAM-1, NF-KB, ROS, cell senescence rate and apoptosis rate were gradually decreased, the Bax protein and phosphorylation level of NF-κB p65 were gradually decreased, the Bcl-2 protein and p- AKT/AKT were gradually increased （all P〈0.05）. The results of the above indexes in AKT action group were contrary to those of Klotho protein groups. Conclusions Anti-aging Klotbo protein can enhance the HUVEC survival rate after H2O2 induced oxidative damage, promote cell function and antioxidation, reduce cell inflammatory reaction, aging and apoptosis. It plays roles through inhibiting Bax, NF-KB p65 and promoting Bcl-2, p-AKT/AKT.