葛根素对过氧化氢诱导的血管内皮细胞损伤的保护作用

Protective effect of Puerarin on the injury of human vascular endothelial cells induced by hydrogen peroxide in vitro

目的探讨葛根素对过氧化氢诱导人血管内皮细胞(HUVECs)损伤的保护作用及其机制。方法培养HUVECs并分组:正常组(不加任何干预因素);过氧化氢组(在细胞培养体系中加入终浓度为0.5mmol/L过氧化氢);葛根素25mg/L、50mg/L、100mg/L、200mg/L组(先分别加入相应质量浓度葛根素干预30min后,再加入过氧化氢)。各组培养24h后,通过MTT法检测细胞增殖能力;检测纤溶酶原激活物(t-PA)及纤溶酶原激活剂抑制物(PAI)活性观察HUVECs功能;取培养上清液测定超氧化物歧化酶(SOD)活性及丙二醛(MDA)的含量;采用Annexin V/PI染色后,应用流式细胞仪检测HUVECs凋亡率,并检测caspase-3表达水平与线粒体膜电位变化。结果与正常组比较,过氧化氢可明显降低HUVECs增殖活性(P<0.01),t-PA活性下降及PAI活性增加;而预防应用不同浓度葛根素能够不同程度对抗其作用,且最佳保护浓度是100mg/L。与过氧化氢组比较,葛根素能够显著降低HUVECs的MDA水平、细胞凋亡率、caspase-3阳性率及线粒体损伤率,能够显著提高HUVECs的SOD水平(P<0.05)。结论葛根素对氧化应激诱导HUVECs损伤具有一定的保护作用,其机制可能与其抗氧化、维持线粒体膜电位、减低caspase-3表达而抑制细胞凋亡有关。

Objective To study the effect of Puerarin on the injury of human vascular endothelial cells(HUVECs) induced by hydrogen peroxide in vitro and its regulation mechanisms. Methods HUVECs were treated with 0.5 mmol/L hydrogen peroxide,therefore,a cell model for oxidative damage was established.Puerarin before H2O2-treatment was as an appropriate intervention.HUVECs were divided into three groups: normal control,hydrogen peroxide treatment,and Puerarin pretreatment groups.The proliferation was determined by the MTT assay.Functions of HUVECs were detected by assaying the activity of t-PA and PAI-1.SOD and MDA was also detected.After annexin V/PI staining,apoptotic rate of HUVECs was detected by flow cytometry(FCM) and expression of caspase-3 and membrane potential of mitochondria were detected by FCM. Results Puerarin could protect hydrogen peroxide induced HUVECs injury in the dose-dependent manner.The Puerarin concentration of 100 mg/L was determined as the optimal effective concentration.Compared with hydrogen peroxide groups,Puerarin decreased evidently MDA level,apoptotic rate,caspase-3 positive rate,and mitochondrial injury rate of HUVECs(P<0.05).And puerarin markedly elevated SOD activity(P<0.05). Conclusion Puerarin can protect HUVECs against hydrogen peroxide-induced injury.The mechanisms could be related to anti-oxidative effect,maintenance of membrane potential of mitochondria,reduction of caspase-3 activity and apoptosis.