【摘要】：目的觀察睡眠剝奪(sleep deprivation,SD)對內(nèi)側(cè)前額葉皮層(medial prefrontal cortex,m PFC)神經(jīng)元自噬(autophagy)活動的影響及機制。方法實驗使用成年(8~10周)雄性C57BL/6小鼠,體重23-26g,隨機分為睡眠剝奪6 h(SD6 h)組與其對照組(n=10-12只/組)、睡眠剝奪12 h(SD12 h)組與其對照組(n=10-12只/組)。采用輕柔刺激法建立SD6 h和SD12 h模型。蛋白免疫印跡(Western blot)檢測內(nèi)側(cè)前額葉神經(jīng)元皮層超氧化物歧化酶2(superoxide dismutase,SOD2)、過氧化氫酶(catalase)、沉默信息調(diào)節(jié)因子3(Silent information regulator 3,Sirt3)、微管相關(guān)蛋白1輕鏈3-Ⅱ(LC3-Ⅱ)、P62(也稱SQSTM1)、Beclin-1、腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)、雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)、p-AMPK、p-m TOR蛋白的表達水平。免疫熒光染色觀察內(nèi)側(cè)前額葉皮層神經(jīng)元溶酶體膜相關(guān)蛋白1(lysosomal-associated membrane protein 1,LAMP-1)的表達情況,Dihydroethidine(DHE)染色檢測m PFC神經(jīng)元O2-的水平。結(jié)果1.在正常睡眠覺醒周期中,與白天相比,夜間自噬流活性較高,P62蛋白表達較低,Beclin-1蛋白表達相對較高。2.與對照組相比,SD6 h后m PFC神經(jīng)元SOD2(P0.05)、Catalase(P0.05)和Sirt3(P0.05)蛋白表達均顯著上調(diào),O2-的水平同時也相對升高(P0.05);而SD12 h后,m PFC神經(jīng)元SOD2、Catalase和Sirt3蛋白表達水平與對照組相比均無顯著差異,但O2-的水平相對升高。3.與對照組相比,SD6 h與SD12 h均使m PFC神經(jīng)元LC3-Ⅱ(P0.05)和P62的表達水平升高(P0.05),進一步研究發(fā)現(xiàn),Beclin-1(P0.05)和LAMP-1(P0.05)的表達水平均相對升高。4.SD6 h后,與對照組相比,m PFC神經(jīng)元AMPK和m TOR蛋白水平無顯著變化;SD12 h后,與對照組相比,m PFC神經(jīng)元m TOR蛋白表達水平顯著下調(diào)(P0.05),但AMPK蛋白表達無明顯變化。結(jié)論1.睡眠期間,m PFC神經(jīng)元自噬活動較低,而覺醒期間m PFC神經(jīng)元自噬活動較高。2.睡眠剝奪后,m PFC神經(jīng)元氧化水平升高,而抗氧化水平只產(chǎn)生一過性升高。3.無論是SD6 h還是SD12 h,均使m PFC神經(jīng)元自噬活動增強。4.SD12 h使m PFC神經(jīng)元m TOR信號通路被抑制,進而誘導自噬活動上調(diào)。
[Abstract]:Objective to investigate the effect and mechanism of sleep deprivation (sleep deprivation,SD) on autophagy (autophagy) activity of (medial prefrontal cortex,m PFC neurons in medial prefrontal cortex. Methods male C57BL/6 mice were randomly divided into sleep deprivation group (SD6 h) and control group (n = 10-12), sleep deprivation for 12 h (SD12 h) and control group (n = 10-12). SD6 h and SD12 h models were established by soft stimulation. Western blot (Western blot) detection of superoxide dismutase 2 (superoxide dismutase,SOD2), catalase (catalase), silencing signaling factor 3 (Silent information regulator 3 sirt3, microtubule-associated protein 1 light chain 3- 鈪，

本文編號：2265470