基于Keap-1/Nrf2/ARE信号通路探讨片仔癀改善MCAO大鼠氧化应激损伤的作用

Effect of Pien Tze Huang on improving oxidative stress damage in MCAO rats based on Keap-1/Nrf2/ARE signaling pathway

目的:基于Keap-1/Nrf2/ARE信号通路探讨片仔癀(PTH)减轻大脑中动脉阻塞(MCAO)大鼠氧化应激损伤的作用及其机制。方法:36只SD大鼠随机分为假手术组、模型组和PTH组,每组12只。用线栓法制备MCAO大鼠模型。Zea-Longa法进行神经功能缺损评分;MRI法检测大鼠脑梗死体积;HE染色法观察大鼠脑组织病理形态;DCFH-DA探针法检测活性氧(ROS)含量;比色法测定丙二醛(MDA)、超氧化物歧化酶(SOD)含量;Western Blot法检测Keap-1、Nrf2、HO-1、NQO-1蛋白的表达。采用侧脑室注射Nrf2抑制剂Brusatol,30 min后造模,检测上述指标的表达。结果:与模型组比较,PTH组能显著改善大鼠神经功能损伤(P<0.05),改善大鼠脑组织病理状态,降低脑内MDA、ROS含量(P<0.01),升高SOD含量(P<0.01),同时下调Keap-1(P<0.05)、上调HO-1、NQO-1蛋白及核内Nrf2蛋白的表达(P<0.01)。经侧脑室注射Brusatol,逆转了PTH对以上指标的作用。结论:PTH可显著改善MCAO大鼠脑内氧化应激损伤,其机制主要与激活Keap-1/Nrf2/ARE通路有关。

Objective:To investigate the effect of Pien Tze Huang(PTH)against oxidative stress responses in MCAO rats by activating Keap-1/Nrf2/ARE signaling pathway.Methods:Thirty-six SD rats were randomly divided into Sham group,MCAO group and PTH group,12 in each group.Rat models of ischemic stroke were prepared by middle cerebral artery occlusion using suture method.Zea-Longa method was used to evaluate neurological deficit scores;MRI was used to observe cerebral infarction volume;the pathological morphology was observed by HE staining;the ROS level was detected by DCFH-DA probe method;the content of malondialdehyde(MDA)and superoxide dismutase(SOD)was determined by colorimetry;the protein expression of Keap-1,Nrf2,HO-1 and NQO-1 was detected by Western Blot.Meanwhile,MCAO rats were established after an intracerebroventricular injection of Brusatol,an inhibitor of Nrf2,for 30 minutes.The above indicators were measured.Results:Compared with the MCAO group,PTH group could significantly improve the neurological damage in rats(P<0.05),the neurons in the brain of PTH group were arranged neatly and the content of MDA,ROS in the brain was significantly reduced(P<0.01),while the content of SOD was significantly increased(P<0.01),PTH group inhibited the protein expression of Keap-1(P<0.05)and promoted the proteins expression of HO-1,NQO-1 and nuclear Nrf2(P<0.01).After injecting Brusatol,the effect of PTH on the above indicators was reversed.Conclusion:PTH can significantly inhibit oxidative stress injury in MCAO rats,and its underlying mechanism is mainly related to regulating Keap-1/Nrf2/ARE signaling pathway.