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; 别玉龙; 王江; 高静; 杨冰月; 万海同

doi:10.24920/J1001-9294.2017.048

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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

中国医学科学杂志(英文版) 2017年32卷第4期页码：239-247

ORIGINAL ARTICLE | Updated：2024-04-10

- 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
- Affiliations：
  
  1. 1Department of Basic Medicine, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, China
  2. 2Department of Pharmacy, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, China
  3. 3Institute of Cardio-Cerebrovascular Disease, Zhejiang Chinese Medical University, Hangzhou 310053, China
- Author bio：
  
  *Corresponding author Tel: 86-571-86613711, E-mail: wanhaitong@zcmu.edu.cn
- Funds：
  
  △Fund supported by National Science Foundation of China (NSFC) 81503491,81374053, 81630105.
- DOI：10.24920/J1001-9294.2017.048
  中图分类号：
- 收稿日期：2017-03-16，
  
  网络出版日期：2017-12-30，
  
  纸质出版日期：2017-12
- Accepted：
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朱慧渊, 别玉龙, 王江, 等. 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[J]. 中国医学科学杂志(英文版), 2017,32(4):239-247.

Zhu Huiyuan, Bie Yulong, Wang Jiang, et al. Experimental Study on the Protection of Agrimony Extracts from Different Extracting Methods against Cerebral Ischemia-Reperfusion Injury^△[J]. Chinese medical sciences journal, 2017, 32(4): 239-247.
朱慧渊, 别玉龙, 王江, 等. 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[J]. 中国医学科学杂志(英文版), 2017,32(4):239-247. DOI： 10.24920/J1001-9294.2017.048.

Zhu Huiyuan, Bie Yulong, Wang Jiang, et al. Experimental Study on the Protection of Agrimony Extracts from Different Extracting Methods against Cerebral Ischemia-Reperfusion Injury^△[J]. Chinese medical sciences journal, 2017, 32(4): 239-247. DOI： 10.24920/J1001-9294.2017.048.

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、Ca

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（

0.01）;治疗组中

仅WEA组的神经功能评分明显低于MCAO组（

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％（

=4.75

0.01）

19.90±6.90％（

=5.23

0.01）

20.40±5.30％（

= 4.68

0.01）

22.50±10.50％（

= 6.25

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

-ATPase活性（3.67±0.48）和Ca

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

-ATPase活性（7.56±0.85

=12.65

=0.010）和Ca

-ATPase酶活性（3.59±0.22

=8.32

= 0.041）明显高于MCAO组。MCAO模型组的P53 mRNA（

0.01）和HSP70 mRNA（

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（

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。

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)

-ATPase

-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 (

0.01). The WEA group showed a significantly lower neurological score than the MCAO group (

0.05)

indicating the protective effect of WEA on neurological deficits. The mean infarction volumes of WEA (13.5±6.6%

=4.75

0.01)

EEA (19.90±6.90%

=5.23

0.01)

PEA (20.40±5.30%

=4.68

0.01) and EAEA (22.50±10.50%

=6.25

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

-ATPase and Ca

-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

=12.65

=0.010; 3.59±0.22 U/mg

=8.32

=0.041

respectively). The MCAO group showed significantly elevated P53 and HSP70 mRNA expressions compared to the sham group (

0.01

0.05). P53 mRNA expressions in Agrimony extracts treated groups were significantly lower than that of the MCAO group (all

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.

关键词

Keywords

references

Gao HJ , Liu PF , Li PW , Huang ZY , Yu FB , Lei T , et al . Ligustrazine monomer against cerebral ischemia/reperfusion injury. Neural Regen Res 2015 ; 10 : 832 - 40 . doi: 10.4103/1673-5374.156991 https://doi.org/10.4103/1673-5374.156991 .

Fisher M. New approaches to neuroprotective drug development. Stroke 2011 ; 42 : S24 - 27 . doi: 10.1161/STROKEAHA. 110.592394 https://doi.org/10.1161/STROKEAHA.110.592394 .

Langhorne P , Bernhardt J , Kwakkel G. Stroke rehabilitation. Lancet 2011 ; 377 : 1693 - 702 . doi: 10.1016/S0140-6736(11)60325-5 https://doi.org/10.1016/S0140-6736(11)60325-5 .

Lo EH , Dalkara T , Moskowitz MA. Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci 2003 ; 4 : 399 - 415 . doi: 10.1038/nrn1106 https://doi.org/10.1038/nrn1106 .

Ivanova D , Vankova D , Nashar M. Agrimonia eupatoria tea consumption in relation to markers of inflammation, oxidative status and lipid metabolism in healthy subjects. Arch Physiol Biochem 2013 ; 119 : 32 - 7 . doi: 10.3109/13813455.2012.729844 https://doi.org/10.3109/13813455.2012.729844 .

Nho KJ , Chun JM , Kim HK. Agrimonia pilosa ethanol extract induces apoptotic cell death in HepG2 cells. J Ethnopharmacol 2011 ; 138 : 358 - 63 . doi: 10.1016/j.jep.2011.09.008 https://doi.org/10.1016/j.jep.2011.09.008 .

Pan Y , Liu HX , Zhuang YL , Ding L , Chen LX . Qiu F. Studies on isolation and identification of flavonoids in herbs of Agrimonia pilosa . Zhongguo Zhong Yao Za Zhi . 2008 ; 33: 2925-8. Chinese.

Correia HS , Batista MT , Dinis TC. The activity of an extract and fraction of Agrimonia eupatoria L. against reactive species. Biofactors 2007 ; 29 : 91 - 104 . doi: 10.1002/biof.552029209 https://doi.org/10.1002/biof.552029209 .

Seo UM , Nguyen DH , Zhao BT , Min BS , Woo MH. Flavanonol glucosides from the aerial parts of Agrimonia pilosa Ledeb and their acetylcholinesterase inhibitory effects. Carbohydr Res 2017 ; 445 : 75 - 9 . doi: 10.1016/j.carres. 2017.04.014 https://doi.org/10.1016/j.carres.2017.04.014 .

Zhu L , Chen J , Tan J , Liu X , Wang B. Flavonoids from Agrimonia pilosa Ledeb: Free Radical Scavenging and DNA Oxidative Damage Protection Activities and Analysis of Bioactivity-Structure Relationship Based on Molecular and Electronic Structures. Molecules 2017 ; 22 : 195 . doi: 10.3390/molecules22030195 https://doi.org/10.3390/molecules22030195 .

Kim SB , Hwang SH , Suh HW , Lim SS. Phytochemical Analysis of Agrimonia pilosa Ledeb, Its Antioxidant Activity and AldoseReductase Inhibitory Potential. Int J Mol Sci 2017 ; 18 : 379 . doi: 10.3390/ijms18020379 https://doi.org/10.3390/ijms18020379 .

Liu X , Zhu L , Tan J , Zhou X , Xiao L , Yang X , et al. Glucosidase inhibitory activity and antioxidant activity of flavonoid compound and triterpenoid compound from Agrimonia Pilosa Ledeb. BMC Complement Altern Med 2014 ; 14 : 12 . doi: 10.1186/1472-6882-14-12 https://doi.org/10.1186/1472-6882-14-12 .

Wang J , Chao F , Han F , Zhang G , Xi Q , Li J , et al. PET demonstrates functional recovery after transplantation of induced pluripotent stem cells in a rat model of cerebral ischemic injury. J Nucl Med 2013 ; 54 : 785 - 92 . doi: 10.2967/jnumed.112.111112 https://doi.org/10.2967/jnumed.112.111112 .

Longa EZ , Weinstein PR , Carlson S , Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 1989 ; 20 : 84 - 91 . doi: 10.1161/01.str. 20.1.84 https://doi.org/10.1161/01.str.20.1.84 .

Wang Z , Song F , Li J , Zhang Y , Ye Y , Yang J , et al. PET Demonstrates Functional Recovery after Treatment by Danhong Injection in a Rat Model of Cerebral Ischemic-Reperfusion Injury. Evid Based Complement Alternat Med 2014 ; 430757 . doi: 10.1155/2014/430757 https://doi.org/10.1155/2014/430757 .

Sun L , Jin Y , Dong L , Sumi R , Jahan R , Li Z. The neuroprotective effects of Coccomyxa gloeobotrydiformis on the ischemic stroke in a rat model. Int J Biol Sci 2013 ; 9 : 811 - 17 . doi: 10.7150/ijbs.6734 https://doi.org/10.7150/ijbs.6734 .

Wan L , Cheng Y , Luo Z , Guo H , Zhao W , Gu Q , et al. Neuroprotection, learning and memory improvement of a standardized extract from Renshen Shouwu against neuronal injury and vascular dementia in rats with brain ischemia. J Ethnopharmacol 2015 ; 165 : 118 - 26 . doi: 10.1016/ j.jep.2015.02.027 https://doi.org/10.1016/j.jep.2015.02.027 .

Guo Z , Cao G , Yang H , Zhou H , Li L , Cao Z , et al. A combination of four active compounds alleviates cerebral ischemia-reperfusion injury in correlation with inhibition of autophagy and modulation of AMPK/mTOR and JNK pathways. J Neurosci Res 2014 ; 92 : 1295 - 306 . doi: 10.1002/jnr.23400 https://doi.org/10.1002/jnr.23400 .

Chen L , Zhao Y , Zhang T , Dang X , Xie R , Li Z , et al. Protective effect of Sheng-Nao-Kang decoction on focal cerebral ischemia-reperfusion injury in rats. J Ethnopharmacol 2014 ; 151 : 228 - 36 . doi: 10.1016/j.jep.2013.10.015 https://doi.org/10.1016/j.jep.2013.10.015 .

Liu Y , Ling Y , Hu W , Xie L , Yu L , Qian X , et al. The herb medicine formula "chong lou fu fang" increases the cytotoxicity of chemotherapeutic agents and down-regulates the expression of chemotherapeutic agent resistance-related genes in human gastric cancer cells in vitro . Evid Based Complement Alternat Med 2011 ; 2011 : 834231 . doi: 10.1093/ecam/nep175 https://doi.org/10.1093/ecam/nep175 .

Chaturvedi RK , Beal MF. Mitochondrial approaches for neuroprotection. Ann N Y Acad Sci 2008 ; 1147 : 395 - 412 . doi: 10.1196/annals.1427.027 https://doi.org/10.1196/annals.1427.027 .

Ying W. NAD+ and NADH in brain functions, brain diseases and brain aging. Front Biosci 2007 ; 12 : 1863 - 88 . doi: 10.2741/2194 https://doi.org/10.2741/2194 .

Liu D , Gharavi R , Pitta M , Gleichmann M , Mattson MP. Nicotinamide prevents NAD+ depletion and protects neurons against excitotoxicity and cerebral ischemia: NAD+ consumption by SIRT1 may endanger energetically compromised neurons. Neuromolecular Med 2009 ; 11 : 28 - 42 . doi: 10.1007/s12017-009-8058-1 https://doi.org/10.1007/s12017-009-8058-1 .

Chan PH. Role of oxidants in ischemic brain damage . Stroke. 1996 ; 27 : 1124 - 29 . doi: 10.1161/01.STR.27.6.1124 https://doi.org/10.1161/01.STR.27.6.1124 .

Yamada J , Yoshimura S , Yamakawa H , Sawada M , Nakagawa M , Hara S , et al. Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation. Neurosci Res 2003 ; 45 : 1 - 8 . doi: 10.1016/S0168-0102(02)00196-7 https://doi.org/10.1016/S0168-0102(02)00196-7 .

Song D , Xu J , Du T , Yan E , Hertz L , Walz W. Inhibition of brain swelling after ischemia-reperfusion by beta-adrenergic antagonists: correlation with increased K+ and decreased Ca 2 + concentrations in extracellular fluid . Biomed Res Int 2014 ; 2014 : 873590 . doi: 10.1155/2014/873590 https://doi.org/10.1155/2014/873590 .

de Lores Arnaiz GR , Ordieres MG . Brain Na(+), K(+)-ATPase Activity In Aging and Disease. Int J Biomed Sci 2014 ; 10 : 85 - 102 .

Song T , Liu J , Tao X , Deng JG. Protection effect of atorvastatin in cerebral ischemia-reperfusion injury rats by blocking the mitochondrial permeability transition pore. Genet Mol Res 2014 ; 13 : 10632 - 42 . doi: 10.4238/2014.December.18.5 https://doi.org/10.4238/2014.December.18.5 .

Christophe M , Nicolas S. Mitochondria: a target for neuroprotective interventions in cerebral ischemia-reperfusion. Curr Pharm Des 2006 ; 12 : 739 - 57 . doi: 10.2174/138161206775474242 https://doi.org/10.2174/138161206775474242 .

Macmanus JP , Buchan AM , Hill IE , Rasquinha I , Preston E. Global-ischemia can cause DNA fragmentation indicative of apoptosis in rat brain. Neuroscience Letters 1993 ; 164 : 89 - 92 . doi: 10.1016/0304-3940(93)90864-h https://doi.org/10.1016/0304-3940(93)90864-h .

Wang LY , Liu J , Li Y , Li B , Zhang YY , Jing ZW. Time-dependent variation of pathways and networks in a 24-hour window after cerebral ischemia-reperfusion injury. BMC Syst Biol 2015 ; 9 : 11 . doi: 10.1186/s12918-015-0152-4 https://doi.org/10.1186/s12918-015-0152-4 .

32 . Guo C, Yin Y, Duan J, Zhu Y, Yan J,Wei G. Neuroprotective effect and underlying mechanism of sodium danshensu [3-(3,4-dihydroxyphenyl) lactic acid from Radix and Rhizoma Salviae miltiorrhizae = Danshen] against cerebral ischemia and reperfusion injury in rats. Phytomedicine 2015 ; 22 : 283 - 9 . doi: 10.1016/j.phymed.2014.12.001 https://doi.org/10.1016/j.phymed.2014.12.001 .

Nho KJ , Chun JM , Kim HK. Agrimonia pilosa ethanol extract induces apoptotic cell death in HepG2 cells. J Ethnopharmacol 2011 ; 138 : 358 - 63 . doi: 10.1016/j.jep. 2011.09.008 https://doi.org/10.1016/j.jep.2011.09.008 .

Yang X , Zhang L , Jiang SQ , Gong PL , Zeng FD. Effect of dauricine on apoptosis and expression of apoptogenic protein after transient focal cerebral ischemia-reperfusion injury in rats. Zhongguo Zhong Yao Za Zhi 2009 ; 34 : 78 - 83 .

Tounai H , Hayakawa N , Kato H , Araki T. Immunohistochemical study on distribution of NF-kappaB and p53 in gerbil hippocampus after transient cerebral ischemia: effect of pitavastatin. Metab Brain Dis 2007 ; 22 : 89 - 104 . doi: 10.1007/s11011-006-9040-3 https://doi.org/10.1007/s11011-006-9040-3 .

Gu ZT , Li L , Wu F , Zhao P , Yang H , Liu YS. Heat stress induced apoptosis is triggered by transcription-independent p53, Ca 2 + dyshomeostasis and the subsequent Bax mitochondrial translocation. Sci Rep 2015 ; 5 : 11497 . doi: 10.1038/srep11497 https://doi.org/10.1038/srep11497 .

Mihara M , Erster S , Zaika A , Petrenko O , Chittenden T , Pancoska P. p53 has a direct apoptogenic role at the mitochondria. Molecular Cell 2003 ; 11 : 577 - 90 . doi: 10.1016/S1097-2765(03)00050-9 https://doi.org/10.1016/S1097-2765(03)00050-9 .

Gong JL , Zhu BM , Murshid A , Adachi H , Song B , Lee A. T Cell Activation by Heat Shock Protein 70 Vaccine Requires TLR Signaling and Scavenger Receptor Expressed by Endothelial Cells-1. J Immunol 2009 ; 183 : 3092 - 8 . doi: 10.4049/jimmunol.0901235 https://doi.org/10.4049/jimmunol.0901235 .

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