Objective: To investigate the effects of Cathepsin K(CatK) on spatial learning and memory in rat hippocampus and its mechanisms.
Methods: Twenty male SD rats were randomly divided into Control group and CatK inhibitor group(CatKⅡ group), which were microinjected with Cathepsin K specific inhibitor(0.5 μg/μl) and artificial cerebrospinal fluid in hippocampal DG area respectively with 5 days. The cultured hippocampal neuron cells were divided into control group (CON group), negative control group(NC group), siRNA interference group(siCatK group). Three re-wells were set for each group, and samples were collected 18～20 h after siRNA transfection. Morris water maze was used to evaluate spatial learning and memory function of rats. Meanwhile, dynamic changes of glutamate(Glu) content in extracellular fluid of DG region during learning and memory were observed by microdialysis and high performance liquid chromatography in conscious rats. Western blot was used to detect CatK-mediated Notch1 activation and other signal molecules.
Results: Animal experiments showed that compared with the control group, the spatial learning and memory ability were decreased significantly in CatKII group, and the hippocampus protein expressions of c-Notch1, p-Akt, p-CREB and BDNF were also decreased significantly(P＜0.05); the levels of Glu in DG area of control group and CatK II group were increased significantly with Morris water maze training days, but the increase of CatK II group was significantly weaker than that of control group(P＜ 0.05). The results of cell experiment showed that the expressions of CatK, c-Notch1, p-CREB and BDNF in siCatK group were significantly lower than other groups (P＜0.05).
Conclusion: CatK can affect the spatial learning and memory function of rats by activating Notch1 and its memory related signal protein in hippocampus.
目的: 探讨组织蛋白酶K(CatK)对大鼠海马空间学习和记忆功能的影响及其神经化学机制。方法: SD雄性大鼠随机分为对照组(Control组)和CatK阻断组(CatK Ⅱ组)每组各10只,分别于海马DG区微量注射组织蛋白酶K特异性阻断剂(0.5 μg/μl)和等量人工脑脊液5 d;原代培养海马神经元细胞,设对照组(CON组)、阴性对照组(NC组)和siRNA干扰组(siCatK组),每组设3个复孔,siRNA导入18～20 h后收集样本。应用Morris水迷宫评估大鼠空间学习和记忆功能,同时在大鼠清醒、自由活动状态下,结合脑部微量透析法和高效液相色谱法观察学习记忆过程中DG区细胞外液中谷氨酸(Glu)含量的动态变化;采用蛋白免疫印迹法检测海马组织和神经元细胞中c-Notch1、Notch1、p-CREB、BDNF等相关信号蛋白的表达情况。结果: 动物实验显示,与Control组相比,CatK Ⅱ组空间学习记忆能力明显减弱,并伴随着海马组织的c-Notch1、p-Akt、p-CREB和BDNF蛋白表达明显下降(P＜ 0.05);Control组和CatK II组DG区Glu含量均随水迷宫训练天数的增加而明显升高,但CatK Ⅱ组升高幅度均明显弱于Control组(P＜0.05)。细胞实验结果显示,与CON组和NC组相比,siCatK组CatK、c-Notch1、p-CREB和BDNF蛋白表达明显减少(P＜0.05)。结论: CatK可通过激活海马Notch1及其记忆相关信号蛋白,影响大鼠空间学习记忆功能。.
Keywords: Cathepsin K; Notch1; cell culture; hippocampus; learning and memory; rat.