虾青素对缺氧缺血性脑损伤新生大鼠模型的神经保护作用

doi:10.3969/j.issn.2095-4344.2015.40.016

摘要/Abstract

摘要：

背景：研究发现虾青素有良好的神经保护作用，但是对于其在新生儿缺氧缺血性损伤中的治疗作用，目前尚无相关报道。

目的：构建缺氧缺血性脑损伤新生大鼠模型，观察虾青素对其产生的神经保护作用及作用的途径。

方法：从98只7 d龄的SD乳鼠中随机取30只作为假手术组，其余大鼠结扎左颈总动脉2 h后，置于体积分数92%的特种标准气体、8%的氧气缺氧舱2 h建立缺血缺氧性脑损伤模型。假手术组仅分离颈总动脉，不予缺血缺氧处理。将造模成功的大鼠随机分为脑缺血缺氧组和虾青素治疗组，各30只。虾青素治疗组大鼠在脑缺血缺氧模型建成后立即通过腹腔注射80 mg/kg虾青素。

结果与结论：与假手术组相比，脑缺血缺氧组大鼠缺血损伤区顶叶皮质中p-Akt、p-GSK3β、cleaved-caspase3蛋白的表达水平显著增加，Bcl-2蛋白的表达水平显著减少(P < 0.05)；与脑缺血缺氧组相比，虾青素治疗可以显著减少凋亡相关蛋白cleaved-caspase3蛋白的表达水平(P < 0.05)，显著上调Bcl-2蛋白的表达水平(P < 0.05)，明显减少凋亡细胞的数量(P < 0.05)。提示虾青素可以显著改善新生大鼠缺氧缺血性脑损伤的预后及作用途径与上调Akt/GSK3β信号通路相关。

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

关键词: 实验动物, 脑及脊髓损伤动物模型, 虾青素, 组织工程, 新生大鼠, 缺血缺氧性损伤, p-Akt, p-GSK3β, Bcl-2, cleaved-caspase3, 凋亡

Abstract:

BACKGROUND: Several studies have demonstrated that astaxanthin has a good neuroprotective effect; however, the treatment effects of astaxanthin on newborns with hypoxic-ischemic brain damage have not been reported.

OBJECTIVE: To build newborn rat models of hypoxic-ischemic brain damage, and investigate the neuroprotective effects of astaxanthin and the ways of action.

METHODS: Thirty newborn Sprague-Dawley rats aged 7 days out of 98 were randomly taken as sham-operated group. The rest of rats were subjected to ligature of the left carotid artery for 2 hours and then placed in the hypoxic box containing 92% special standard gas and 8% oxygen to establish the models of hypoxic-ischemic brain damage. The rats in the sham-operated group only underwent separation of the carotid arteries, without hypoxic-ischemic treatment. Rat models of hypoxic-ischemic brain damage were randomly divided into hypoxic-ischemic brain damage and astaxanthin-treated groups, with 30 rats in each group. The rats in the astaxanthin-treated group were intraperitionally injected with 80 mg/kg astaxanthin after hypoxic-ischemic brain damage.

RESULTS AND CONLUSION: Compared with the sham-operated group, the expression levels of p-Akt, p-GSK3β and cleaved-caspase-3 protein in the parietal cortex of ischemic damage area of rats were significantly increased, whereas Bcl-2 protein expression was significantly decreased in the hypoxic-ischemic brain damage group (all P < 0.05). Compared with the hypoxic-ischemic brain damage group, astaxanthin treatment significantly reduced the expression level of apoptosis-related protein cleaved-caspase 3 protein (P < 0.05), significantly increased the expression level of Bcl-2 protein (P < 0.05), and significantly reduced the number of apoptotic cells in the hypoxic-ischemic brain area(P < 0.05). These findings suggest that astaxanthin can distinctly improve the prognosis and ways of action of hypoxic-ischemic brain damage in newborn rats which associates with up-regulation of Akt/GSK3β signaling pathways.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Cerebral Hypoxic-Ischemia, Carotenoids, Newborn Animal, Brain Injures

中图分类号:

R318

林良烽. 虾青素对缺氧缺血性脑损伤新生大鼠模型的神经保护作用[J]. 中国组织工程研究, 2015, 19(40): 6480-6484.

Lin Liang-feng. The neuroprotective effect of astaxanthin on newborn rat models of hypoxic-ischemic brain damage [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(40): 6480-6484.

图/表（结果） 2

2.1 实验动物数量分析建模时未出现死亡，注射虾青素和溶剂后共死亡8例，其中脑缺血缺氧组死亡5只，虾青素治疗组死亡3只，两组间死亡率差异无显著意义，假手术组新生大鼠未见死亡。最终每组有20只用于免疫印迹法的检测，各10只用于TUNEL的检测。实验流程见图1。 2.2 虾青素对缺氧缺血性脑损伤新生大鼠模型大体行为的影响各组新生鼠在实验前的行为能力均正常，新生大鼠在行缺血手术后进入低氧环境后大约10 min后出现烦躁不安、翻滚，随后逐渐出现四肢颤动、头颤、翻身不能，夹尾左旋，甚至抽搐与嗜睡，假手术组新生鼠均未见上述异常行为。 2.3 虾青素对缺氧缺血性脑损伤新生大鼠模型缺血损伤区顶叶皮质中p-Akt/GSK3β信号通路的影响与假手术组相比，脑缺血缺氧组新生大鼠缺血损伤区顶叶皮质中p-Akt和p-GSK3β的表达水平显著增加(P < 0.05)；与脑缺血缺氧组相比，虾青素可以显著上调新生大鼠缺血损伤区顶叶皮质中p-Akt和p-GSK3β蛋白的表达水平(P < 0.05)，见图2，表1。 2.4 虾青素对缺氧缺血性脑损伤新生大鼠模型缺血损伤区顶叶皮质中cleaved-caspase-3和Bcl-2表达水平的影响如图2，表1所示，与假手术组相比，脑缺血缺氧组新生大鼠缺血损伤区顶叶皮质中cleaved-caspase-3的表达水平显著增加(P < 0.05)，Bcl-2的表达水平则显著降低(P < 0.05)；与脑缺血缺氧组相比，虾青素可以显著抑制新生大鼠缺血损伤区顶叶皮质中cleaved-caspase-3的表达(P < 0.05)，同时显著增加Bcl-2的表达(P < 0.05)。 2.4 虾青素对缺氧缺血性脑损伤新生大鼠模型脑组织中细胞凋亡的影响 TUNEL染色结果显示，脑缺血缺氧组新生大鼠脑组织中凋亡细胞的数量较假手术组明显增加(P < 0.05)；与脑缺血缺氧组相比，虾青素治疗后可以显著减少新生大鼠脑组织中凋亡细胞的数量(P < 0.05)，见图3，表2。

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引言

材料方法

1.1 造模动物及材料 98只新生清洁级SD雄性乳鼠，鼠龄7 d，体质量12-18 g，由福建医科大学实验动物中心提供，动物许可证号SCXK(闽)2004-0002。每只大鼠给予24 h昼夜灯光照射控制及严格、规范的卡片登记管理，24 ℃条件下封闭群养。

虾青素粉剂购自美国Sigma公司，临用前溶于体积分数10%的DMSO溶剂中。

1.2 造模方法及干预实验于2013年5月至2014年5月在福建医科大学附属第二医院医学实验中心完成。

分组及造模方法：从98只乳鼠中随机取30只作为假手术组，其余大鼠建立缺血缺氧性脑损伤模型。新生大鼠缺血缺氧性脑损伤的模型制作参照以往文献^[24]：使用乙醚将乳鼠麻醉后，仰卧位固定于手术板上，常规消毒颈部皮肤，行颈正中切口，游离左颈总动脉并用灭菌丝线分别结扎远心端及近心端，然后逐层缝合切口并消毒切口及周围皮肤，将乳鼠放置于复温毯上恢复2 h后，将其置于有机玻璃缺氧舱内，先通入氮气使缺氧舱内氧体积分数达到8%，然后以1.5-2.5 L/min输入体积分数92%的特种标准气体、8%的氧气，维持舱内氧体积分数为8%，持续2 h后，将实验动物取出放回原饲养环境由母鼠喂养。

造模成功标准：在缺血缺氧后出现肌力下降、平衡失常、行动障碍，以及翻身困难或者翻身不能、夹尾左旋等行为。假手术组乳鼠仅切开颈部皮肤。将造模成功大鼠随机分为脑缺血缺氧组和虾青素治疗组，各30只。

虾青素治疗：虾青素治疗组在模型制作成功后立即腹腔注射虾青素80 mg/kg，12 h后再注射1次^[25]。

1.3 Western blot检测给药后12 h，将大鼠麻醉，经左心尖灌注100 mL的生理盐水，取出缺血损伤区顶叶皮质放入-80 ℃冰箱中保存待用。总蛋白的提取参照试剂盒说明书(碧云天)进行，Bradford法蛋白定量，按1∶4比例加入5×loading沸水中煮10 min，变性，按每孔加入35 μg蛋白，电泳、转膜后封闭1 h，加入β-actin，p-Akt，p-GSK-3β (Ser9)，cleaved-caspase-3和Bcl-2(1∶1 000；美国Cell signaling公司)一抗稀释液，4 ℃摇床过夜，加入二抗后，洗膜，滴加ECL显影液，Image J软件(美国国立卫生院)行灰度分析。

1.4 TUNEL法检测 TUNEL检测方法参照试剂盒(美国Roche公司)说明书及以往文献^[26]：在缺血再灌注损伤后 24 h，经左心尖灌注生理盐水和多聚甲醛进行鼠脑的固定，断头取脑，常规石蜡包埋，将脑组织切片常规脱蜡脱水，在经蒸馏水浸泡及PBS洗涤后滴加H₂O₂，然后将切片放入 37 ℃烤箱中放置40 min，取出后使用PBS洗涤，每次 5 min，共3遍，滴加蛋白酶K后放入37 ℃烘箱中孵育 10 min，取出后使用PBS洗涤，每次5 min，共3遍。将凋亡检测试剂盒中的A试剂与B试剂配成TUNEL反应混合物，充分混匀后滴加在切片上，后放入37 ℃烤箱置60 min，取出后PBS洗涤，滴加转化剂POD，37 ℃烤箱放置30 min后洗涤，然后采用DAB显色，苏木精复染，脱水，透明，封固。由2位有经验的病理科医生采用双盲法在400倍显微镜下观察切片并统计凋亡细胞的数量，每张切片取10个视野，计算平均值。

1.5 主要观察指标新生大鼠缺血损伤区顶叶皮质中 β-actin，p-Akt，p-GSK-3β(Ser9)，cleaved-caspase-3和Bcl-2蛋白的表达水平及神经元凋亡情况。

1.6 统计学分析采用SPSS 15.0软件(美国IBM公司)进行结果的处理，数据以x(_)±s表示，多组间比较采用单因素方差分析进行。

讨论

Akt是磷脂酰肌醇3激酶信号通路中的重要激酶，又被称为蛋白激酶B。Akt通过Ser473残基位点磷酸化激活后，可以进一步磷酸化激活下游的Bad、GSK3β、Caspase9蛋白^[27]；此外，活化的Akt还可以与GSK-3β结合，使其N端的Ser9位点发生磷酸化，进而导致 GSK-3β失活，最终影响下游的底物NF-κB和c-Jun转录因子的转录活性，从而发挥显著的抗凋亡作用^[28-30]。以往在脑一过性缺血和局部脑缺血性脑损伤中研究发现，p-Akt的表达水平在神经损伤后明显升高^[31]，使用PI3K/Akt的选择性抑制剂可以显著加重脑损伤的程度^[13]，而使用上调p-Akt表达的药物则可以抑制GSK-3β的活性^[32]、抑制凋亡蛋白cleaved-caspase3蛋白的表达，增加抗凋亡蛋白Bcl-2的表达从而显著减少神经元的凋亡，改善神经功能^[33-34]。鉴于Akt可通过调节众多底物而阻止凋亡的发生，靶向Akt治疗脑缺血缺氧性损伤已经成为目前研究的热点和焦点。以往大量研究表明虾青素具有抑制肿瘤发生、抗炎、抗氧化、增强免疫和预防心脑血管疾病的作用^[35-39]，提示虾青素在中枢神经系统损伤性疾病中有良好的神经保护作用^[40-41]，但是对于其在新生大鼠缺氧缺血性脑损伤中的保护作用目前尚缺乏相关研究，本实验结果发现虾青素治疗可以显著减少缺血缺氧组新生鼠的神经细胞凋亡个数，进一步行机制研究发现，虾青素可以显著上调脑缺血、缺氧脑组织中的p-Akt水平，这与以往在蛛网膜下腔出血脑损伤中观察到的神经保护作用具有一致性^[41-42]，提示虾青素可以应用于多种神经系统损伤性疾病的治疗；此外研究还发现，虾青素可以增加p-GS3β(Ser9)的表达水平，显著抑制凋亡关键执行蛋白cleaved-caspase-3蛋白的表达，增加抗凋亡蛋白Bcl-2蛋白的表达，这可能是其发挥显著神经保护作用的机制之一。综上所述，虾青素在新生大鼠脑缺血缺氧性损伤中发挥显著地神经保护作用，其机制可能与上调Akt/GSK3β信号通路相关，提示虾青素可以作为新生儿缺血缺氧性损伤的潜在性治疗药物。

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