文献类型：期刊文献

中文题名：基于RAS/RAF/MEK/ERK信号通路探讨黑逍遥散干预AD模型大鼠氧化应激的作用机制

英文题名：Hei Xiaoyaosan Regulates RAS/RAF/MEK/ERK Signaling Pathway to Ameliorate Oxidative Stress in Rat Model of AD

作者：王虎平[1,2,3];吕育洁[1];胡韵韵[1];孟志鹏[1];杨娇[1];陈怡琴[1]

第一作者：王虎平

机构：[1]甘肃中医药大学,兰州730000;[2]甘肃省中医方药挖掘与创新转化重点实验室,兰州730000;[3]甘肃省中药新产品创制工程实验室,兰州730000

第一机构：甘肃中医药大学

年份：2024

卷号：30

期号：17

起止页码：35

中文期刊名：中国实验方剂学杂志

外文期刊名：Chinese Journal of Experimental Traditional Medical Formulae

收录：CSTPCD;;Scopus;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；

基金：国家自然科学基金项目(81960828,82160862);兰州市科技发展指导性计划项目(2020-ZD-53);第五批全国中医临床优秀人才研修项目(国中医药人教函〔2022〕239号);首批陇原青年英才项目(中共甘肃省委人才工作领导小组〔2022〕5号)。

语种：中文

中文关键词：黑逍遥散;阿尔茨海默病;RAS蛋白(RAS)/RAF激酶(RAF)/有丝分裂原活化蛋白激酶激酶(MEK)/细胞外信号调节激酶(ERK)信号通路;氧化应激

外文关键词：Hei Xiaoyaosan;Alzheimer's disease;rat sarcoma(RAS)/rapidly accelerating fibrosarcoma(RAF)/mitogen-activated protein kinase kinase(MEK)/extracellular signal-regulated kinase(ERK)signaling pathway;oxidative stress

摘要：目的:探讨黑逍遥散调控RAS蛋白(RAS)/RAF激酶(RAF)/有丝分裂原活化蛋白激酶激酶(MEK)/细胞外信号调节激酶(ERK)信号通路干预阿尔茨海默病(AD)大鼠氧化应激的作用及其机制。方法:4月龄SPF级Wistar雄性大鼠100只,随机选取10只作为空白组,10只作为假手术组(双侧海马注射生理盐水1μL),其余80只双侧海马注射β淀粉样蛋白1-42(Aβ_(1-42))溶液1μL复制AD模型。遴选造模合格大鼠50只,随机分为模型组、盐酸多奈哌齐组(0.5 mg·kg^(-1))及黑逍遥散高、中、低剂量组(15.30、7.65、3.82 g·kg^(-1))。连续灌胃42 d,每天1次。灌胃结束后,Morris水迷宫实验测试大鼠学习记忆能力,尼式染色法检测海马CA3区神经元病理结构改变,免疫荧光观察Aβ沉积和tau蛋白磷酸化水平,蛋白免疫印迹法(Western blot)检测海马组织RAS、RAF、磷酸化(p)-RAF、MEK、p-MEK、ERK、p-ERK蛋白表达,生化法检测海马组织活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)水平。结果:与假手术组比较,模型组大鼠第5天逃避潜伏期显著延长(P<0.01),目标象限游泳距离显著缩短(P<0.01),尼氏小体数量减少,染色不均;Aβ、p-tau荧光强度显著增强(P<0.01),海马组织中RAS、p-RAF、p-MEK、p-ERK蛋白表达显著增高(P<0.01),ROS和MDA含量显著增高(P<0.01),SOD活性显著降低(P<0.01);与模型组比较,盐酸多奈哌齐组和黑逍遥散高、中、低剂量组大鼠第5天逃避潜伏期显著缩短(P<0.01),目标象限游泳距离显著增加(P<0.01);尼氏染色显示神经元排列整齐,细胞形态、结构完整,尼氏小体清晰可见,盐酸多奈哌齐组和黑逍遥散高、中、低剂量组Aβ、p-tau荧光强度显著下调(P<0.01),RAS、p-RAF、p-MEK、p-ERK蛋白表达明显增高(P<0.05,P<0.01),ROS、MDA含量显著降低(P<0.01),SOD活性显著增高(P<0.01)。结论:黑逍遥散可能通过RAS/RAF/MEK/ERK信号通路干预氧化应激,降低Aβ和p-tau水平,抑制海马神经元损伤,从而改善学习记忆能力。
Objective:To investigate the role and mechanism of Hei Xiaoyaosan in intervening in oxidative stress in the rat model of Alzheimer's disease(AD)via modulating the rat sarcoma(RAS)/rapidly accelerating fibrosarcoma(RAF)/mitogen-activated protein kinase kinase(MEK)/extracellular signal-regulated kinase(ERK)signaling pathway.Method:One hundred 4-month-old SPF-grade Wistar male rats were randomly grouped as follows:10 in the blank group,10 in the sham group(bilateral hippocampus injected with 1μL normal saline),and 80 in the modeling group[bilateral hippocampus injected with 1μL amyloid beta protein 1-42(Aβ_(1-42))solution for the modeling of AD].Fifty rats qualified for modeling were selected and randomized into the model,donepezil hydrochloride(0.5 mg·kg^(-1)),and high-,medium-,and low-dose(15.30,7.65,3.82 g·kg^(-1),respectively)Hei Xiaoyaosan groups.The rats were administrated with corresponding drugs by gavage once a day for 42 consecutive days.At the end of gavage,Morris water maze test was performed to examine the learning and memory abilities of the rats,and Nissl staining was used to observe the pathological changes of neurons in CA3 region of the hippocampus.The immunofluorescence assay was used to observe Aβdeposition and tau phosphorylation.Western blot was employed to determine the protein levels of RAS,RAF,phosphorylated(p)-RAF,MEK,p-MEK,ERK,and p-ERK in the hippocampal tissue.Biochemical methods were used to determine the levels of reactive oxygen species(ROS),malondialdehyde(MDA),and superoxide dismutase(SOD)in the hippocampal tissue.Result:Compared with the sham group,the model group showed prolonged escape latency(P<0.01),shortened swimming distance in the target quadrant(P<0.01),reduced and uneven stained Nissl bodies,enhanced fluorescence intensity of Aβand p-tau(P<0.01),up-regulated protein levels of RAS,p-RAF,p-MEK,and p-ERK in the hippocampal tissue(P<0.01),increased ROS and MDA content(P<0.01),and decreased SOD activity(P<0.01)on day 5.Compared with the model group,donepezil hydrochloride and high-,medium-,and low-dose Hei Xiaoyaosan shortened the escape latency(P<0.01),increased the swimming distance in the target quadrant(P<0.01),improved the arrangement,morphology,and structures of neurons and the number and distribution of Nissl bodies,decreased the fluorescence intensity of Aβand p-tau(P<0.01),up-regulated the protein levels of RAS,p-RAF,p-MEK,and p-ERK(P<0.05,P<0.01),decreased the ROS and MDA content(P<0.01),and increased the SOD activity(P<0.01)on day 5.Conclusion:Hei Xiaoyaosan may ameliorate oxidative stress,reduce Aβand p-tau levels,and inhibit hippocampal neuronal damage by regulating the RAS/RAF/MEK/ERK signaling pathway,thus improving learning and memory abilities.