亚甲蓝预处理对大鼠局灶性脑缺血再灌注损伤的影响

Effects of methylene blue pretreatment on focal cerebral ischemia-reperfusion injury in rats

摘要:

目的：研究亚甲蓝（methylene blue，MB）预处理对大鼠局灶性脑缺血再灌注（ischemia-reperfusion，I/R）损伤的影响及其可能机制。方法：64只健康成年雄性SD大鼠随机分为4组（n=16）：假手术（S）组、亚甲蓝对照（S+M）组、局灶性脑缺血再灌注（I/R）组、亚甲蓝预处理（I/R+M）组，采用大脑中动脉线栓法制备大鼠局灶性脑缺血再灌注模型。S组于假手术后腹腔注射生理盐水0.5 mL/kg，S+M组于假手术后腹腔注射亚甲蓝5 mg/kg，I/R组于再灌注前5 min腹腔注射生理盐水0.5 mL/kg，I/R+M组于再灌注前5 min腹腔注射亚甲蓝5 mg/kg。各组于再灌注前5 min和再灌注24 h时采用Longa评分法行大鼠神经功能缺失评分；再灌注24 h时2,3,5-氯化三苯基四氮唑（2,3,5-triphenyl tetrazolium chloride，TTC）染色后计算脑梗死体积及其百分比；HE染色观察缺血侧脑皮质病理学变化；化学发光法测定缺血侧脑组织活性氧（reactive oxygen species，ROS）含量；Western blot、免疫组织化学法检测缺血侧脑组织核因子E2相关因子（nuclear factor erythroid 2-related factor 2，Nrf2）和血红素氧合酶-1（heme oxyge－nase 1，HO-1）蛋白表达水平。结果：与S组和S+M组相比，I/R组和I/R+M组的神经功能缺失评分、脑梗死体积及其百分比、脑组织ROS含量均升高，Nrf2和HO-1蛋白表达上调（P<0.01），缺血侧脑皮质有明显病理学损伤。与I/R组相比较，I/R+M组的神经功能缺失评分、脑梗死体积及其百分比、脑组织ROS含量均降低，Nrf2和HO-1蛋白表达上调（P<0.01），缺血侧脑皮质病理学损伤较轻；S组和S+M组之间上述指标差异无统计学意义（P>0.05）。结论：亚甲蓝预处理可以改善大鼠局灶性脑缺血再灌注损伤，其机制可能与上调Nrf2和H0-1的表达、降低脑ROS的含量、产生抗氧化应激作用有关。

Abstract:

Objective：To research the effect of methylene blue（MB） pretreatment on focal cerebral ischemia-reperfusion（I/R） injury in rats and its possibly mechanism. Methods：Sixty-four adult male Sprague-Dawley rats were randomly divided into four groups（n=16）：sham group（group S），methylene blue control group （group S+M），focal cerebral ischemia-reperfusion group（group I/R），methy－lene blue pretreatment group （group I/R+M）. The rat model of focal cerebral ischemia-reperfusion was established by middle cerebral artery occlusion. The neurological deficit scores were performed with Longa’s scale at 5 min before reperfusion and at 24 h after reperfusion. The pathological changes in pallium of ischemic area were observed by HE staining. Reactive oxygen species（ROS） in ischemic brain tissue were measured by chemiluminescent. The expressions of nuclear factor erythroid 2-related factor 2（Nrf2） and heme oxygenase 1（HO-1） in ischemic brain tissue were detected by Western blot and immunohistochemistry staining. Results：The neurological deficit scores，the cerebral infarct volume and the infarct volume fraction，the content of ROS，the expression of Nrf2 and HO-1 were all obviously higher in group I/R and I/R+M than in group S and S+M respectively（P<0.01）. The neurological deficit scores，the cerebral infarct volume and the infarct volume fraction，the content of ROS were all obviously lower in group I/R+M than in group I/R（P<0.01）. The expressions of Nrf2 and HO-1 were obviously increased in group I/R+M than in group I/R（P<0.01）. The pathological changes in pallium of ischemic area were significantly ameliorated in group I/R+M than in group I/R. The difference between group S and group S+M was not statistical significant（P>0.05）. Conclusion：The pretreatment of methylene blue can improve the focal cerebral ischemia-reperfusion injury in rats，which might be related to upregulated the expression of Nrf2 and HO-1，reduce the content of ROS in brain，produce antiox－idant stress.