盐酸纳美芬对大脑中动脉缺血再灌注模型大鼠脑组织的作用及其机制研究

Neuroprotective effect of nalmefene hydrochloride in rat models of middle cerebral artery occlusion and reperfusion

摘要:

目的 探究盐酸纳美芬对大脑中动脉缺血再灌注（MCAO/R）模型大鼠的神经保护作用。方法 将36只大鼠随机分为假手术组、模型组、盐酸纳美芬组，各12只。盐酸纳美芬组大鼠复制MCAO/R模型前10 min尾静脉注射20 μg/kg盐酸纳美芬注射液，假手术组和模型组注射等体积生理盐水。随后除假手术组外，其余两组采用线栓法复制MCAO/R模型。采用Longa评分标准评估大鼠神经功能，酶联免疫吸附试验（ELISA）检测脑组织超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量，苏木精-伊红（HE）染色和TUNEL染色观察脑组织病理变化和细胞凋亡情况，Western blotting检测脑组织Caspase-3、Bcl-2、Bax蛋白的表达。结果 假手术组大鼠神经功能评分低于模型组和盐酸纳美芬组（P <0.05）；盐酸纳美芬组大鼠神经功能评分低于模型组（P <0.05）。模型组、盐酸纳美芬组SOD活性低于假手术组（P <0.05），MDA含量高于假手术组（P <0.05）；与模型组比较，盐酸纳美芬组SOD活性升高（P <0.05），MDA含量降低（P <0.05）。HE染色结果显示，假手术组细胞轮廓清晰，排列有序；模型组细胞破损，数量减少，炎症浸润严重；盐酸纳美芬组细胞数量较多，轮廓清晰，炎症浸润减少。模型组、盐酸纳美芬组凋亡细胞数多于假手术组（P <0.05）；盐酸纳美芬组凋亡细胞数少于模型组（P <0.05）。模型组、盐酸纳美芬组Caspase-3、Bax蛋白相对表达量高于假手术组（P <0.05），Bcl-2蛋白相对表达量低于假手术组（P <0.05）；与模型组相比，盐酸纳美芬组Caspase-3、Bax蛋白相对表达量降低（P <0.05），Bcl-2蛋白相对表达量升高（P <0.05）。结论 盐酸纳美芬可以降低MCAO/R大鼠脑组织氧化应激水平，减少神经细胞凋亡，具有良好的神经保护作用，其作用机制与调节Caspase-3/Bcl-1/Bax通路有关。

Abstract:

Objective To investigate the neuroprotective effect of nalmefene hydrochloride in rat models of middle cerebral artery occlusion and reperfusion.Methods Thirty-six rats were randomly divided into sham operation group, model control group and nalmefene hydrochloride group, with twelve rats in each group. In the nalmefene hydrochloride group, rats were administrated with 20 μg/kg of nalmefene hydrochloride via tail vein injection 10 minutes before the establishment of the models. The rats in the sham operation group and the model control group were injected with an equal volume of normal saline. Then the middle cerebral artery occlusion and reperfusion model was established by intraluminal suture method in the model control group and the nalmefene hydrochloride group. The neurological function of rats was assessed via the Longa scores. The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in brain tissues were measured by enzyme-linked immunosorbent assay (ELISA). The pathological changes and cell apoptosis of the brain tissues were observed by hematoxylin and eosin (HE) staining and TUNEL staining, respectively. The protein expressions of caspase-3, B cell lymphoma 2 (Bcl-2) and Bax were detected by Western blotting.Results The neurological function scores of the sham operation group were lower than those of the model control group and the nalmefene hydrochloride group (P < 0.05). Compared with the model control group, the neurological function scores in the nalmefene hydrochloride group decreased (P < 0.05). The contents of SOD and MDA were higher in the model control group and the nalmefene hydrochloride group than those in the sham operation group (P < 0.05). Compared with the model control group, the content of SOD increased and the content of MDA decreased in the nalmefene hydrochloride group (P < 0.05). The HE staining showed that the cells in the sham operation group were well-defined and regularly-arranged. In the model control group, the cells were destructed, the number of cells was reduced and the inflammatory infiltration was severe. In contrast, the number of cells was relatively higher, the cells were well-defined and the inflammatory infiltration was reduced in the nalmefene hydrochloride group. Compared with the sham operation group, a larger number of apoptotic cells were observed in the model control group and the nalmefene hydrochloride group (P < 0.05). Compared with the model control group, the number of apoptotic cells decreased in the nalmefene hydrochloride group (P < 0.05). The protein expressions of caspase-3 and Bax were higher and the protein expression of Bcl-2 was lower in the model control group and the nalmefene hydrochloride group relative to the sham operation group (P < 0.05). Compared with the model control group, the protein expressions of caspase-3 and Bax were decreased and the protein expression of Bcl-2 was increased in the nalmefene hydrochloride group (P < 0.05).Conclusions Nalmefene hydrochloride can ameliorate the oxidative stress in brain tissues and reduce the neuronal cell apoptosis in rats models of middle cerebral artery occlusion and reperfusion. Thus, nalmefene hydrochloride plays a neuroprotective role, and the underlying mechanisms may be related to the regulation of caspase-3/Bcl-1/Bax pathway.