辛芍组方对缺血再灌注损伤后神经细胞氧化应激及 NF-κB 信号通路的影响

摘要

目的：观察辛芍组方对缺血再灌注损伤后神经细胞的氧化应激反应及核因子κB（NF-κB）信号通路蛋白表达的影响，探讨辛芍组方发挥脑保护作用的机制。方法培养 PC12细胞，分为正常对照组、模型组及辛芍组方低、中高剂量组。模型组及辛芍组方各剂量组均以氧糖剥夺／复氧损伤方法建立缺血再灌注损伤模型，造模后辛芍组方低、中、高剂量组分别加入辛芍组方0．31、0．62、1．25 mg／L，均干预4 h。正常对照组及模型组不干预。采用ELISA 法检测各组细胞内超氧化物歧化酶（SOD）及丙二醛（MDA），采用 Western blot 法检测各组细胞内诱导型一氧化氮合酶（iNOS）、环氧合酶2（COX-2）及 NF-κB 信号通路相关蛋白 IκBα、p-IκBα、p65蛋白表达。结果与正常对照组比较，模型组细胞内 SOD 水平降低、MDA 水平升高（P 均＜0．01）。与模型组比较，辛芍组方中、高剂量组细胞内 SOD 水平均升高（P ＜0．05或0．01），辛芍组方各剂量组细胞内 MDA 水平均降低（P ＜0．05或0．01）。与正常对照组比较，模型组 iNOS、COX-2蛋白表达均升高（P 均＜0．01）；与模型组比较，辛芍组方各剂量组 iNOS、COX-2蛋白表达均降低（P ＜0．05或0．01）。与正常对照组比较，模型组细胞质 p-IκBα／IκBα值及细胞核内 p65蛋白表达均增加（P 均＜0．01）；与模型组比较，辛芍组方各剂量组 p-IκBα／IκBα值、p65蛋白表达均降低（P ＜0．05或0．01）。结论辛芍组方对缺血再灌注损伤后的 PC12细胞具有保护作用，其机制可能与减轻细胞内氧化应激反应、抑制NF-κB 通路的活化有关。%Objective To observe the effect of the formula of Herba Erigerontis and Radix Paeoniae Rubra in oxygen-glucose deprivation/reoxygenation (OGD/R)on oxidative stress and nuclear factor-κB (NF-κB)signaling pathway of nerve cells (PC12 cells)after ischemia-reperfusion injury and to investigate its mechanism of protective effect.Methods The PC12 cells were cultured and divided into the control group,model group and low-dose,medium-dose and high-dose formu-la groups.The models of ischemia-reperfusion injury in the model group and formula groups were established by OGD/R. Then the rats in the low-dose,medium-dose and high-dose formula groups were treated with 0.31,0.62,and 1.25 mg/L formula of Herba Erigerontis and Radix Paeoniae Rubra,and all were intervened for 4 h.The control and model groups were treated with DMEMonly.The activity of superoxide dismutase (SOD)and the level of malondialdehyde (MDA)in cell supernatant were detected by ELISA.The protein expression of NO synthase (iNOS),cyclooxygenase-2 (COX-2)and the related proteins (IκBα,p-IκBαand p65)of NF-κB signaling pathway was examined by Western blotting.Results Compared with the control group,the level of SOD was decreased,the level of MDA was increased in the model group (all P <0.01).Compared with the model group,the level of SOD was increased in the medium-dose and high-dose formula groups (P <0.05 or P <0.01).The level of MDA was decreased in all formula groups (P <0.05 or P <0.01).Com-pared with the control group,the protein expression of iNOS and COX-2 was decreased in the model group (all P <0.01). Compared with the model group,the protein expression of iNOS and COX-2 was decreased in all formula groups (P <0.05 or P <0.01).Compared with the control group,the value of p-IκBα/IκBαin cytoplasm,and p65 in nucleus of the model group was increased (all P <0.01).Compared with the model group,the value of p-IκBα/IκBαin cytoplasm and p65 in nucleus of all formula groups (P <0.05 or P <0.01).Conclusions The formula has protective effect on PC12 cells after ischemia-reperfusion injury.The mechanism may be related to the reduction of intracellular oxidative stress response and inhibition of NF-κB pathway activation.