JNK/c-Jun信号通路通过调控细胞骨架重构参与大鼠垂体柄损毁后精氨酸加压素神经元修复

JNK/c-Jun signaling pathway mediates arginine vasopressin neuron regeneration by promoting cytoskeleton reconstruction in rats with electrical lesions of the pituitary stalk

目的探究大鼠垂体柄损毁后精氨酸加压素(AVP)神经元通过双皮质素(DCX)促进神经元骨架修复的机制。方法取32只SD大鼠,随机分为实验组(损毁后1d组,n=4;损毁进神经元骨架恢后3d组,n=7;损毁后7d组,n=7;损毁后14d组,n=7)和对照组(假手术组,n=7),经颅顶损毁垂体柄,记录尿量、饮水量和尿比重,通过免疫荧光染色比较各组视上核(SON)AVP神经元中Ⅲ型β微管蛋白(Tuj1)、DCX和caspase3表达情况,并通过蛋白质印迹法探讨其调控机制。结果垂体柄损毁后,饮水量、尿量和尿比重出现了典型的三相性尿崩症(损毁后1~2d为第一相,术后3~5d为第二相,6d以后为第三相)。免疫荧光的结果提示了AVP神经元损伤后修复现象,主要表现为DCX表达在损毁后不同程度的升高。AVP神经元中DCX阳性率分别为:假手术组,(37.93±1.83)%;损毁后1d,(44.67±1.79)%;损毁后3d,(48.09±2.96)%;损毁后7d,(71.34±2.18)%;损毁后14d,(61.08±2.15)%,(P<0.01)。同时,神经元骨架蛋白Tuj1也呈现出相同时间规律,即垂体柄损毁后7d最高[假手术组,(42.82±2.02)%;损毁后7d,(60.70±1.28)%,P<0.01]。另一方面,神经元凋亡现象与骨架修复呈现相反的时间规律,主要表现为caspase3+DCX+双阳性细胞占DCX阳性细胞的比例逐渐下降:损毁后3d,(66.03±3.58)%,损毁后7d,(43.42±4.45)%,损毁后14d,(35.11±1.73)%。通过蛋白质印迹技术,进一步研究发现,神经元损伤相关通路(JNK/c-Jun)在损毁后不同时间点出现上调,表现为:p-JNK仅在损毁后3d时表达上调(P<0.05),c-Jun,p-c-Jun表达于损毁后3d时表达上调(P<0.05),并于损毁后7d时达到高峰(P<0.01),这分别与神经元凋亡与修复的时间规律相符合。结论垂体柄损毁后,JNK/c-Jun信号通路激活,一方面诱发早期AVP神经元凋亡,另一方面可能通过诱导DCX表达,促进Tuj1合成从而修复细胞骨架,保护神经元免受凋亡以及促进神经内分泌功能重建。

Objective To investigate the mechanism by which doublecortin promotes the recovery of cytoskeleton in arginine vasopressin (AVP) neurons in rats with electrical lesions of the pituitary stalk (PEL).Methods Thirty-two SD rats were randomized into PEL group with electrical lesions of the pituitary stalk through the floor of the skull base (n=25) and sham operation group (n=7),and the daily water consumption (DWC),daily urine volume (DUV) and urine specific gravity (USG) of the rats were recorded.Four rats on day 1 and 7 rats on each of days 3,7 and 14 after PEL as well as the sham-operated rats were sacrificed for detection of the expressions of β-Tubulin (Tuj1),doublecortin and caspase- 3 in the AVP neurons of the supraoptic nucleus using immunofluorescence assay and Western blotting.Results After PEL,the rats exhibited a typical triphasic pattern of diabetes insipidus,with the postoperative days 1-2 as the phase one,days 3-5 as the phase two,and days 6-14 as the phase three.Immunofluorescent results indicated the repair of the AVP neurons evidenced by significantly increased doublecortin expressions in the AVP neurons following PEL;similarly,the expression of Tuj1 also increased progressively after PEL,reaching the peak level on day 7 after PEL.The apoptotic rates of the AVP neurons exhibited a reverse pattern of variation,peaking on postoperative day 3 followed by progressive reduction till day 14.Western blotting showed that the expressions of c-Jun and p-c-Jun were up-regulated significantly on day 3 (P<0.05) and 7 (P<0.01) after PEL,while an upregulated p-JNK expression was detected only on day 3 (P<0.05),as was consistent with the time-courses of neuronal recovery and apoptosis after PEL.Conclusion JNK/c-Jun pathway is activated after PEL to induce apoptosis of AVP neurons in the acute phase and to promote the repair of neuronal cytoskeleton by up-regulation of doublecortin and Tuj1 expressions.