活性氧自由基清除剂EUK-134对脑缺血再灌注损伤大鼠线粒体相关蛋白PGC-1α和VDAC1的影响

doi:10.3969/j.issn.1006-7795.2024.06.011

摘要/Abstract

摘要： 目的研究活性氧自由基(reactive oxygen species, ROS)清除剂Eukarion-134 (EUK-134)对大脑中动脉梗死（middle cerebral artery occlusion, MCAO）再灌注大鼠脑内线粒体生物发生相关蛋白——过氧化物酶体增殖物激活受体γ辅激活因子-1α（peroxisome proliferator activated receptor gamma coactivator-1α，PGC-1α），以及线粒体电压依赖性阴离子通道1（voltage-dependent anion channel 1，VDAC1）的影响，探讨脑缺血后ROS含量变化对线粒体结构和功能的影响。方法将健康雄性SD大鼠采用随机数字表法分为3组：假手术（Sham）组、MCAO组和MCAO+EUK-134 （EUK-134）组，每组6只。使用改良线栓法构建大鼠中动脉梗死模型，模拟缺血90 min后拔出线栓进行再灌注24 h。手术期间持续监测大鼠心率、平均动脉压以及肛温，确保其生理指标维持在正常范围内。EUK-134组大鼠通过皮下注射的方式注射EUK-134（10 mg/kg），Sham及MCAO组大鼠给予等体积溶剂。采用Western blotting法检测大鼠缺血脑组织中PGC-1α的表达。应用免疫荧光染色检测大鼠脑缺血半暗带区VDAC1的表达，利用氧化感应荧光探针(2′,7′-dichlorofluorescin diacetate, H2DCF-DA)染色法标记ROS阳性细胞，并将VDAC1与ROS进行荧光双标染色。结果 ① MCAO组大鼠再灌注24 h缺血侧脑组织PGC-1α蛋白水平比Sham组明显降低（P <0.05）；与MCAO组相比，EUK-134组缺血侧脑组织PGC-1α蛋白水平明显升高（P<0.05）。② 与Sham组相比，MCAO组大鼠再灌注24 h缺血半暗带区VDAC1阳性细胞数量明显增多（P<0.05），而给予EUK-134处理的脑缺血再灌注大鼠缺血半暗带区VDAC1阳性细胞数量显著低于MCAO组（P<0.05）。VDAC1与神经元标志物NeuN共定位。 ③在MCAO组大鼠缺血半暗带区，VDAC1阳性细胞与ROS共定位，且VDAC1/ROS双阳性细胞数量显著高于Sham组（P<0.05），给予EUK-134的缺血大鼠VDAC1/ROS双阳性细胞数量较MCAO组明显降低（P<0.05）。结论 ROS清除剂EUK-134可以影响线粒体生物发生调节因子PGC-1α和物质运输通道VDAC1的表达，维持线粒体功能，改善脑缺血再灌注损伤。

关键词: 脑缺血再灌注, 活性氧自由基, EUK-134, PGC-1α, VDAC1

Abstract: Objective To investigate the effects of Eukarion-134 (EUK-134), a scavenger of reactive oxygen species (ROS), on peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α) and mitochondrial voltage-dependent anion channel 1 (VDAC1) in middle cerebral artery occlusion (MCAO) and reperfusion rats, and to explore the impact of ROS generation on mitochondrial structure and function after cerebral ischemia. Methods Healthy male Sprague Dawley (SD) rats were divided into three groups with a random number table method: Sham group, MCAO group, and MCAO+EUK-134 (EUK-134) group, with 6 rats in each group. A model of MCAO was induced with the intraluminal suture method. The rats underwent 90 min of MCAO and then were reperfused for 24 h by withdrawal of the filament. The heart rate, mean arterial pressure, and temperature were maintained in the normal range during surgery. EUK-134(10 mg/kg) was injected subcutaneously in rats of the EUK-134 group, and rats in the sham and MCAO groups received equal volumes of solvent. The expression of PGC-1α in ischemic brain tissue was assessed by Western blotting. Immunofluorescence staining was applied to detect the expression of VDAC1 in the ischemic penumbra of rats. The ROS positive cells were labeled with an oxidant-sensing fluorescent probe (2′,7′-dichlorofluorescin diacetate, H2DCF-DA) staining, and double staining for VDAC1 and ROS were performed. Results ① The expression of PGC-1α protein level in the ischemic brain tissue of rats in the MCAO group after 24 h reperfusion was significantly lower than that of the sham group (P<0.05). The PGC-1α protein level in the ischemic brain tissue of the EUK-134 group was significantly higher than that of the MCAO group (P<0.05). ② After 24 h reperfusion, the number of VDAC1 positive cells in the ischemic penumbra of the MCAO group increased significantly compared to the sham group (P<0.05), while the number of VDAC1 positive cells in the ischemic penumbra of the EUK-134 group was significantly lower than that of the MCAO group (P<0.05). VDAC1 positive cells were colocalized with the neuronal marker NeuN in the brain ischemic penumbra. ③ In the MCAO group, VDAC1 positive cells were colocalized with ROS in the ischemic penumbra, and the number of VDAC1/ROS double positive cells was considerably increased compared to that of the sham group (P<0.05). The number of VDAC1/ROS double positive cells in the penumbra of EUK-134 treated rats decreased significantly compared to the MCAO group (P<0.05). Conclusions The ROS scavenger EUK-134 could maintains mitochondrial function by affecting the expression of the mitochondrial biogenic regulator PGC-1α and the substance transport channel VDAC1, thereby ameliorating cerebral ischemia-reperfusion injury.

Key words: cerebral ischemia/reperfusion, reactive oxygen species, EUK-134, PGC-1α, VDAC1

中图分类号:

R743.3

李亚堃, 李蔚, 杨雪琦, 师文娟, 朱玥荃, 赵咏梅. 活性氧自由基清除剂EUK-134对脑缺血再灌注损伤大鼠线粒体相关蛋白PGC-1α和VDAC1的影响[J]. 首都医科大学学报, 2024, 45(6): 1016-1022.

Li Yakun, Li Wei, Yang Xueqi, Shi Wenjuan, Zhu Yuequan, Zhao Yongmei. Effect of mitochondrial reactive oxygen species inhibitor EUK-134 on mitochondrial associated proteins PGC-1α and VDAC1 in rats with cerebral ischemia-reperfusion injury[J]. Journal of Capital Medical University, 2024, 45(6): 1016-1022.

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