基于突触和突触蛋白探讨复方金思维对拟散发性阿尔茨海默病小鼠早期突触传递和功能的影响

Discussion on effects of compound Jinsiwei on early synaptic transmission and function in mice with sporadic AD based on synaptic and its proteins

目的通过观察复方金思维干预后拟散发性阿尔茨海默病(Alzheimer’s disease,AD)小鼠海马突触形态以及α7尼古丁乙酰胆碱受体(α7-nicotinic acetylcholine receptors,α7-nAChRs)和代谢性谷氨酸受体5(metabotropic glutamate receptors 5,mGluR5)两种突触相关蛋白的变化,探讨复方金思维对拟散发性AD小鼠早期学习记忆的影响。方法将3月龄C57/BL6J小鼠随机分为对照组、模型组、多奈哌齐组[0.92 mg/(kg·d)]及复方金思维大、中、小剂量组[20 mg/(kg·d),10 mg/(kg·d),5 mg/(kg·d)],对照组双侧侧脑室注射人工脑脊液,其余各组小鼠双侧侧脑室注射链脲佐菌素建立散发性AD模型,造模1个月后连续灌胃3个月,进行Morris水迷宫行为检测,之后取小鼠海马对其CA1区突触进行超微结构观察,并采用免疫组织化学和Western blot技术对海马CA1区α7-nAChRs和mGluR5分布和表达进行检测。结果Morris水迷宫结果显示,随着训练次数的增加每组小鼠定向航行潜伏期均缩短。电镜结果显示,与模型组相比,各干预组小鼠突触超微结构均有一定程度改善。免疫组化结果显示,与模型组比较,多奈哌齐组和复方金思维大剂量组α7-nAChRs的阳性细胞数均有所增加(P<0.05),多奈哌齐组及复方金思维大、中剂量组mGluR5的阳性细胞数均有所减少(P<0.01或P<0.05);Western-blot检测mGluR5结果与免疫组化结果一致。结论复方金思维可能通过改善拟散发性AD小鼠海马CA1区突触结构,调节突触相关蛋白α7-nAChRs和mGluR5表达起到早期神经保护作用,进而提高其学习记忆能力。

Objective To explore effects of Compound Jinsiwei on early learning and memory in mice with sporadic Alzheimer’s disease(AD), by observing the changes of synaptic morphology in hippocampus and α7 nicotinic acetylcholine receptors(α7-nAChRs)and metabolic glutamate receptor 5(mGluR5)in AD mice after intervention with compound Jinsiwei . Methods The 3-month-old C57/BL6J mice were divided into 6groups, including control group, model group, donepizil group (0.92 mg·kg^-1 ·d^-1 ), low, medium and high dose of compound Jinsiwei groups (20 mg·kg^-1 ·d^-1 , 10 mg·kg^-1 ·d^-1 and 5 mg·kg^-1 ·d^-1 reapectively). In the control group, bilateral lateral ventricles were injected with artificial cerebrospinal fluid, and bilateral lateral ventricles were injected with STZ in the rest groups to establish mice model with sporadic AD. After one month of modeling, continuous gavage for 3 months were performed, then the mice’s behavior was detected by Morris water maze teset. After that, the hippocampus of mice were taken to observe synapse’s ultrastructure in CA1 region. The distribution and expression of α7-nAChRs and mGluR5 in in CA1 region of hippocampus were detected by by immunohistochemistry and Western blot. Results The Morris water maze results showed that the directional navigation latency of each group decreased with the increase of training times. The results of electron microscopy showed that the synaptic ultrastructure of mice in each intervention group was improved to some extent compared with the model group. Immunohistochemical results showed that, compared with the model group, the number of positive cells of α7-nAChRs increased in both donepezil group and high dose of compound Jinsiwei group ( P <0.05 ), while the number of positive cells of mGluR5 in donepezil group and high and medium dose of compound Jinsiwei all decreased ( P <0.01 or P <0.05). Western blot results of mGluR5 were consistent with immunohistochemical results. Immunohistochemical results showed that compared with the control group, the number of positive cells in the hippocampal CA1 region of α7-nAChRs decreased significantly and the number of positive cells in mGluR5 increased significantly ( P <0.01).Compared with the model group, the number of positive cells of α7-nAChRs in both the donepezil group and Chinese medicine compound Jinsiwei high dose group increased ( P <0.05), and the number of positive cells of mGluR5 in both the Chinese medicine compound Jinsiwei high dose group and middle dose group decreased ( P <0.01 or P <0.05).The consequences of western-blot analysis of mGluR5 were consistent with the immunohistochemical results. Conclusion Compound Jinsiwei may play an early neuroprotective role, by improving the synapses’s structure in hippocampus CA1 region of sporadic AD mice and regulating the expression of synapse associated proteins α7-nAChRs and mGluR5, thus improving their learning and memory ability.