慢性睡眠剥夺对大鼠海马超微结构和海马内多巴胺D1受体下游信号通路的影响

Effect of chronic sleep deprivation on ultrastructure and dopamine D_1 receptor downstream signaling pathway in hippocampus of rats

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 explore the effects of chronic sleep deprivation（CSD）on the ultrastructure and downstream signaling pathway of dopamine D_1 receptor of the hippocampus in rats.Methods Thirty-five male SD rats were selected in this experiment,of which 11 with the lightest weight,shortest weight-bearing swimming time or no finding the platform within 90 sin Morris water maze experiment were excluded.The other 24 rats were randomly divided into tank control（TC）group,CSD group and CSD＋dopamine D_1 receptor agonist SKF38393（SKF）group.The CSD rat model was established by modified multi-platform water environment,and then the rats in the SKF group were intraperitoneally injected with SKF38393（1mg/kg）at 15-21 dof CSD.At 21 dafter CSD,the ultrastructure of hippocampus was observed by transmission electron microscopy,and the expression of key factors in dopamine D_1receptor-related signal pathway in the hippocampus was detected by Western blotting and qPCR.Results The mitochondrial swelling,degeneration and destruction of membrane structure of the hippocampus neurons induced by CSD were improved by SKF38393.Compared with the TC group,the mRNA expression levels of adenylate cyclase 5（Adcy5）,protein kinase cAMP-dependent catalytic α（Prkacα）,dopamine and cAMP-regulated phosphoprotein（Darpp32）,Ras-related protein（Rap）1a,extracellular signal regulated kinase 1and 2（ERK1/2）,phospholipase Cβ1（PLCβ1）,calcium/calmodulin-dependent protein kinase Ⅱa and Ⅳ（CaMKⅡa,CaMKⅣ）in the CSD group were significantly decreased（P〈0.05）,and the protein levels of total and phosphorylatied protein kinase A catalyzes subunitα（PKAcα）,phosphorylated ERK1/2,phosphorylated PLCβ1,and phosphorylated CaMKⅣ were significantly decreased（P〈0.05）.Compared with the CSD group,the mRNA expressions of Prkacα,Darpp32,Rap1 a,Rap1b,ERK1 and CaMKⅣin the SKF group were significantly increased（P〈0.05）,and the protein levels of total and phosphorylated PKAcαand phosphorylated CaMKⅣ were significantly increased（P〈0.05）,while the expression of PLCβ1and total CaMKⅣ was similar in the two groups.Conclusion CSD damages the ultrastructure of the hippocampus neurons in rats,which can be effectively improved by dopamine D_1 receptor agonist SKF38393,and the protective mechanism may be related to the PKA pathway and phosphoinositol pathway.