知母-黄柏药对改善D-半乳糖诱导衰老小鼠认知障碍的机制研究

Study on the mechanism of Anemarrhenae Rhizoma⁃Phellodendri Chinensis Cortex herb pair in improving the cognitive impairment of D⁃galactose⁃induced aging mice

目的:研究知母-黄柏药对(简称“知柏”)对D-半乳糖(D-gal)诱导衰老模型小鼠认知障碍的改善作用,并探讨其作用机制。方法:将75只C57BL/6J小鼠随机分为空白组、模型组(D-gal 125 mg/kg)、吡拉西坦组(468 mg/kg)、知柏组(4.5 g/kg)和雷帕霉素哺乳靶蛋白(mTOR)抑制剂组(依维莫司,RAD001,3mg/kg),每组15只。通过D-gal建立小鼠衰老模型,并连续8周灌胃给予知柏水煎液。采用Morris水迷宫和新物体识别测试评价衰老模型小鼠的认知功能;HE染色法观察各组小鼠海马组织病理变化情况;尼氏染色和高尔基染色观察海马区神经元和突触的病理形态变化;收集海马组织以测定脑内三磷酸腺苷(ATP)含量,Western Blot法检测海马中泛素结合蛋白62(P62)、肌球蛋白样BCL2结合蛋白(Beclin-1)、突触素(Syn)、突触后密度蛋白-95(PSD-95)、腺苷酸活化蛋白激酶(AMPK)、mTOR的蛋白表达水平;实时荧光定量PCR(qRT-PCR)法检测海马中Syn、AMPK、mTOR基因表达水平。结果:与空白组相比,模型组小鼠在Morris水迷宫和新物体识别测试中表现出认知功能显著降低(P<0.01)。在模型组中,海马区的细胞数量减少,细胞排列松散,神经元形态不规则,尼氏小体数量明显减少,树突分支数量以及树突棘密度也显著减少。模型组小鼠海马中ATP水平、P62、Syn、PSD-95、mTOR蛋白表达显著降低(P<0.01),Beclin-1表达显著升高(P<0.01),pAMPK/AMPK蛋白含量比值和AMPK基因表达显著升高(P<0.01),Syn和mTOR基因表达显著降低(P<0.01)。与模型组相比,吡拉西坦组和知柏组小鼠的认知功能得到了显著改善,表现为Morris水迷宫测试中潜伏期的显著缩短(P<0.01),穿越平台次数和靶象限停留时间显著增加(P<0.01),以及新物体识别指数的显著升高(P<0.01),海马区树突棘密度显著增加(P<0.01),神经元损伤也在一定程度上得到了缓解。海马中ATP水平、P62、Syn、PSD-95和mTOR蛋白表达显著升高(P<0.01,P<0.05),Beclin-1蛋白表达显著降低(P<0.01),pAMPK/AMPK蛋白含量比值和AMPK基因表达显著降低(P<0.01,P<0.05),Syn、mTOR基因表达显著升高(P<0.01)。抑制剂RAD001组与模型组结果相似,经知柏治疗后上述指标均有所回调。结论:知柏可能通过激活AMPK/mTOR信号通路抑制神经元过度自噬,增强突触可塑性,改善由D-gal引起的小鼠认知障碍。

Objective:To study the improving effect of Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex herb pair(Zhi Bai)on cognitive impairment in D-galactose(D-gal)administered mice,and to explore the mechanism of Zhi Bai in improving cognitive impairment through the AMPK/mTOR signaling pathway.Methods:Seventy-five C57BL/6J mice were randomly divided into five groups:the control group,the model group(D-gal 125 mg/kg),the piracetam group(468 mg/kg),the Zhi Bai group(4.5 g/kg),and the mTOR inhibitor group(everolimus,RAD001,3 mg/kg),with 15 mice in each group.The aging mouse model was established using D-gal,followed by 8 weeks of oral administration of Zhi Bai decoction.Cognitive functions in aging mice were assessed using the Morris water maze and novel object recognition tests.Hippocampal tissue pathology was observed using hematoxylin and eosin(H&E)staining;neuronal and synaptic pathological morphology in the hippocampal region was exam-ined with Nissl and Golgi staining.Hippocampal tissues were collected to determine the levels of adenosine triphosphate(ATP)in the brain.The protein expression levels of ubiquitin-binding protein 62(P62),Beclin-1 associated with myosin,light chain kinase BCL2(Beclin-1),synaptophysin(Syn),postsynaptic density protein-95(PSD-95),AMP-activated protein kinase(AMPK),and mTOR in the hippocampus were detected by Western blot.The gene expression levels of Syn,AMPK,and mTOR in the hippocampus were measured using quantitative real-time PCR(qRT-PCR).Results:Compared to the control group,the model group mice showed significant cognitive impairment in the Morris water maze and novel object recognition tests(P<0.01).In the model group,there was a reduction in cell numbers,loose cell arrangement,and irregular neuronal morphology,a significant decrease in Nissl bodies,and a decrease in dendritic branch and spine density in the hippocampal region.ATP levels,as well as P62,Syn,PSD-95,and mTOR protein expressions in the hippocampus of the model group,were significantly reduced(P<0.01),while Beclin-1 expression was significantly increased(P<0.01).The ratio of pAMPK to AMPK protein content and the expression of AMPK genes were significantly elevated(P<0.01),whereas Syn and mTOR gene expressions were significantly decreased(P<0.01).Compared to the model group,cognitive function was significantly improved in the piracetam and Zhi Bai groups,as evidenced by a significant reduction in the latency period in the Morris water maze test(P<0.01),increased platform crossing numbers and target quadrant dwell time(P<0.01),and a significant increase in the novel object recognition index(P<0.01).Dendritic spine density in the hippocampal region was significantly increased(P<0.01),and neuronal damage was alleviated to some extent.ATP levels and P62,Syn,PSD-95,and mTOR protein expressions in the hippocampus were significantly increased(P<0.01,P<0.05),Beclin-1 protein expression was significantly reduced(P<0.01),and the ratio of pAMPK to AMPK protein content and AMPK gene expression were significantly decreased(P<0.01,P<0.05),with Syn and mTOR gene expressions significantly elevated(P<0.01).The results for the RAD001 inhibitor group were similar to the model group,but Zhi Bai treatment led to a recalibration of these indicators.Conclusion:Zhi Bai may inhibit excessive autophagy of neurons,enhance synaptic plasticity and improve D-gal-induced cognitive impairment in mice by activating the AMPK/mTOR signaling pathway.