山奈酚联合环孢素A抑制人滋养细胞氧化应激损伤的研究

Study of integrated kaempferol and cyclosporin A in preventing oxidative stress injury of human trophoblast

目的探讨山奈酚联合环孢素A(Cs A)对人滋养细胞氧化应激损伤的影响及分子机制。方法以人绒毛膜上皮癌细胞系JEG-3为载体,构建滋养细胞氧化应激模型。实验分为对照组、H_2O_2组、Cs A组(Cs A+H_2O_2)、山奈酚组(山奈酚+H_2O_2)、山奈酚联合Cs A组(山奈酚联合Cs A+H_2O_2)。MTT和Transwell法检测细胞活力和侵袭力;试剂盒法检测总过氧化物歧化酶(TSOD)、过氧化氢酶(CAT)、活性氧(ROS)及丙二醛(MDA)水平;Western blot法检测核因子E2相关因子2(Nrf2)及血红素加氧酶1(HO-1)的表达水平;小干扰RNA沉默Nrf2表达。结果 1山奈酚联合Cs A组的细胞活力为(88.3±1.4)%,显著高于Cs A组[(78.7±2.8)%]和山奈酚组[(76.7±2.1)%](P<0.05);2山奈酚联合Cs A组的细胞侵袭力为(71.4±4.0)%,显著高于Cs A组[(64.4±2.5)%]和山奈酚组[(60.9±2.9)%](P<0.05);3与Cs A组和山奈酚组相比,山奈酚联合Cs A组TSOD、CAT、Nrf2和HO-1水平显著升高(P<0.05),MDA和ROS水平降低(P<0.05);4沉默Nrf2会显著降低山奈酚联合Cs A对细胞活力和侵袭力的促进作用(P<0.05)。结论山奈酚联合Cs A对滋养细胞的保护作用优于单独给药治疗,其机制与上调Nrf2蛋白表达有关。

Objective To investigate the effects of integrated kaempferol and cyclosporine A（Cs A） on oxidative stressinduced injury of human trophoblast and the molecular mechanism. Methods Oxidative stress model was established using human choriocarcinoma cell line JEG-3. The experiments were divided into the control group, H2O2 group, Cs A group（Cs A ＋H2O2）, kaempferol group（kaempferol ＋H2O2） and integrated kaempferol and Cs A group（integrated kaempferol and Cs A ＋H2O2）. Then cell viability and invasion ability were detected by MTT and Transwell invasion assay, respectively. Levels of total superoxide dismutase（TSOD）, catalase（CAT）, reactive oxygen species（ROS） and malondialdehyde（MDA） were measured using respective kits. Protein levels of nuclear factor-erythroid 2 related factor 2（Nrf2） and heme oxygenase-1（HO-1） were analyzed by Western blot. In addition, Nrf2 gene was silenced by small interference RNA. Results 1Cell viability value of integrated kaempferol and Cs A group [（88.3±1.4）%] was higher than that of Cs A group [（78.7 ±2.8）%] and kaempferol group [（76.7 ±2.1）% ]（P 〈 0.05）. 2 Invasion ability of integrated kaempferol and Cs A group [（71.4 ±4.0）% ] was higher than that of Cs A group [（64.4 ±2.5）% ] and kaempferol group[（60.9±2.9）%]（P 〈 0.05）. 3Compared with kaempferol group and Cs A group（P 〈 0.05）, levels of TSOD, CAT, Nrf2 and HO-1 significantly increased in integrated kaempferol and Cs A group（P 〈 0.05）, whereas levels of MDA and ROS decreased（P 〈 0.05）. 4Knockdown of Nrf2 significantly blocked the promotive effect of integrated kaempferol and Cs A on cell viability and invasion ability（P 〈 0.05）. Conclusion Compared to single drug treatment, integrated kaempferol and Cs A can protect trophoblast from injury more effectively, which may be associated with up-regulation of Nrf2 protein expression.