靶向调节Nrf2基因对急性重症一氧化碳中毒大鼠脑损伤的神经保护作用

The neuroprotective effect of targeted regulation Nrf2 gene on injured brain caused by acute severe carbon monoxide poisoning in rats

目的探讨靶向调节核因子E2相关因子2（nuclear factor E2 related factor2，Nrf2）基因对急性重症一氧化碳（CO）中毒大鼠脑损伤的神经保护作用。方法按随机数字表法将180只健康雄性SD大鼠分为正常对照组、CO中毒组、慢病毒组和Nrf2治疗组，每组45只。采用高压氧舱吸入法建立急性重症CO中毒动物模型。慢病毒组在脑立体定向仪指导下用微量注射器向纹状体内局部注射慢病毒稀释液（4×10^6TU／μl），Nrf2治疗组给予同等剂量重组Nrf2基因慢病毒稀释液，正常对照组和CO中毒组给予等量缓冲液。各组分别于造模后1d、7d、14d各取5只大鼠，用JC-1法检测脑组织神经细胞线粒体膜电位（mitochondrial membrane potential，MMP），用免疫组化法及Western blot法观察脑组织Nrf2和谷氨酸-半胱氨酸连接酶催化亚基（glutamate-cysteine ligase catalytic subunit，GCLC）蛋白的表达变化。结果造模1d，与正常对照组[皮质区：（75.3±6.8），海马区：（76.4±7.1），纹状体区：（73.8±7.3）]比较，CO中毒组[皮质区：（34.5±6.7），海马区：（30.3±5.6），纹状体区：（41.5±6.1）]和慢病毒组[皮质区：（36.8±6.2），海马区：（30.8±6.0），纹状体区：（42.7±6.3）]大鼠脑组织神经细胞MMP明显下降，差异有统计学意义（P〈0.05）。但CO中毒组和慢病毒组比较，差异无统计学意义（P〉0.05）。正常对照组大鼠脑组织有少量Nrf2蛋白（0.22±0.05）和GCLC蛋白（0.24＋0.04）表达，CO中毒组和慢病毒组脑组织Nrf2蛋白（0.31±0.06，0.31±0.05）和GCLC蛋白（0.30±0.04，0.31±0.07）的表达略增高（P〈0.05）。但CO中毒组和慢病毒组比较，差异无统计学意义（P〉0.05）。Nrf2治疗组大鼠脑组织神经细胞MMP[皮质区：（53.3±5.3），海马区：（56.9±6.1），纹状体区：（60.6±6.0）]，明显高于同一时间点CO中毒组及慢病毒组（P〈0.05）；脑组织Nrf2的表达明显增高（0.59±0.05），与同一时间点CO中毒组及慢病毒组比较，差异有统计学意义（P〈0.05）；GCLC蛋白略有升高（0.37±0.06），但与CO中毒组及慢病毒组比较差异无统计学意义（P〉0.05）。结论CO中毒能诱发机体氧化应激反应，损伤神经细胞线粒体功能；靶向调节Nrf2的活性状态，能明显增强大鼠抗氧化能力，对急性重症CO中毒引起的脑损伤起积极的保护作用。

Objective To explore the neuroprotective effect of targeted regulation Nrf2 gene on rats with brain injury caused by acute severe carbon monoxide （CO） poisoning. Methods A total of 180 healthy adult SD rats were divided into 4 groups at random：normal control group （NC group）, CO poisoning group（CO group）,lentivirus group （LV group） and Nrf2 gene therapy group （Nrf2 group ）, and 45 rats in each group. An acute CO toxic rat model was established by inhalation in a hyperbaric oxygen tank. The lentivirus group was directly injected with lentivirus dilution （4×10^6 TU/μl） into striatum with a microsy-ringe guided by a stereotactic apparatus, and the Nrf2 gene therapy group was administrated the same dose of recombinant Nrf2 gene lentivirus dilution, while rats in the normal control group and the CO poisoning group were received the same amount of normal saline. Five rats were taken and decapitated at day 1, day 7 and week 2 from each group ,respectively. The mitochondrial membrane potential （MMP） of neurons in brain tissue was detected by JC-1 method,and the expressions of Nrf2 and GCLC proteins were observed by immunohistochemistry and Western Blot. Results Compared with the NC group （cortex： （75.3±6. 8） ;hippocampus： （76.4±7.1） ;striatum： （73.8±7.3）） at the same time point,the MMPs of neurons in CO group （ cortex： （34.5±6.7） ;hippocampus ： （30. 3±5.6） ;striatum： （41.5±6. 1） and LV group （cortex： （36.8±6.2） ; hippocampus ： （30.8±6.0） ; striatum： （42.7±6. 3 ） ） were significantly decreased, and the difference was sig- nificant（P〈0.05）. However, there was no significant difference between the CO poisoning group and the lentivirns group （P〉0.05）. A small amount of Nrf2 protein （0.22±0.05） and GCLC protein （0.24±0.04） were expressed in the brain tissue of normal control rats. The expressions of Nrf2 protein （0. 31±0.06,0. 31±0.05） and GCLC protein （0.30±0.04,0.31±0.07） in CO group and LV group were slightly increased （P〈0.05）. Similarly, there was no significant difference between the CO poisoning group and the lentivirus group （P〉0.05）. The MMPs value of nerve cells in the Nrf2 group （cortex： （53.3±5.3） ;hippocampus： （56.9±6.1）;striatum： （60.6±6.0）） also decreased,but it was significantly higher than that in the CO group and the LV group at the same time point （P〈0.05）. The expression of Nrf2 in brain tissue was significantly increased （0.59±0.05）, and there was significant difference between CO group and LV group at the same time point （P〈0.05） ;GCLC protein increased slightly （0.37±0.06）, but there was no statistical difference compared with CO poisoning group and lentivirus group （P〉0.05）. Conclusion CO poisoning could induce oxidative stress and damage mitochondrial function of nerve cells. The active state of targeted regulation Nrf2 could significantly enhance the antioxidant capacity of rats and positively protect rats against brain injury induced by acute severe CO poisoning.