目的 探讨银杏内酯K(ginkgolide K,GK)对中动脉阻塞(middle cerebral artery occlusion,MCAO)损伤大鼠的脑损伤保护作用是否依赖于线粒体Ca2+单向转运体(mitochondrial calcium uniporter,MCU)的作用机制。方法 成年健康SD大鼠,随机分成5大组:假手术组、模型组、GK+钌红(ruthenium red,RR,MCU拮抗剂)组、GK组和GK+精胺(spermine,SM,MCU兴奋剂)组。采用栓线法制备脑中动脉缺血2 h再灌注22 h的大鼠脑损伤模型;采用Zea Longa 5分制对各组大鼠进行行为学评分;重量法测定脑含水量;2,3,5-氯化三苯基四氮唑(TTC)染色测定脑梗死面积;利用酶联免疫吸附法(ELISA)测定超氧化物歧化酶(superoxide dismutase,SOD)活性、丙二醛(malondialdehyde,MDA)、一氧化氮(nitric oxide,NO)含量、一氧化氮合酶(nitric oxide synthase,NOS)活性;采用荧光法检测线粒体内i浓度;采用苏木精-伊红(HE)染色观察损伤脑组织形态学的改变;利用Western blot法检测caspase-3/8/9蛋白的表达水平。结果 与GK组相比,联合GK+RR组中大鼠神经缺损评分、脑含水量、脑梗死面积比显著降低(P<0.01);脑组织MDA、NOS和NO含量减少、SOD活性升高、i显著下降(P<0.01),caspase-3/8/9蛋白表达显著降低,然而,联合GK+SM组与GK+RR组药理作用相反。结论 RR能够提高GK对局灶性脑缺血再灌注损伤大鼠的保护作用,而SM能够减弱GK对局灶性脑缺血再灌注损伤大鼠的保护作用,其GK作用机制可能与RR或者SM竞争性调节MCU转运能力来抑制线粒体Ca2+内流有关。
Abstract
OBJECTIVE To explore whether the protective mechanism of ginkgolide K on cerebral focal ischemia reperfusion injury in rats induced by middle cerebral artery occlusion (MCAO) was associated with the amelioration of mitochondrial calcium uniporter (MCU) or not. METHODS Sprague Dawley (SD) rats were divided into 5 big groups randomly:sham operation group, MCAO group, GK added into RR group, GK group and GK added into SM group. The MCAO rat model were established after cerebral artery ischemia for 2 h and reperfusion for 22 h. Zea Longa 5 score system was used to evaluate neurological deficit score; Determination of brain water content and cerebral infarction areas were determined using gravimetric method and by triphenyltetrazolium chloride(TTC) staining method, respectively. In addition, malondialdehyde (MDA) and superoxide dismutase (SOD), nitric oxide synthase (NOS), nitric oxide (NO) were detected by Elisa. Additionally, mitochondrial i concentration was estimated with the fluorescence spectrophotomete. The morphological change of the injured brains were observed by HE staining. The expression of caspase-3/8/9 protein were detected by Western blot. RESULTS Compared with GK group, GK+RR group relieved obviously the neurological deficit score and reduced the cerebral infarction areas, brain water content, mitochondrial i concentration and MDA, caspase-3/8/9 protein expression while enhance SOD activity. However, the effect of SM on the GK protective activity in MCAO rat injury was the opposite in comparison to GK+RR group. CONCLUSION The stimulative effect of RR and the inhibitory effect of SM on the GK protection in MCAO rat had proves that the protective mechanism of GK on MCAO rat injury is associate with its down-regulation of the transport capacity of MCU, leading the attenuation of mitochondrial i influx.
关键词
脑缺血再灌注 /
银杏内酯K /
线粒体Ca2+单向转运体 /
caspase蛋白
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Key words
cerebral ischemia-reperfusion /
ginkgolide K /
mitochondrial calcium uniporter (MCU) /
caspase protein
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中图分类号：
R965
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参考文献
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脚注
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基金
浙江省中医管理局项目资助(2015ZB105);浙江医药高等专科学校校级项目资助(ZPCSR2015008);国家自然科学基金项目资助(81403067);黑龙江省高校基本科研业务费资助(UNPYSCT-2017160;135209306)
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