活性氧介导的线粒体途径细胞凋亡在多次七氟烷麻醉诱发新生大鼠远期认知功能障碍中的作用

Role of reactive oxygen species-mediated mitochondrial pathway of apoptosis in long-term cognitive impairment induced by multiple exposures to sevoflurane in neonatal rats

目的评价活性氧(ROS)介导的线粒体途径细胞凋亡在多次七氟烷麻醉诱发新生大鼠远期认知功能障碍中的作用。方法SPF级健康新生SD大鼠60只,体重12~20 g,采用随机数字表法分为3组(n=20):对照组(C组)、多次七氟烷麻醉组(S组)和ROS抑制剂组(A组)。S组和A组于出生后6、7和8 d时吸入3%七氟烷2 h,C组吸入空气。A组大鼠于每次七氟烷麻醉前腹腔注射ROS抑制剂N-乙酰半胱氨酸150 mg/kg。于出生后35 d时行旷场实验评估大鼠的自发活动能力,于出生后36 d时行Morris水迷宫实验检测认知功能,水迷宫实验结束后处死大鼠分离海马组织,采用流式细胞术检测海马神经元凋亡率、ROS及线粒体膜电位(MMP)水平,采用Western blot法检测细胞色素c(Cyt c)、裂解的caspase-9和caspase-3水平。采用RT-PCR法检测Bcl-2和Bax的mRNA表达水平。透射电镜下观察海马神经元线粒体的超微结构。结果与C组比较,S组逃避潜伏期延长,穿越原平台次数减少,海马神经元凋亡率、ROS与MMP水平升高,Cyt c、裂解的caspase-9和caspase-3、Bax mRNA表达上调,Bcl-2 mRNA表达下调,Bax/Bcl-2比值升高(P<0.05),线粒体肿胀、线粒体嵴结构断裂。与S组比较,A组逃避潜伏期缩短,穿越原平台次数增加,海马神经元凋亡率、ROS与MMP水平下降,Cyt c、裂解的caspase-9和caspase-3、Bax mRNA表达下调,Bcl-2 mRNA表达上调,Bax/Bcl-2比值降低(P<0.05),线粒体肿胀及嵴结构断裂的情况改善。结论多次七氟烷麻醉诱发新生大鼠远期认知功能障碍的机制可能与激活ROS介导的线粒体途径细胞凋亡有关。

Objective To evaluate the role of reactive oxygen species(ROS)-mediated mitochondrial pathway of apoptosis in long-term cognitive impairment induced by multiple exposures to sevoflurane in the neonatal rats.Methods Sixty SPF healthy neonatal Sprague-Dawley rats,weighing 12-20 g,were divided into 3 groups(n=20 each)using a random number table method:control group(group C),multiple exposures to sevoflurane for anesthesia group(group S)and ROS inhibitor group(group A).Group S and group A inhaled 3%sevoflurane for 2 h starting from 6,7 and 8 days after birth,while group C inhaled air.In group A,ROS inhibitor N-acetylcysteine(NAC)150 mg/kg was intraperitoneally injected before each anesthesia with sevoflurane.The spontaneous activity was evaluated by open field test on day 35 after birth.The cognitive function was determined by Morris water maze test on day 36 after birth.The rats were sacrificed after the end of Morris water maze test,and the hippocampal tissues were obtained for determination of the apoptosis rate of hippocampal neurons,reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)(by flow cytometry)and levels of Cyt c and cleaved caspase-9 and caspase-3(by Western blot).The expression of Bcl-2 and Bax mRNA was detected by real-time polymerase chain reaction.The ultrastructure of mitochondria in hippocampal neurons was observed with a transmission electron microscope.Results Compared with group C,the escape latency was significantly prolonged,the number of crossing the original platform was reduced,the apoptosis rate of hippocampal neurons and levels of ROS and MMP were increased,the expression of Cyt c,cleaved caspase-9,cleaved caspase-3 and Bax mRNA was up-regulated,the expression of Bcl-2 mRNA was down-regulated,the ratio of Bax/Bcl-2 was increased(P<0.05),mitochondria were swollen,and mitochondrial cristae structure was broken in group S.Compared with group S,the escape latency was significantly shortened,the number of crossing the original platform was increased,the apoptosis rate of hippocampal neurons and levels of ROS and MMP were decreased,the expression of Cyt c,cleaved caspase-9,cleaved caspase-3 and Bax mRNA was down-regulated,the expression of Bcl-2 mRNA was up-regulated,the ratio of Bax/Bcl-2 was decreased(P<0.05),and the mitochondrial swelling and rupture of cristae structure were improved in group A.Conclusion The mechanism by which multiple exposures to sevoflurane induce long-term cognitive impairment may be related to activating the ROS-mediated mitochondrial pathway of apoptosis in neonatal rats.