瑞芬太尼对过氧化氢处理脐静脉内皮细胞的抗氧化应激作用

摘要

Objective To establish oxidative stress model of hydrogen peroxide treatment by using human umbilical vein endothelial cells (ECS) as cell model to study the protective mechanism of anti oxidative stress and determine the signal transduction pathway of remifentanil.Methods Primary cultured human umbilical vein endothelial cells which were incubated with 0.1 M hydrogen peroxide to establish injury model to study remifentanil protection and related pathways.The experiment was divided into nine groups: control group (group C), Hydrogen peroxide group (group H1), Hydrogen peroxide+SP600125 group (group H2), Hydrogen peroxide+SB203580 group (group H3), Hydrogen peroxide+PD98059 group (group H4), hydrogen peroxide+remifentanil group (group HR1), hydrogen peroxide+remifentanil+SP600125 group (group HR2), hydrogen peroxide+remifentanil+SB203580 group (group HR3), hydrogen peroxide+remifentanil+PD98059 group (group HR4).Groups H1, H2, H3 and H4 only performed MAPK pathway blockade experiments.Groups HR1, HR2, HR3 and HR4 individually added remifentanil 10 ng/ml to protect 1 h.SOD activity, MDA level, Caspase-3 activity were detected and anti oxidative stress of remifentanil observed to confirm preliminary transduction pathway;Using RT-PCR expression levels of c-Jun before was observed before and after treated with remifentail 10 ng/ml.The aim was to determine the transduction pathway of the signaling molecules.Results Compared with group C, SOD activity were decreased significantly, MDA performance level were increased significantly in groups H1, H2, H3 and H4 (P<0.05).Compared with group H1, SOD activity was increased significantly, MDA performance level was decreased significantly in group HR1 (P<0.05).SOD activity difference and MDA performance level of groups HR2 and H2 had no statistical significance.Compared with group H3, SOD activity was increased significantly, MDA performance level was decreased siginificantly in group HR3 (P<0.05).Compared with group H4, SOD activity was increased significantly, MDA performance level was decreased significantly in group HR4 (P<0.05).Caspase-3 activity of groups H1, H2, H3 and H4 were higher significantly than that of group C (P<0.05).The level of C-Jun mRNA in group H1 was significantly higher than that of group C;But it was higher in group HR1 than that of group C, it was significantly lower than that of group H1 (P<0.05).Conclusion By activating the JNK pathway and its downstream signaling molecule c-Jun, remifentanil 10 ng/ml has the effect of increasing SOD activity, reducing the level of MDA expression and playing a role in anti oxidative stress.%目的 以人脐静脉内皮细胞为细胞模型,建立过氧化氢处理的氧化应激模型,研究瑞芬太尼的抗氧化应激保护机制,并确定信号转导通路.方法 过氧化氢0.1 mol/L孵育原代培养的人脐静脉内皮细胞,建立细胞损伤模型,然后进行瑞芬太尼保护及相关通路研究.实验共分为九组:空白对照组(C组)、过氧化氢组(H1组)、过氧化氢+SP600125组(H2组)、过氧化氢+SB203580组(H3组)、过氧化氢+PD98059组(H4组);过氧化氢+瑞芬太尼组(HR1组)、过氧化氢+瑞芬太尼+SP600125组(HR2组)、过氧化氢+瑞芬太尼+SB203580组(HR3组)、过氧化氢+瑞芬太尼+PD98059组(HR4组).H1组、H2组、H3组和H4组仅进行MAPK通路阻断实验,HR1组、HR2组、HR3组和HR4组分别加入瑞芬太尼10 ng/ml保护1 h.随后分别检测超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量及Caspase-3活性,观察瑞芬太尼抗氧化应激作用并初步确定转导通路;利用RT-PCR观察瑞芬太尼10 ng/ml处理前后c-Jun的表达水平,确定转导通路的信号分子.结果 H1、H2、H3、H4组SOD活性明显低于C组,MDA含量明显高于C组(P<0.05);HR1组SOD活性明显高于H1组,MDA含量明显低于H1组(P<0.05);HR2组与H2组SOD活性及MDA含量差异无统计学意义;HR3组SOD活性明显高于H3组,MDA含量明显低于H3组(P<0.05);HR4组SOD活性明显高于H4组,MDA含量明显低于H4组(P<0.05).H1、H2、H3、H4组Caspase-3活性明显高于C组(P<0.05).H1组和HR1组c-Jun mRNA表达量明显高于C组,且HR1组明显低于H1组(P<0.05).结论 瑞芬太尼10 ng/ml可激活JNK通路及其下游信号分子c-Jun,上调SOD活性,降低MDA含量,进而起到抗氧化应激作用.