氧自由基/JNK信号通路在蛛网膜下腔出血大鼠神经细胞自噬中的作用机制

The mechanism of oxygen free radical/JNK signaling pathway on neuronal autophagy after subarachnoid hemorrhage in rats

目的自噬是真核细胞中产生的一种细胞防御机制,决定应激损伤后细胞的恢复、修复、损伤、加重或死亡。探讨氧自由基/c-Jun氨基端激酶(JNK)信号通路在蛛网膜下腔出血(SAH)大鼠神经细胞自噬中的作用。方法 160只雄性SD大鼠随机数字表法分成假手术组、SAH组、低剂量依达拉奉组、高剂量依达拉奉组。除假手术组外,其余组采用枕大池二次注血法制作SAH大鼠模型,假手术组向枕大池2次注入0.3 mL等渗盐水,余操作与SAH组一致;低剂量依达拉奉组、高剂量依达拉奉组在建模成功后经尾静脉分别给予依达拉奉(5 mg/kg,10 mg/kg,1次/24 h)。光镜观察海马区神经细胞形态结构,采用硫代巴比妥酸法检测大脑组织丙二醛水平;免疫组织化学法观察磷酸化JNK、自噬标志蛋白(Beclin-1和LC3-II)变化趋势,实时荧光定量PCR检海马区JNK mRNA、Beclin-1 mRNA、LC3 mRNA表达水平。结果 SAH组海马区可见坏死神经细胞,表现为细胞出现核溶解、核碎裂或核消失结构不清;与假手术组比较,SAH组各时间点神经细胞存活率均明显减少(P<0.05)。低剂量依达拉奉组、高剂量依达拉奉组神经细胞形态结构损伤均减轻,视野中死亡神经细胞减少;与SAH组比较,低剂量依达拉奉组、高剂量依达拉奉组各时间点神经元存活率均明显增多(P<0.05)。与假手术组比较,SAH组各时间点丙二醛均明显增多(P<0.05);与SAH组6、24、48、72 h丙二醛水平[(4.98±0.72、6.38±0.86、7.86±0.82、9.26±0.96)mmol/L]比较,低剂量依达拉奉组[(3.08±0.60、4.02±0.72、4.96±0.91、5.01±0.74)mmol/L]、高剂量依达拉奉组[(2.16±0.52、2.74±0.66、3.02±0.59、3.24±0.60)mmol/L]均降低(P<0.05)。与假手术组比较,SAH组各磷酸化JNK、Beclin-1和LC3-II免疫阳性反应增强。与SAH组比较,低剂量依达拉奉组、高剂量依达拉奉组磷酸化JNK免疫阳性反应减弱,Beclin-1和LC3-II免疫阳性反应增强。与假手术组比较,SAH组各JNK mRNA、Beclin-1 mRNA和LC3-II mRNA表达水平增加(P<0.05)。与SAH组比较,低剂量依达拉奉组、高剂量依达拉奉组JNK mRNA表达水平降低,Beclin-1 mRNA和LC3-II mRNA表达水平增强(P<0.05)。结论 SAH后氧自由基激活JNK信号通路,调控Beclin-1和LC3-Ⅱ表达介导神经细胞丢失。

Objective To investigate the mechanisms of oxygen free radical/JNK signaling pathway on neuronal autophagy after subarachnoid hemorrhage（ SAH） in rats. Methods 160 male Sprague-Dawley rats were randomly divided into four groups ： sham group, SAH model group, low dose Edaravone group and high dose Edaravone group. The SAH model was established by autologous blood injection into cisterna magna twice, while the rats in the sham group were injected with isotonic saline （ 0. 3 mL/time） . The high dose of edaravone group and low dose of edaravone group were given 5 mg/kg or lOmg/kg of edaravone, respectively, once daily with tail intrave-nous injection after the models were established. The morphological changes of hippocampus neural cells were detected by light micro-scope. The malondialdehyde （MDA） level in brain tissue was determined with thiobarbituric acid. The changes of phosphorylated JNK and autophagic biomarkers （Beclin-1 and LC3-II）were detected by immunohistochemical method. The expressions of JNK mRNA,Bec- lin-1 mRNA and LC3 mRNA in hippocampus was detected by Real time-quantitative PCR. Results The necrotic nerve cells were seen in the hippocampus of SAH group in terms of nuclear dissolution, nuclear fragmentation or nuclear disappearance.Compared with Sham group, the level of MDA and the number of dead neurons, the expression of JNK mRNA, Beclin-1 mRNA and LC3-II mRNA were increased in the SAH group （P〈0.05）. The survival rate of nerve cells in the SAH group was lower than that in the sham group. The immunoreactivity of phosphorylated JNK、 Beclin-1 and LC3-II in the SAH group was enhanced than that in the sham group. How-ever the damage of the morphological structure of nerve cells was relatively decreased in both doses groups. Compared with SAH group, the level of MDA and the expression of JNK mRNA in low dose Edaravone group and high dose Edaravone group were decreased. The expression of Beclin-1 mRNA and LC-3 mRNA was higher （P〈0.05）. Furthermore, the survival rates of nerve cells in both doses- groups were higher than that in the SAH group （P〈0.05） . Meanwhile, the immunoreactivity of phosphorylated JNK in both doses groups was weakened than that in the SAH group. The mRNA expression of Beclin-1 and LC3-II was increased （ P 〈 0. 05 ）. Conclusion Oxygen free radical played an important role in process of neuronloss by activating the JNK signaling pathway to regulate Beclin-1 and LC3-II expression.