摘要
目的:研究黑逍遥散是否可以通过调控激活Wntβ-连环蛋白(β-catenin)信号通路抑制阿尔茨海默病(AD)大鼠海马区炎症反应,改善AD大鼠认知和记忆功能障碍。方法:90只雄性Wistar大鼠先随机分别选择12只作为空白组和假手术组,剩余大鼠在左右两侧海马区注射β淀粉样蛋白42(Aβ42)制造AD模型大鼠,随机分为模型组、黑逍遥散低、中、高剂量组和多奈哌齐组,每组12只,并连续42 d给予相应剂量黑逍遥散和多奈哌齐灌胃。尼氏染色观察海马CA1区病理形态学变化;Morris水迷宫实验检测1~5 d大鼠逃避潜伏期的变化,第6天的空间探索能力。酶联免疫吸附测定法(ELISA)检测大鼠海马组织和血清中白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)的表达,蛋白免疫印迹法(Western blot)检测检测海马中海马区糖原合成激酶-3β(GSK-3β)、β-catenin、过氧化物酶体增殖物激活受体γ(PPARγ)的蛋白表达水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠GSK-3β、β-catenin、PPARγmRNA的水平。结果:与空白组比较,模型组大鼠海马CA1区神经元数量明显减少,排布不均,细胞体损坏比较明显,尼式小体不清;模型组大鼠第3~5天的逃避潜伏期均相比治疗组显著延长(P<0.01),跨台次数显著减少(P<0.01);模型组大鼠血清和海马组织中IL-10水平显著降低,IL-6、TNF-α水平显著升高(P<0.01);模型组GSK-3β蛋白和mRNA显著升高,β-catenin、PPARγ蛋白表达显著降低(P<0.01),差异较为明显;与模型组比较,黑逍遥散中、高剂量组和盐酸多奈哌齐组大鼠神经元细胞数量分布较多,排列整齐,结构完整,尼式小体清晰明确;黑逍遥散中、高剂量组和多奈哌齐组的第3~5天逃避潜伏期显著缩短(P<0.01),跨越平台次数显著增多(P<0.01);大鼠海马组织和血清中IL-10的表达水平明显升高,IL-6和TNF-α显著降低(P<0.01);大鼠海马中GSK-3β蛋白和GSK-3βmRNA表达明显降低,β-catenin、PPARγ蛋白和β-catenin、PPARγmRNA表达水平显著升高(P<0.01)。盐酸多奈哌齐组与黑逍遥散高剂量组之间各指标比较,差异无统计学意义。结论:黑逍遥散可以通过调控Wnt/β-catenin信号通路抑制AD大鼠海马区炎症反应,改善AD模型大鼠认知和记忆障碍。
Objective:To explore whether Hei Xiaoyaosan can inhibit the inflammatory response in the hippocampi of Alzheimer’s disease(AD)rats by regulating and activating the Wnt/β-catenin signaling pathway to improve the cognitive and memory dysfunction.Method:Among the 90 male Wistar rats,12 were randomly selected as the blank group(normal saline)and 12 as the sham operation group(normal saline).For the remainder,amyloidβ-protein_(42)(Aβ_(42))was injected in the left and right hippocampus to induce AD,and then the AD rats were randomized into model group,low-,medium-,and high-dose Hei Xiaoyaosan groups(corresponding doses of Hei Xiaoyaosan,ig),and donepezil group(donepezil hydrochloride,ig),with 12 in each group.The administration lasted 42 days.The pathological changes of hippocampal CA1 region was observed based on Nissl staining.The escape latency on the 1^(st) to 5^(th) day in Morris water maze was recorded and the spatial memory on the 6th day was tested.Enzyme-linked immunosorbent assay(ELISA)was employed to detect the expression of interleukin(IL)-6,IL-10,and tumor necrosis factor-α(TNF-α)in rat hippocampus and serum,Western blotting to examine the protein expression of glycogen synthase kinase-3β(GSK-3β),β-catenin,and peroxisome proliferator-activated receptor gamma(PPARγ),and real-time polymerase chain reaction(Real-time PCR)to determine the mRNA expression of rat GSK-3β,β-catenin,and PPARγ.Result:Compared with the blank group,the number of neurons in the hippocampal CA1 area of model group was significantly reduced,the arrangement was uneven,the cell body was damaged more obviously,and the Neisser body was unclear.The treatment group was significantly prolonged(P<0.01),and the number of crossing stations was significantly reduced(P<0.01),the levels of IL-10 in serum and hippocampus of rats in the model group were significantly decreased,while the levels of IL-6 and TNF-αwere significantly increased(P<0.01),the GSK-3βprotein and mRNA in the model group were significantly increased,and the protein expressions ofβ-catenin and PPARγwere significantly decreased(P<0.01),and the difference was more obvious.The number of neurons in the donepezil group was more distributed,neatly arranged,the structure was intact,and the Nissl bodies were clear and definite,the escape latency on the 3^(rd) to 5^(th) days in middle and high dose groups of Hei Xiaoyaosan and the donepezil group was significantly shortened(P<0.01),the number of crossing platforms increased significantly(P<0.01),the expression levels of IL-10 in the rat hippocampus and serum were significantly increased,while IL-6 and TNF-αwere significantly decreased(P<0.01),GSK-3βin the rat hippocampus was significantly increased.The expressions of GSK-3βprotein and mRNA were significantly decreased,while the expression levels ofβ-catenin and PPARγprotein and mRNA were significantly increased(P<0.01).There was no significant difference in each index between the donepezil hydrochloride group and the high-dose Hei Xiaoyaosan group.Conclusion:Hei Xiaoyaosan can inhibit the inflammatory response in the hippocampus of AD rats by regulating the Wnt/β-catenin signaling pathway,thereby alleviating the cognitive and memory impairment of AD rats.
作者
李晓
胡亦明
韦春昕
安耀荣
LI Xiao;HU Yi-ming;WEI Chun-xin;AN Yao-rong(Key Laboratory of Chinese Herbs and Prescription Mining and Innovation and Transformation in Gansu Province,Gansu University of Chinese Medicine,Lanzhou 730000,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第11期33-41,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
甘肃省中医方药挖掘与创新转化重点实验室项目(40160302)
甘肃省中医药研究中心开放性课题(zxzy-2020-01)。
