目的 基于Kelch样ECH关联蛋白1/核因子E2相关因子2/抗氧化反应元件(Keap1/Nrf2/ARE)信号通路探讨康复训练联合胞磷胆碱对小鼠脑出血模型的神经保护作用及潜在作用机制.方法 将C57B76雄性小鼠采用随机数字表法分为假手术组(假手术处理)、模型组(用Ⅶ型胶原酶右侧尾壳核脑出血模型)、胞磷胆碱组(模型+胞磷胆碱64 mg·kg-1)、康复训练组(康复训练)及联合组(模型+康复训练+胞磷胆碱64 mg·kg-1).观察脑出血小鼠改良神经损害程度评分(mNSS),用比色法检测脑组织丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的表达,用蛋白质印迹法和逆转录-定量聚合酶链反应检测脑组织Keap1、Nrf2、血红素氧合酶-1(HO-1)、醌氧化还原酶1(NQO1)蛋白和mRNA的表达.结果 假手术组、模型组、胞磷胆碱组、康复训练组及联合组mNSS评分分别为0、(1.56±0.73)、(1.00±0.00)、(0.78±0.44)和(0.67±0.50)分,MDA 水平 分别为(6.93±0.92)、(22.97±0.77)、(19.26±1.73)、(13.21±0.78)和(7.25±0.97)nmol·mgprot-1,Keap1 蛋白相对表达水平分别为 0.79±0.03、1.02±0.04、0.95±0.10、0.90±0.09和0.86±0.05,Nrf2蛋白相对表达水平分别为0.94±0.12、0.71±0.08、0.90±0.07、0.98±0.12 和 1.33±0.25.模型组上述指标与假手术组比较,在统计学上差异均有统计学意义(P＜0.05,P＜0.01);胞磷胆碱组、康复训练组上述指标与模型组比较,在统计学上差异均有统计学意义(P＜0.05,P＜0.01);联合组上述指标与胞磷胆碱组、康复训练组比较,除Keap1蛋白相对表达水平外,在统计学上差异均有统计学意义(均P＜0.01).结论 康复训练、胞磷胆碱均可减轻脑出血小鼠的神经功能缺损症状,作用机制可能为激活Keap1/Nrf2/ARE信号通路发挥抗氧化应激作用,且联合使用效果最佳.
Objective To investigate the neuroprotective effect and potential mechanism of rehabilitation training combined with citicoline on cerebral hemorrhage model in mice based on Keap1/Nrf2/ARE signaling pathway.Methods The C57BL/6 male mice were randomly divided into sham operation group(sham operation treatment),model group(right caudate putamen hemorrhage model induced by type Ⅶcollagenase),choline group(model+choline 64 mg·kg-1),rehabilitation training group(rehabilitation training)and combined group(model+rehabilitation training+choline 64 mg·kg-1).The study observed the modified neurological severity score(mNSS)in mice with cerebral hemorrhage;colorimetric assays were used to detect the expression of malondialdehyde(MDA),superoxide dismutase(SOD)and catalase(CAT)in brain tissues;protein imprinting and reverse transcription-quantitative polymerase chain reaction were employed to assess the expression of Kelch-like ECH-associated protein 1(Keap1),nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE),heme oxygenase-1(HO-1),quinone oxidoreductase 1(NQ01)proteins and mRNA in brain tissues.Results The mNSS scores of sham operation group,model group,citicoline group,rehabilitation training group and combined group were 0,1.56±0.73,1.00±0.00,0.78±0.44 and 0.67±0.50;the MDA contents were(6.93±0.92),(22.97±0.77),(19.26±1.73),(13.21±0.78)and(7.25±0.97)nmol·mgprot-1;the relative expression of Keap1 protein were 0.79±0.03,1.02±0.04,0.95±0.10,0.90±0.09 and 0.86±0.05;the relative expression levels of Nrf2 protein were 0.94±0.12,0.71±0.08,0.90±0.07,0.98±0.12 and 1.33±0.25.There were significant differences in the above indexes between the model group and the sham operation group(P＜0.05,P＜0.01);there were significant differences between the citicoline group and the rehabilitation training group,the model group(P＜0.05,P＜0.01);there were significant differences between the combined group and the citicoline group,the rehabilitation training group except for protein expression of Keap1(all P＜0.01).Conclusion Rehabilitation training and citicoline can reduce the symptoms of neurological deficits in mice with cerebral hemorrhage.The mechanism way be that they can activate the Keap1/Nrf2/ARE signaling pathway to exert anti-oxidative stress,and the combined effect is the best.
卢苇;尤红;张敏;何宝峰;马瑞;高雅雅;马鑫宇
甘肃中医药大学第一临床医学院,甘肃兰州 730000甘肃省人民医院中法神经康复科,甘肃兰州 730000甘肃中医药大学药学院,甘肃兰州 730000
药学
胞磷胆碱;Kelch样ECH关联蛋白1/核因子E2相关因子2/抗氧化反应元件信号通路;康复训练;神经保护
citicoline;Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2/antioxidant response element signaling pathway;rehabilitation training;neuroprotection
《中国临床药理学杂志》 2024 (008)
1175-1179 / 5
甘肃省自然科学基金资助项目(21JR1RA020,21JR7RA597);甘肃省人民医院院内基金资助项目(22GSSYD-65,22GSSYD-66,22GSSYB-9)
10.13699/j.cnki.1001-6821.2024.08.018
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