Chinese Medical Sciences Journal ›› 2017, Vol. 32 ›› Issue (4): 239-247.doi: 10.24920/J1001-9294.2017.048

• 论著 • 上一篇    下一篇

仙鹤草不同方式提取产物抗脑缺血再灌注损伤保护作用的实验研究

朱慧渊1,3, 别玉龙1, 王江1, 高静2, 杨冰月2, 万海同3,*()   

  1. 1陕西中医药大学,基础医学院,咸阳,陕西 712046
    2陕西中医药大学,药学院,咸阳,陕西 712046
    3浙江中医药大学,心脑血管研究所,杭州, 310053
  • 收稿日期:2017-03-16 出版日期:2017-12-30 发布日期:2017-12-30
  • 通讯作者: 万海同 E-mail:wanhaitong@zcmu.edu.cn

Experimental Study on the Protection of Agrimony Extracts from Different Extracting Methods against Cerebral Ischemia-Reperfusion Injury

Zhu Huiyuan1,3, Bie Yulong1, Wang Jiang1, Gao Jing2, Yang Bingyue2, Wan Haitong3,*()   

  1. 1Department of Basic Medicine, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, China
    2Department of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, China
    3Institute of Cardio-Cerebrovascular Disease, Zhejiang Chinese Medical University, Hangzhou 310053, China
  • Received:2017-03-16 Published:2017-12-30 Online:2017-12-30
  • Contact: Wan Haitong E-mail:wanhaitong@zcmu.edu.cn

摘要: 目的

研究仙鹤草不同提取方式抗大鼠脑缺血再灌注损伤的保护作用,进而优化仙鹤草提取方案。

方法

将SD大鼠随机分为假手术组、模型组、仙鹤草石油醚提取物组,仙鹤草乙酸乙酯提取物组,仙鹤草乙醇提取物组,仙鹤草水提取物组,尼莫地平组。采用改良的MCAO建立大鼠脑缺血再灌损伤模型,分别于再灌0时、6时分组i.g给药,24小时后观察各组大鼠的神经功能缺损症状。处死大鼠后进行脑梗死体积测量及脑组织病理学染色,采用改良 Svoboda and Mosinger法检测脑组织中Na+-K+ATP酶、Ca2+ATP酶的调控作用;通过定量Real-time PCR检测各组细胞凋亡相关基因P53与Hsp70表达。

结果

MCAO模型组与假手术组比较具有显著高的神经功能评分(P<0.01);治疗组中,仅WEA组的神经功能评分明显低于MCAO组(P<0.05)。与MCAO组脑梗死体积(29.40±6.50%)相比,WEA,EEA,PEA和EAEA的梗死体积分别为13.50±6.60%(F=4.75,P <0.01),19.90±6.90%(F=5.23,P<0.01),20.40±5.30%(F= 4.68,P<0.01),22.50±10.50%(F= 6.25,P<0.05)。病理组织学观察发现MCAO组脑细胞肿胀,细胞核染色轻,细胞分布不规则,神经元数量减少等改变较各治疗组相比均更为明显。MCAO组的Na+/K+-ATPase活性(3.67±0.48)和Ca2+ ATPase活性(1.28±0.26)显著低于假手术组;在治疗组各组中,WEA组Na+/K+-ATPase活性(7.56±0.85,F=12.65,P=0.010)和Ca2+-ATPase酶活性(3.59±0.22,F=8.32,P= 0.041)明显高于MCAO组。MCAO模型组的P53 mRNA(P<0.01)和HSP70 mRNA(P<0.05)表达较假手术组明显升高,P53 mRNA表达在仙鹤草提取物治疗的各组均明显低于MCAO组(P<0.01),而WEA组与MCAO组差异较其他各组最为显著。 而HSP70 mRNA水平在仙鹤草提取物治疗各组与MCAO组无显着差异。

结论

仙鹤草水提取物的神经保护作用较其他方法提取物为优。其机制可能与抗氧化作用及其改善缺血再灌注损伤后的脑神经细胞能量代谢有关。

关键词: 仙鹤草, 脑中动脉阻塞, 能量代谢, 脑缺血再灌注损伤, 大鼠

Abstract: Objective

To study the protective effect of agrimony extracts from different extracting methods on cerebral ischemia-reperfusion injury in rats, in order to optimize the extraction scheme of agrimony.

Methods

Male rats were randomly assigned into seven groups: 1. Sham-operated group, 2. Untreated MCAO group (MCAO), 3. Petroleum ether extract of Agrimonia pilosa treated MCAO group (PEA), 4. Ethyl acetate extract of Agrimonia pilosa treated MCAO group (EAEA), 5. Ethanol extract of Agrimonia pilosa treated MCAO group (EEA), 6. Water extract of Agrimonia pilosa treated MCAO group (WEA), 7. Nimodipine treated MCAO group (NP). Intragastrical drug administration (i.g) was performed at 0 and 6 hours after MCAO. Neurological function tests were performed after reperfusion for 24 hours, then the brain was removed for the evaluations of the cerebral infarction volume (percentage of total brain volume) by immunohistochemistry, histological changes (hematoxylin-eosin staining), Na+/K+-ATPase, Ca2+-ATPase (modified method of Svoboda and Mosinger), mRNA expression of Tumor suppressor gene (P53) and hot shock protein (HSP70) (quantitative real-time PCR).

Results

The neurological function of MCAO group had significantly higher scores than the sham group (P<0.01). The WEA group showed a significantly lower neurological score than the MCAO group (P<0.05), indicating the protective effect of WEA on neurological deficits. The mean infarction volumes of WEA (13.5±6.6%, F=4.75, P<0.01), EEA (19.90±6.90%, F=5.23, P<0.01), PEA (20.40±5.30%, F=4.68, P<0.01) and EAEA (22.50±10.50%, F=6.25, P<0.05) group were all significantly smaller than that of MCAO group (29.40±6.50%). HE staining demonstrated that, compared to the treated groups, the infarcted cerebral tissue of MCAO group had more swelling neural cells, lighter stained nucleus, fewer and irregularly distributed neurons. The activity of Na+/K+-ATPase and Ca2+-ATPase reduced in the MCAO group (3.67±0.48 U/mg, 1.28±0.26 U/mg, respectively), and were significantly higher in WEA group (7.56±0.85 U/mg, F=12.65, P=0.010; 3.59±0.22 U/mg, F=8.32, P=0.041, respectively). The MCAO group showed significantly elevated P53 and HSP70 mRNA expressions compared to the sham group (P<0.01, P<0.05). P53 mRNA expressions in Agrimony extracts treated groups were significantly lower than that of the MCAO group (all P<0.01), with the WEA group showing the greatest difference from MCAO group. The HSP70 mRNA level of the treated groups were not significantly different from that of the MCAO group.

Conclusions

Treatment using water extracts of agrimony can promote the best functional and metabolic recovery for rat model of cerebral ischemia-reperfusion injury, which maybe relate with the upregulation of energy metabolism in nerve cells after MCAO.

Key words: Agrimonia pilosa, middle cerebral artery occlusion (MCAO), energy metabolism, ischemia-reperfusion injury, rat

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