黄精丸对学习记忆障碍小鼠海马神经元自噬的影响

Effect of Huangjingwan on Hippocampal Autophagy in Mouse Model of Learning and Memory Impairments

目的:观察黄精丸(CHP)对D-半乳糖联合东莨菪碱所致学习记忆障碍小鼠海马神经自噬的影响,探讨其可能的作用机制。方法:将90只8周龄雄性昆明小鼠随机分为正常组、模型组、多奈哌齐组、CHP低、中、高剂量组。除正常组外,前3周各组小鼠每日颈背部皮下注射1次0.14 g·kg^(-1)D-半乳糖、后2周改为每日腹腔注射1次2 mg·kg^(-1)东莨菪碱复制学习记忆障碍小鼠以模拟阿尔茨海默病(AD)。造模开始1周后,多奈哌齐组给予0.65 mg·kg^(-1)多奈哌齐,CHP低、中、高剂量组分别给予1.25、2.5、7.5 g·kg^(-1)CHP提取液,模型组给予生理盐水0.5 mL,每天灌胃1次,连续4周。以水迷宫装置观察各小鼠行为学改变,尼氏染色分析各小鼠海马形态学变化,实时荧光定量聚合酶链式反应(Real-time PCR)检测各小鼠海马组织哺乳动物雷帕霉素靶蛋白(mTOR)、自噬效应蛋白-1(Beclin-1)及泛素结合蛋白p62(p62)的mRNA表达水平差异,蛋白免疫印迹法(Western blot)检测各组小鼠海马组织mTOR、Beclin-1、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、微管相关蛋白1轻链3Ⅰ(LC3Ⅰ)、p62的蛋白表达水平差异,免疫组化探测各组小鼠海马神经元Beclin-1阳性表达。结果:与正常组比较,模型组小鼠学习记忆障碍、痴呆症状明显,其海马结构紊乱、神经元数量减少(P<0.01),mTOR、Beclin-1的mRNA及蛋白表达水平均显著降低,LC3Ⅱ/LC3Ⅰ显著降低,p62的mRNA及蛋白表达水平显著升高(P<0.01);与模型组比较,多奈哌齐组及CHP各组小鼠痴呆症状及学习记忆好转,海马神经结构紊乱有所改善、神经元数量增加(P<0.05,P<0.01),mTOR、Beclin-1的mRNA及蛋白表达水平均明显升高,LC3Ⅱ/LC3Ⅰ明显升高,p62的mRNA及蛋白表达水平均明显降低(P<0.05,P<0.01)。结论:CHP能促进海马神经元自噬发挥治疗AD的作用,其机制可能与激活mTOR信号通路有关。

Objective:To observe the effect of Huangjingwan(CHP)on hippocampal autophagy in the mice model of learning and memory impairments induced by D-galactose combined with scopolamine and decipher the underlying mechanism.Method:Ninety 8-week-old male Kunming mice were randomized into the normal,model,donepezil(0.65 mg·kg^(-1)),and low-,medium-,and high-dose(1.25,2.5,7.5 g·kg^(-1),respectively)CHP groups.The mice in other groups except the normal group were subcutaneously injected with 0.14 g·kg^(-1)D-galactose once daily for the first 3 weeks and then intraperitoneally injected with 2 mg·kg^(-1)scopolamine once daily for the next 2 weeks to induce learning and memory impairments and simulating Alzheimer's disease(AD).One week after modeling,mice were administrated with corresponding drugs by gavage once daily for 4 consecutive weeks.The learning and memory abilities of mice in all groups were examined by Morris water maze,and the hippocampal neural structure was observed by Nissl staining.The mRNA levels of mammalian target of rapamycin(mTOR),Beclin-1,and p62 in the hippocampus of mice were determined by real-time fluorescence quantitative polymerase chain reaction.The protein levels of mTOR,Beclin-1,microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ),microtubule-associated protein light chain 3Ⅰ(LC3Ⅰ),and p62 in the hippocampus of mice were measured by Western blot.The positive expression of Beclin-1 in the hippocampus of mice was detected by immunohistochemistry.Result:Compared with the normal group,the model group showed obvious dementia,poor learning and memory,disarrangement of hippocampal neural structure,and reduced number of neurons(P<0.01).In addition,the modeling downregulated the mRNA and protein levels of mTOR and Beclin-1,decreased the protein ratio of LC3Ⅱ/LC3Ⅰ,and up-regulated the mRNA and protein levels of p62(P<0.01).Compared with the model group,donepezil and CHP alleviated dementia,improved the learning and memory performance,mitigated the disarrangement of hippocampal neural structure,and increased the number of neurons(P<0.05,P<0.01).Furthermore,donepezil and CHP up-regulated the mRNA and protein levels of mTOR and Beclin-1,increased the protein ratio of LC3Ⅱ/LC3Ⅰ,and down-regulated the mRNA and protein levels of p62(P<0.05,P<0.01).Conclusion:CHP promotes hippocampal autophagy to treat AD by activating the mTOR signaling pathway.