摘要
目的 研究补肾启智方通过调控Kelch样环氧氯丙烷相关蛋白1(Keap1)/核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)信号通路改善缺血性卒中大鼠认知功能的作用机制。方法 采用大脑中动脉栓塞法建立缺血性卒中大鼠模型。将雄性SD大鼠随机分为假手术组、模型组、补肾启智方低剂量组(3.6 g/kg)、补肾启智方高剂量组(14.4 g/kg),每组9只,连续灌胃14 d。采用Garcia JH法评价大鼠神经功能缺损程度;Morris水迷宫实验检测大鼠空间学习和记忆能力;Western blotting法检测大鼠缺血侧海马组织中Keap1、Nrf2及血红素氧合酶1(HO-1)的蛋白表达水平;TBA法、WST-1法、微板法分别检测大鼠缺血侧海马组织中丙二醛(MDA)含量、超氧化物歧化酶(SOD)活力及谷胱甘肽(GSH)含量;酶联免疫吸附测定法检测大鼠缺血侧海马组织中肿瘤坏死因子-α(TNF-α)、白细胞介素-18(IL-18)、干扰素诱导蛋白10(CXCL10)水平。结果 与假手术组比较,模型组大鼠神经功能评分降低(P<0.01),逃避潜伏期时间延长(P<0.01),目标象限停留时间及穿越平台次数均减少(P<0.05,P<0.01),海马组织中Keap1、Nrf2、HO-1蛋白表达水平、MDA含量以及TNF-α、IL-18、CXCL10水平均升高(P<0.05,P<0.01),SOD活力、GSH含量降低(P<0.01)。与模型组比较,补肾启智方高、低剂量组大鼠的神经功能评分升高(P<0.01),其中补肾启智方高剂量组大鼠逃避潜伏期时间缩短(P<0.01),目标象限停留时间及穿越平台次数均增多(P<0.05,P<0.01),补肾启智方低剂量组大鼠目标象限停留时间增多(P<0.05);补肾启智方高、低剂量组海马组织中Keap1蛋白表达、MDA含量及TNF-α、IL-18、CXCL10水平均降低(P<0.05,P<0.01),Nrf2蛋白表达水平及SOD活力、GSH含量均升高(P<0.05,P<0.01),补肾启智方高剂量组HO-1蛋白表达水平升高(P<0.05)。与补肾启智方低剂量组比较,补肾启智方高剂量组大鼠神经功能评分升高(P<0.01),海马组织中Keap1蛋白表达水平、MDA含量及TNF-α、IL-18水平降低(P<0.05,P<0.01),SOD活力及GSH含量升高(P<0.05)。结论 补肾启智方改善缺血性卒中大鼠神经功能缺损及认知功能损伤的作用机制,可能与激活Keap1/Nrf2/ARE信号通路、抑制海马氧化应激及炎症反应有关。
Objective We aimed to study the mechanisms underlying the positive effects of Bushen Qizhi formula on cognitive function in ischemic stroke rats by the Keap1/Nrf2/ARE signaling pathway.Methods Ischemic stroke was induced in male SD rats by thread occlusion.The rats were randomly divided into the sham group, the model group, the Bushen Qizhi formula low-dose(BSQZ-L) group(3.6 g/kg), and the Bushen Qizhi formula high-dose(BSQZ-H) group(14.4 g/kg),nine rats in each group. Treatment lasted for 14 days.The Garcia JH method was used to evaluate the degree of neurological deficit.The Morris water maze test was used to evaluate spatial learning and memory.Western blotting was used to detect the protein expression levels of Keap1, Nrf2, and HO-1 in the ischemic hippocampus.The TBA test, the WST-1 assay, and the microplate method were used to determine MDA levels, SOD activity, and GSH levels in the ischemic hippocampus.ELISA was used to detect TNF-α, IL-18, and CXCL10 levels in the ischemic hippocampus.Results In the model group, the neurological score was lower(P<0.01), the time of escape latency was higher(P<0.01), the time in the target quadrant and the time spent crossing the platform were both lower(P<0.05, P<0.01), and the protein expression levels of Keap1, Nrf2, and HO-1, the MDA content, and TNF-α, IL-18, and CXCL10 levels in the hippocampus were all higher(P<0.05, P<0.01), and SOD activity and GSH levels were both lower(P<0.01) compared with the sham group.Compared with the model group, the neurological scores were higher in the BSQZ-H and BSQZ-L groups(P<0.01), the escape latency was reduced in the BSQZ-H group(P<0.01), the time in the target quadrant and the time spent crossing the platform were higher in the BSQZ-H group(P<0.05, P<0.01), the time in the target quadrant was higher in the BSQZ-L group(P<0.05), the protein expression level of Keap1, MDA content, and the TNF-α, IL-18, and CXCL10 levels in the hippocampus were lower in the BSQZ-L and BSQZ-H groups(P<0.05, P<0.01), the protein expression levels of Nrf2, GSH levels, and SOD activity were all higher in the BSQZ-L and BSQZ-H groups(P<0.05, P<0.01), and the protein expression level of HO-1 was higher in the BSQZ-H group(P<0.05).Compared with the BSQZ-L group, the neurological score was higher(P<0.01), the protein expression level of Keap1, MDA content, and TNF-α and IL-18 levels in the hippocampus were all lower(P<0.05, P<0.01), and GSH level and SOD activity were both higher(P<0.05) in the BSQZ-H group.Conclusion The mechanisms underlying the positive effects of Bushen Qizhi formula on neurological deficit and cognitive impairment in rats after ischemic stroke may be related to the activation of the Keap1/Nrf2/ARE signaling pathway, the reduction of hippocampal oxidative stress, and the inhibition of the inflammatory response.
作者
吴艺帆
傅晨
罗超琴
刘孟涵
王翎沣
刘雪梅
WU Yifan;FU Chen;LUO Chaoqin;LIU Menghan;WANG Lingfeng;LIU Xuemei(Dongfang Hospital of Beijing University of Chinese Medicine,Beijing 100078,China)
出处
《北京中医药大学学报》
CAS
CSCD
北大核心
2022年第7期654-662,共9页
Journal of Beijing University of Traditional Chinese Medicine
基金
国家自然科学基金项目(No.82104808,No.81973788)
国家中医药管理局中医药创新团队及人才支持计划项目(No.ZYYCXTD-C-202007)
中医药防治脑病传承创新团队项目(No.CI2021B006)。
