自噬反应对大鼠创伤性颅脑损伤后神经功能的影响及机制研究

Effect of autophagy response on neurological functions and its mechanism in rats after traumatic brain injury

目的探讨自噬反应对大鼠创伤性颅脑损伤（TBI）后神经功能修复的影响及丝裂素活化蛋白激酶家族（MAPKs）信号通路在其中的作用。方法健康雄性SD大鼠54只按随机数字表法分为假手术组、TBI组、TBI＋3-甲基腺嘌呤（3-MA）组，每组18只；后2组大鼠采用改良Feeney法建立TBI模型，假手术组大鼠仅开骨窗不予撞击。TBI＋3-MA组大鼠于造模后30min腹腔注射3-MA（5mg／kg），其余2组大鼠腹腔注射等量生理盐水。造模后1、3、7d酶联免疫吸附法检测3组大鼠血清中S100B和神经元特异性烯醇化酶（NSE）蛋白的浓度，采用改良神经功能评分（mNSS）对大鼠进行运动、感觉和反射功能检查并测定脑组织含水量；造模后3dWestern blotting检测大鼠脑组织自噬相关因子微管相关蛋白轻链3-Ⅱ（LC3-Ⅱ）、自噬相关基因-1（Beclin-1）以及c-jun氨基末端激酶（YNK）、磷酸化JNK（p-JNK）、细胞外调节蛋白激酶1／2（ERK1／2）、p-ERK1／2、p38MAPK、p—p38MAPK蛋白的表达。结果与假手术组比较，TBI组、TBI＋3-MA组大鼠造模后1、3、7d血清神经损伤标志物S100B和NSE蛋白、mNSS评分、脑组织含水量升高，差异有统计学意义（P＜0．05）。TBI＋3-MA组造模后3、7dS100B和NSE蛋白，造模后1、3、7dmNSS评分、脑组织含水量均低于TBI组，差异有统计学意义（P＜0．05）。其中造模后3d假手术组、TBI组、TBI＋3-MA组大鼠mNSS评分分别为1．10±0．27、11．76±0．35、10．71±0．33；与假手术组比较，TBI组、TBI＋3-MA组大鼠造模后3d脑组织LC3-Ⅱ、Beclin-1蛋白以及JNK、p-JNK、ERK1／2、p-ERK1／2、p38MAPK、p-p38MAPK蛋白表达上调，差异有统计学意义（P＜0．05）。与TBI组比较，TBI＋3-MA组大鼠造模后3d脑组织LC3-Ⅱ、Beclin-1蛋白，p-JNK和p-p38MAPK蛋白表达减少，差异有统计学意义（P＜0．05）。结论抑制自噬反应能改善颅脑损伤后的神经功能，起到神经保护作用，其机制可能是通过抑制MAPKs家族中的JNK和p38MAPK信号传导通路来实现。

Objective To investigate the effect of autophagy response on neurological functions and the role of mitogen-activated protein kinases （MAPKs） signaling pathway in rats after traumatic brain injury （TBI）. Methods Fifty-four healthy male SD rats were randomly divided into sham-operated group, TBI group and TBI＋autophagy inhibitor 3-methyladenine （3-MA） group （n=18）. TBI animal models of the later two groups were established using Feeney＇s method. Rats in the sham-operated group were only performed bone window opening without knock; rats in the TBI＋3-MA group were given intraperitoneal injection of 3-MA（5 mg/kg） 30 rain after modeling and rats in the other two groups were given the same volume of normal saline. Three and 7 d after modeling, the protein levels of S100B and neuron specific enolase （NSE） in serum were tested with enzyme linked immunosorbent assay （ELISA）; modified neurologic severity scale （mNSS） was used to detect the movement, sense and reflex functions; brain water content was measured with wet-dry weight method. The autophagy related factors （LC3-Ⅱ and Beclin-1） and MAPKs signaling pathway related factors （c-Jun N-terminal kinase [JNK], phosphorylated [p]-JNK, extracellular signal-regulated kinase [ERK]1/2, p- ERK1/2, p38MAPK and p-p38MAPK） protein expressions in TBI cerebral cortex were determined by Western blotting. Results As compared with those in the sham-operated group, the brain edema level, mNSS scores, and S100B and NSE protein levels in the TBI group and TBI＋3-MA group were significantly increased （P〈0.05）; TBI＋3-MA group had significantly lower brain edema level, mNSS scores, and S100B and NSE protein levels than TBI group （P〈0.05）. The expression levels of autophagy and MAPKs signaling pathway related factors in the TBI group and TBI＋3-MA group were significantly higher as compared with those in the sham-operated group （P〈0.05）. As compared with the TBI group, TBI＋3-MA group had significantly decreased levels of LC3-Ⅱ, Beclin-1 and activation of JNK and p-p38MAPK signaling pathways （P〈0.05）. Conclusion Suppressing autophagy response markedly improves neurological outcomes after TBI, possibly mediated by inhibiting activation of JNK and p38MAPK signaling pathways.