缺血再灌注损伤时iNOS通过激活BAD诱发脊髓神经元凋亡

iNOS induces apoptosis via p-BAD dephosphorylation during spinal cord ischemia reperfusion injury

目的:探讨大鼠脊髓缺血再灌注损伤过程中诱导型一氧化氮合酶(iNOS)调控脊髓神经元凋亡的具体机制。方法:42只SD大鼠随机分为假手术组(A组,6只)、缺血再灌注组(B组,18只)和iNOS抑制组(C组,18只)。A组动物麻醉后仅腹腔切开,B组及C组采用腹主动脉阻断法(夹闭腹主动脉1 h后松开,再灌注6、12、24 h)建立脊髓缺血再灌注损伤模型。手术后,A组及B组动物按5 ml/kg腹腔注射5%二甲基亚砜,C组动物按150 mg/kg腹腔注射同等量氨基胍。Tarlov运动功能评分法评估三组大鼠的后肢运动功能。取腰段脊髓组织,透射电镜观察脊髓神经细胞形态学变化,神经元特异性抗体NeuN免疫荧光计算各组脊髓组织中存活神经元数目,Western blot检测iNOS、Bcl-xL/Bcl-2相关死亡启动因子(Bcl-xL/Bcl-2 associated death promoter,BAD)、磷酸化BAD(p-BAD)、14-3-3及细胞色素C的表达,免疫共沉淀检测BAD与14-3-3的结合状态。结果:Tarlov运动功能评分显示A组大鼠后肢运动功能无损害,B组和C组大鼠后肢功能明显损害,但C组大鼠后肢功能好于B组(P<0.05)。NeuN免疫荧光结果显示B组和C组大鼠脊髓神经元数目明显减少,但C组大鼠脊髓神经元数目多于B组(P<0.05)。电镜结果示B组及C组中脊髓组织均有明显凋亡现象,但C组大鼠凋亡程度较轻。Western blot结果表明iNOS和细胞色素C表达量在B组及C组中随再灌注时间延长而增加,但相同再灌注时间C组表达强度明显弱于B组(P<0.05);p-BAD表达量则随着灌注时间延长而下降,但C组中下降程度明显小于B组(P<0.05)。免疫共沉淀结果表明:BAD与14-3-3结合力随着再灌注时间的延长在B组和C组中逐渐降低,但是C组程度更慢(P<0.05)。结论:脊髓缺血再灌注过程中,iNOS通过促进pBAD去磷酸化,降低BAD与14-3-3的结合力,进而诱发脊髓神经元的凋亡。

Objective：To investigate the mechanism of iNOS in ischemia-reperfusion injury of rat spinal cord. Methods：Forty-two Sprague-Dawley rats were randomly divided into three different groups： Sham-operated group （n = 6）,ischemia-reperfusion group （I/R,n = 18）,and iNOS inhibitor group（I/R ＋ AG,n = 18）. In sham-operated group,peritoneotomy was performed without abdominal aortic cross-clamping （AACC）,however,rats in I/R group and iNOS inhibitor group experienced 1 h AACC followed by 6 h,12 h,24 h reperfusion, respectively. After operation,rats in sham-operated and I/R group received an intraperitoneal injection of the carrier solutions （5 ml/kg of 5% DMSO）,while rats in iNOS inhibitor groups received the treatment of AG at the dose of 150 mg/kg （i.p.）. We examined the neurological motor function using ‘Tarlov’s score’ at 6 h,12 h and 24 h,alterations of spinal cord neurons morphologically by transmission electron microscopy （TEM）. Immunofluerescent staining with anti-NeuN,a specific marker for neurons,was performed to observe the number of surviving neurons in different conditions. The level of iNOS,BAD,p-BAD,14-3-3, and cytochrome C were detected by Western blot;the interaction of BAD and 14-3-3 was determined by coimmunoprecipitation analysis. Results：①The motor functions of the hind limbs of the sham-operated rats were normal. Rats in ischemia-reperfusion group were observed with significant reduce on hind limb movements （P 〈 0.05） in every reperfusion time compared to those in inhibitor group. ②The number of NeuN-positive cells （surviving neurons） in I/R group and iNOS inhibitor group was less than that in sham-operated group,while the number in iNOS inhibitor group was more than that in I/R group （P 〈 0.05）. ③In the sham-operated group,no apoptotic neuron was noted. In ischemia-reperfusion group and iNOS inhibitor group,numerous apoptotic neurons could be detected. However the severity of apoptotic neuron in inhibitor group was less than that in I/R group. ④In the sham-operated group,iNOS was hardly detected at an extremely low level and was increased in a time-dependent manner in I/R group and iNOS inhibitor group,but the expression of iNOS in iNOS inhibitor group was lower than that in I/R group at each reperfusion time （6 h,12 h,24 h） （P 〈 0.05）. Simultaneously,the change of cytochrome C was the same as the iNOS expression mode. In addition,the expression of p-BAD started to decrease at 6 h and decrease remarkably at 12 h,24 h in I/R group and iNOS inhibitor group,but the extent of decrease of p-BAD expression in iNOS inhibitor group was lower than that in I/R group （P 〈 0.05）. ⑤In I/R group,the level of p-BAD/14-3-3 dimerization began to decrease since the reperfusion process started. However in the iNOS inhibitor group,the trend was alleviated by AG treatment （P 〈 0.05）. Conclusion：iNOS inducing apoptosis of neurons in spinal cord ischemia-reperfusion injury is involved in dephosphorylating BAD, and it can lessen spinal neurons apoptosis by inhibiting the activity of iNOS.