知母-黄柏对D-gal认知功能障碍模型小鼠海马神经元细胞异常自噬的影响

Effect of Rhizoma⁃Anemarrhenae⁃Cortex Phellodendri on abnormal autophagy of hippocampal neurons in D⁃gal cognitive impairment model mice

目的:通过观察海马内自噬相关蛋白的表达水平探讨知母-黄柏药对(AP)改善D-半乳糖(D-gal)模型小鼠认知功能障碍的作用机制。方法:75只小鼠随机分为空白组、模型组、吡拉西坦组、AP组,自噬诱导剂依维莫司(RAD001)组,除空白组腹腔注射生理盐水外,其余各组均注射0.125 g·kg^(-1)·d^(-1)的D-gal以复制认知功能障碍模型。通过水迷宫实验测试小鼠认知功能障碍情况,HE染色及尼氏染色法观察各组小鼠海马组织神经元状态以及尼氏小体数量,免疫组化法检测海马中LC3及P62阳性细胞数量的表达,Western Blot实验检测海马中与自噬相关蛋白ULK1、LC3、Beclin-1、P62等蛋白相对表达水平,实时荧光定量PCR法检测海马中ULK1、Beclin-1、P62等基因表达。结果:与空白组相比,模型组小鼠的学习记忆能力显著降低,海马CA1区神经元和尼氏体出现严重丢失,且排列紊乱;海马内ULK1、LC3、Beclin-1蛋白及基因表达升高(P<0.01,P<0.05),P62蛋白及基因表达下降(P<0.01)。与模型组相比,吡拉西坦组、AP组小鼠学习记忆能力显著提高,神经元、尼氏体丢失现象减少;海马内ULK1、LC3、Beclin-1蛋白及基因表达下降(P<0.01),P62蛋白及基因表达升高(P<0.01)。RAD001组显示AP给药后可部分逆转细胞过度自噬现象。结论:AP可以改善D-gal模型小鼠的认知功能障碍,其机制可能是通过抑制海马区细胞过度自噬来实现的。

Objective:To explore the mechanism of Rhizoma Anemarrhenae⁃Cortex Phellodendri(AP)in ameliorating cogni⁃tive dysfunction in D⁃galactose(D⁃gal)model mice by observing the expression level of autophagy⁃related proteins in the hippo⁃campus.Methods:Seventy⁃five mice were randomly divided into blank group,model group,piracetam group,AP group and au⁃tophagy inducer everolimus(RAD001)group.Except for the blank group,all the other groups were injected with 0.125 g∙kg^(⁃1)∙d^(⁃1) D⁃gal intraperitoneally to replicate the cognitive impairment model.The cognitive dysfunction of mice was tested by water maze ex⁃periment.The neuron status and Nissl bodies of hippocampus in each group were observed by HE staining and Nissl corpuscle number.The expression of LC3 and P62 positive cells in hippocampus was detected by immunohistochemistry.The relative ex⁃pression levels of autophagy⁃related proteins such as ULK1,LC3,Beclin⁃1 and P62 were detected by Western Blot.The genes such as ULK1,Beclin⁃1 and P62 were detected by real⁃time fluorescence quantitative PCR.Results:Compared with the blank group,the learning memory ability of mice in the model group was significantly reduced,and neurons and Nissl bodies in hippo⁃campal CA1 area were seriously lost and arranged in disorder.The expression of ULK1,LC3,Beclin⁃1 protein and gene in hippo⁃campus increased(P<0.01,P<0.05),while the expression of P62 protein and gene decreased(P<0.01).Compared with the model group,the learning memory ability of mice in the piracetam and AP groups was significantly improved,and the loss of neu⁃rons and Nissl bodies decreased.The expression of ULK1,LC3,Beclin⁃1 protein and gene in hippocampus decreased(P<0.01),while the expression of P62 protein and gene increased(P<0.01).RAD001 group showed that AP could partially reverse the au⁃tophagy of cells after administration.Conclusion:AP can improve the cognitive dysfunction of D⁃gal model mice,and its mecha⁃nism may be achieved by inhibiting autophagy of hippocampal cells.