葡萄籽原花青素提取物对小鼠脑缺血再灌注损伤的抑制作用及其机制研究

doi:10.24920/J1001-9294.2017.020

Chinese Medical Sciences Journal ›› 2017, Vol. 32 ›› Issue (2): 92-99.doi: 10.24920/J1001-9294.2017.020

葡萄籽原花青素提取物对小鼠脑缺血再灌注损伤的抑制作用及其机制研究

孔祥溢^1,², 关健¹, 巩顺^3,⁴, 王任直^1,^*()

1 中国医学科学院北京协和医学院北京协和医院神经外科,中国北京 100730
2 哈佛大学哈佛医学院麻省总医院麻醉与疼痛科,美国马萨诸塞州 02114
3 第二军医大学附属上海长征医院神经外科中国人民解放军神经外科研究所上海市神经外科研究所,中国上海 200003
4 哈佛大学哈佛医学院布莱根与妇女医院放射科,美国马萨诸塞州 02215

收稿日期:2017-02-17 出版日期:2017-06-30 发布日期:2017-06-10
通讯作者: 王任直 E-mail:wrzpumch@163.com

Neuroprotective Effects of Grape Seed Procyanidin Extract on Ischemia-Reperfusion Brain Injury^△

Kong Xiangyi^1,², Guan Jian¹, Gong Shun^3,⁴, Wang Renzhi^1,^*()

¹Department of Neurosurgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China
²Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Harvard Medical School, Harvard University, Massachusetts 02114, USA
³Department of Neurosurgery, Shanghai Institute of Neurosurgery, PLA Institute of Neurosurgery, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
⁴Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Massachusetts 02215, USA

Received:2017-02-17 Published:2017-06-30 Online:2017-06-10
Contact: Wang Renzhi E-mail:wrzpumch@163.com

摘要/Abstract

摘要：

目的氧化应激（oxidative stress, OS）在缺血性脑卒中病程中起到关键作用。葡萄籽原花青素提取物（grape seed procyanidin extract, GSPE）是OS的重要调节剂。我们猜想GSPE可以保护缺血缺氧性脑损伤。本研究旨在探索GSPE能否在小鼠的缺血再灌注脑损伤起保护作用。方法对于接受GSPE注射（MCAOG, n=60）和生理盐水注射（MCAONS, n=60）的小鼠,制作短暂性大脑中动脉栓塞（transient middle cerebral artery occlusion, MCAO）模型。设置假手术组小鼠（GSPE组和生理盐水组）作为对照。采用改良的神经损害评分（mNSS）评估并比较MCAO造模后1小时、24小时、3天、7天的神经功能损害情况。在MCAO造模24小时后行3T动物颅脑核磁共振（T2WI）扫描,评估并比较梗死灶体积差异。在MCAO造模24小时后,对脑组织行神经元细胞核抗体（NeuN）、CD34、Bcl-2和Bax, 以及脑组织中谷胱甘肽过氧化物酶（glutathione peroxidation, GSH-Px)）和丙二醛（MDA）进行免疫组化分析。对以上指标都在各组之间的差异进行统计学分析。结果在MCAO造模24小时及之后,MCAOG组小鼠神经功能恢复好于MCAONS组小鼠（p < 0.05）在MCAO造模24小时,MCAOG组小鼠脑梗塞灶体积明显小于MCAONS组小鼠（22.46 ± 11.45 mm³ vs. 47.84 ± 9.06 mm³, P<0.05）;MCAOG小鼠梗塞灶周边区域NeuN和CD34阳性染色细胞数目明显多于MCAONS小鼠（p < 0.05）。MCAONS组小鼠Bax阳性染色细胞数目明显多于MCAOG组（p < 0.05）。MCAOG组小鼠脑组织中平均MDA水平明显低于MCAONS组（P<0.05）,GSH-Px活力明显高于MCAONS组（p < 0.05）。结论 GSPE可抑制小鼠缺血再灌注脑损伤的病程。其机制可能与GSPE抑制了病程中氧化应激和凋亡反应,促进了微血管生成,以及激活抗氧化酶（GSH-Px）有关。GSPE可能代表了一种治疗缺血再灌注脑损伤的新方向。

关键词: 葡萄籽原花青素提取物, 脑缺血再灌注损伤, 神经保护, 缺血再灌注损伤

Abstract:

Objective Oxidative stress (OS) plays a crucial role in ischemic stroke. Grape seed procyanidin extract (GSPE) was reported to be a critical regulator of OS. We hypothesized that GSPE might also be protective in ischemia-reperfusion brain injury. This study aimed to explore whether GSPE administration can protect mice from ischemia-reperfusion brain injury.Methods Transient middle cerebral artery occlusion (MCAO) was conducted followed by reperfusion for 24 hours to make ischemia-reperfusion brain injury in mice that received GSPE (MCAOG, n=60) or normal saline (MCAONS, n=60). Sham-operated mice (GSPE group and normal saline group) were set as controls. The neurological severity score (NSS) was used to evaluate neural function impairment 1 hour, 24 hour, 3 days and 7 days after MCAO. Mice underwent brain T2WI imaging with a 3T animal MRI scanner 24 hours after reperfusion, and the stroke volume of brains were calculated according to abnormal signal intensity. Immunohistopathological analysis of brain tissues at 24 h after reperfusion was performed for neuronal nuclear antigen (NeuN), CD34, Bcl-2, and Bax. Glutathione peroxidation (GSH-Px) activity and the level of malonaldehyde (MDA) of brain tissue were also examined. The above indexes were compared among the groups statistically.Results Significant functional improvement was observed 24 hours after MCAO in MCAOG group compared to MCAONS group (P<0.05). MCAOG group had smaller cerebral stroke volume (22.46 ± 11.45 mm³ vs. 47.84±9.06 mm³, P<0.05) than MCAONS group 24 hours after MCAO. More mature NeuN-immunoreactive neurons and more CD34-positive cells in peri-infarct zones were observed in brain tissue of MCAOG mice 24 h after MCAO than that of MCAONS mice (both P<0.05). MCAONS mice had significantly higher number of Bax-positive cells in brain tissue than MCAOG (P<0.05). The mean MDA level was significantly lower (P<0.05) and the GSH-Px activity was significantly higher (P<0.05) in brains of MCAOG mice compared to those of MCAONS mice.Conclusion GSPE administration protects mice from ischemia-reperfusion brain injury through attenuating oxidative stress and apoptosis, promoting angiogenesis, and activating antioxidant enzyme GSH-Px. GSPE may represent a new therapeutical direction for the treatment of ischemia-reperfusion brain injury.

Key words: grape seed procyanidin extract, oxidative stress, neuroprotection, ischemia-reperfusion injury

Kong Xiangyi, Guan Jian, Gong Shun, Wang Renzhi. Neuroprotective Effects of Grape Seed Procyanidin Extract on Ischemia-Reperfusion Brain Injury^△[J].Chinese Medical Sciences Journal, 2017, 32(2): 92-99.

图/表 6

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