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目的探讨慢性睡眠剥夺(CSD)对海马超微结构及海马内多巴胺D_1受体下游信号通路的影响。方法选取雄性SD大鼠35只,剔除体质量最轻、负重游泳时间最短和Morris水迷宫实验中90s仍找不到平台的11只大鼠,其余24只随机分为大平台对照(TC)组、CSD组和CSD+多巴胺D_1受体激动剂SKF38393(SKF)组,采用改良多平台水环境法建立大鼠CSD模型,SKF组在CSD 15~21d腹腔注射SKF38393(1mg/kg)。CSD 21d时,采用透射电镜观察各组大鼠海马的超微结构,采用蛋白质印迹法及qPCR检测大鼠CSD后海马内多巴胺D_1受体相关信号通路关键因子的表达。结果 CSD导致的海马神经元线粒体肿胀变性、膜结构破坏可通过使用SKF38393得以改善。与TC组相比,CSD组大鼠海马内腺苷酸环化酶5(Adcy5)、cAMP依赖蛋白激酶α型催化亚基(Prkacα)、多巴胺和cAMP调节的磷蛋白(Darpp32)、Ras相关蛋白(Rap)1a、细胞外信号调节蛋白激酶1和2(ERK1/2)、磷脂酶Cβ1(PLCβ1)、钙/钙调蛋白依赖性蛋白激酶Ⅱa和Ⅳ(CaMKⅡa、CaMKⅣ)mRNA表达均降低(P<0.05),蛋白激酶A催化亚基α(PKAcα)总蛋白及其磷酸化水平、磷酸化ERK1/2、PLCβ1和磷酸化-CaMKⅣ蛋白表达均降低(P<0.05)。与CSD组相比,SKF组Prkacα、Darpp32、Rap1a、Rap1b、ERK1和CaMKⅣmRNA表达均增加(P<0.05);PKAcα总蛋白及其磷酸化均以及磷酸化CaMKⅣ蛋白表达均增加(P<0.05),但PLCβ1和CaMKⅣ总蛋白表达水平无明显变化。结论 CSD可破坏海马神经元超微结构,使用多巴胺D_1受体激动剂SKF38393可有效改善海马超微结构,其机制可能与PKA和磷酸肌醇信号通路的参与有关。
Objective To investigate the effect of chronic sleep deprivation (CSD) on the ultrastructure of hippocampus and the signal transduction pathway of dopamine D_1 receptor in hippocampus. Methods Thirty-five male Sprague-Dawley rats were selected and the rats with the lightest weight and the shortest swimming time under load were excluded. Eleven rats were still not found in the 90-day Morris water maze test. The remaining 24 rats were randomly divided into a large platform control group (TC) CSD group and CSD + dopamine D_1 receptor agonist SKF38393 (SKF) group. CSD model was established by modified multi-platform water environment method. SKF group was injected intraperitoneally with SKF38393 (1 mg / kg) on CSD 15-21 d. CSD 21d, the ultrastructure of hippocampus in each group was observed by transmission electron microscope. The expression of key factors of dopamine D 1 receptor-related signal pathway in hippocampus were detected by Western blotting and qPCR after CSD. Results CSD induced mitochondrial swelling and degeneration of hippocampal neurons. The destruction of membrane structure could be ameliorated by the use of SKF38393. Compared with the TC group, the levels of adenylyl cyclase 5 (Adcy5), cAMP dependent protein kinase α type catalytic subunit (Prkacα), dopamine and cAMP regulated phosphoprotein (Darpp32), Ras related protein (P <0.05). The mRNA expression of ERK1 / 2, phospholipase Cβ1 (PLCβ1), CaMKⅡa and CaMKⅣ were decreased (P <0.05). The protein PKAcαprotein phosphorylation level, phosphorylated ERK1 / 2, PLCβ1and phospho-CaMKⅣprotein were all decreased (P <0.05). Compared with CSD group, the expressions of Prkacα, Darpp32, Rap1a, Rap1b, ERK1 and CaMKⅣmRNA in SKF group were all increased (P <0.05); the expressions of PKAcαprotein and phosphorylated phosphorylated CaMKⅣprotein were increased However, there was no significant change in total protein expression level of PLCβ1 and CaMKⅣ. Conclusion CSD can damage the ultrastructure of hippocampal neurons. Using dopamine D_1 receptor agonist SKF38393 can effectively improve the ultrastructure of hippocampus. The mechanism may be related to the involvement of PKA and phosphoinositide signaling pathway.