The Effect of Dl-3-n-butylphthalide on Expressions of VEGF, Ang-2 Proteins and Angiogenesis in Intracerebral Hemorrhage Rats

doi:10.3969/j.issn.1673-5765.2019.12.006

Abstract

Abstract:

Objective To investigate the possible neuroprotective effect and mechanism of dl-3-nbutylphthalide in intracerebral hemorrhage rats by observing the dynamic expression of vaseular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) protein and the change of neovascularization in brain tissue of rats with intracerebral hemorrhage at different time points after giving different doses of dl-3-n-butylphthalide. Methods Male healthy SD rats were used to prepare cerebral hemorrhage models by injecting autologous blood, and were randomly divided into no-treatment intracerebral hemorrhage model group, the low-dose dl-3-n-butylphthalide group and medium-dose dl-3-n-butylphthalide group, and the sham-operated rats served as control group. Rats in the low and medium dose groups were given dl-3-n-butylphthalide in the dose of 10 mg/kg and 25 mg/kg (twice a day for both groups) respectively, while rats in the sham operation group and no-treatment intracerebral hemorrhage model group were given the same dose of soybean oil at the same time. The following indexes in all groups were assessed at 1, 3, 7 and 15 day after operation: neurological deficit, the expression of CD34, neovascularization and vascular field area, the expressions of VEGF and Ang-2 protein. The volume of hematoma in intracerebral hemorrhage rats were also measured. Results Compared with that in no-treatment model group, the expression of VEGF was up-regulated in the low-dose group at all time points and in the medium-dose group only at 7, 15 days after operation; the expression of Ang-2 protein was up-regulated and the neovascularization count was more in the low-dose and medium-dose group at all time points, and the neurological deficit score was lower in the low-dose group at 3 days after operation and were all lower in the medium-dose group at all time points, and all the above difference had statistical significance. Moreover, there were no significant difference in hematoma volume at 7 and 15 days after operation between the low-dose and medium-dose dl-3-n-butylphthalide groups and no-treatment model group. Conclusions Dl-3-n-butylphthalide can significantly alleviate neurological deficit in cerebral hemorrhage rats, with no increase in the risk of hematoma enlargement. The mechanism of dl-3- n-butylphthalide may be related to up-regulating the expressions of VEGF and Ang-2 protein and increasing the blood vessel density around hematoma.

Key words: Intracerebral hemorrhage; Vascular endothelial growth factor; Angiopoietin-2; Dl-3-n-butylphthalide

摘要：

目的通过观察大鼠脑出血后脑组织中血管内皮生长因子（vaseular endothelial growth factor， VEGF）、血管生成素-2（angiopoietin-2，Ang-2）蛋白在不同时间点的动态表达及不同剂量丁基苯酞干预后其与新生血管数量的变化，探讨丁基苯酞对脑出血大鼠可能的神经保护作用及机制。方法通过自体血注入法制备SD大鼠脑出血模型并随机分为脑出血模型组、丁基苯酞低剂量组及丁基苯酞中剂量组，以及对照的假手术组。丁基苯酞低、中剂量组大鼠分别予以丁基苯酞10 mg/kg、 25 mg/kg灌胃给药（每日2次），假手术组及脑出血模型组大鼠则在相同时间用同等体积大豆油代替灌胃。分别在术后1 d、3 d、7 d、15 d评估大鼠神经功能缺损，采用免疫组化法检测各时间点CD34的表达并进行新生血管计数及血管场面积测定，检测各时间点VEGF、Ang-2蛋白的表达，测定脑出血大鼠血肿体积。结果与脑出血模型组比较，丁基苯酞中剂量组在术后各时间点，低剂量组术后7 d、15 d VEGF蛋白表达上调；丁基苯酞低、中剂量组术后各时间点An g-2蛋白表达均上调，血管计数均增多；丁基苯酞低剂量组术后3 d的神经功能缺损评分较低，丁基苯酞中剂量组术后3 d、7 d、15 d的神经功能缺损评分较低，上述差异均有统计学意义。丁基苯酞低、中剂量组与脑出血模型组，在术后7 d、15 d血肿体积差异无统计学意义。结论丁基苯酞可以显著减轻脑出血大鼠的神经功能缺损，其作用机制可能与上调VEGF、Ang-2蛋白的表达，增加脑出血血肿周围新生血管密度有关，同时未增加血肿增大的风险。

关键词: 脑出血; 血管内皮生长因子; 血管生成素-2; 丁基苯酞

TU E-Wen, LIU Qiu-Ting, TAN Li, ZENG Yan-Xiang, CHEN Qiong. The Effect of Dl-3-n-butylphthalide on Expressions of VEGF, Ang-2 Proteins and Angiogenesis in Intracerebral Hemorrhage Rats[J]. Chinese Journal of Stroke, 2019, 14(12): 1225-1231.

涂鄂文, 刘秋庭, 谭莉, 曾艳香, 陈琼. 丁基苯酞对脑出血大鼠血管内皮生长因子、血管生成素-2蛋白表达及血管新生的影响[J]. 中国卒中杂志, 2019, 14(12): 1225-1231.

References

[1] PRABHAKARAN S，NAIDECH A M. Ischemic

brain injury after intracerebral hemorrhage：a

critical review[J]. Stroke，2012，43（8）：2258-2263.

[2] ARONOWSKI J，ZHAO X. Molecular

pathophysiology of cerebral hemorrhage：secondary

brain injury[J]. Stroke，2011，42（6）：1781-1786.

[3] IADECOLA C. The neurovascular unit coming of

age：a journey through neurovascular coupling in

health and disease[J]. Neuron，2017，96（1）：17-42.

[4] ROSENBERG G A，MUN-BRYCE S，Wesly M，et

al. Collagenase-induced intracerebral hemorrhage in

rats[J]. Stroke，1990，21（5）：801-807.

[5] LONGA E Z，WEINSTEIN P R，CARLSON S，et al.

Reversible middle cerebral artery occlusion without

craniectomy in rats[J]. Stroke，1989，20（1）：84-91.

[6] BERNSTEIN J E，SAVLA P，DONG F，et al.

Inflammatory markers and severity of intracerebral

hemorrhage[J/OL]. Cureus，2018，10（10）：e3529.

https：//doi.org/10.7759/cureus.3529.

[7] ZHANG J，ZOU H，ZHANG Q，et al. Effects of

Xiaoshuan enteric-coated capsule on neurovascular

functions assessed by quantitative multiparametric

MRI in a rat model of permanent cerebral ischemia[J/

OL]. BMC Complement Altern Med，2016，16：198.

https：//doi.org/10.1186/s12906-016-1184-z.

[8] FERRARA N. VEGF：an update on biological and

therapeutic aspects[J]. Curr Opin Biotechnol，2000，

11（6）：617-624.

[9] FOX S B. Tumor angiogenesis and prognosis[J].

Histopathology，1997，30（3）：294-301.

[10] POLVERINI P J. Cellular adhesion molecules.

Newly identified mediators of angiogenesis[J]. Am J

Pathol，1996，148（4）：1023-1029.

[11] PLOUET J，MORO F，BERTAQNOLLI S，et al.

Extracellular cleavage of the vascular endothelial

growth factor189-amino acid form by urokinase

is required for its mitogenic effect[J]. J Biol Chem，

1997，272（12）：13390-13396.

[12] DAS S K，VASUDEAN D M. Essential factors

associated with hepatic angiogenesi[J]. Life Sci，

2007，80（23-24）：1555-1564.

[13] KRUPINSKI J，KALUZA J，KUMAR P，et al. Role

of angiogenesis in patients with cerebral ischemic[J].

stroke，1994，25（9）：1794-1798.

[14] DUAN S，SHAO G Q，YU L，et al. Angiogenesis

contributes to the neuroprotection induced by

hyperbaric oxygen preconditioning against focal

cerebral ischemia in rats[J]. Int J Neurosci，2015，125

（8）：625-634.

[15] FAQIANI F，CHRISTOFORI G. Angiopoietins in

angiogenesis[J]. Cancer Lett，2013，328（1）：18-26.

[16] BEN-SHOSHAN J，STEINVIL A，ARBEL Y，et al.

Sustained elevation of vascular endothelial growth

factor and angiopoietin-2 levels after transcatheter

aortic valve replacement[J]. Can J Cardiol，2016，32

（12）：1454-1461.

[17] CONROY S，KRUYT F A E，WAGEMAKERS

M，et al. IL-8 associates with a pro-angiogenic and

mesenchymal subtype in glioblastoma[J]. Oncotarget，

2018，9（21）：15721-15731.

[18] MOON H S，PARK W I，SUNG S H，et al.

Immunohistochemical and quantitative competitive

PCR analyses of midkine and pleiotrophin expression

in cervical cancer[J]. Gynecol Oncol，2003，88（3）：

289-297.

[19] PENG Z R，YANG A L，YANG Q D. The effect of

hyperbaric oxygen on intracephalic angiogenesis in

rats with intracerebral hemorrhage[J]. J Neurol Sci，

2014，342（1-2）：114-123.

[20] SENN R，ELKIND M S，MONTANER J，et al.

Potential role of blood biomarkers in the management

of nontraumatic intracerebral hemorrhage[J].

Cerebrovasc Dis，2014，38（6）：395-409.

[21] QIN C，ZHOU P，WANG L，et al. Dl-3-nbutylphthalide

attenuates ischemic reperfusion

injury by improving the function of cerebral artery

and circulation[J]. J Cereb Blood Flow Metab，2019，

39（10）：2011-2021.

[22] HE Z，ZHOU Y，HUANG Y，et al. Dl-3-nbutylphthalide

improves functional recovery in rats

with spinal cord injury by inhibiting endoplasmic

reticulum stress-induced apoptosis[J]. Am J Transl

Res，2017，9（3）：1075-1087.

[23] YANG X D，CEN Z D，CHENG H P，et al. L-3-nbutylphthalide

protects HSPB8 K141N mutationinduced

oxidative stress by modulating the

mitochondrial apoptotic and Nrf2 pathways[J/

OL]. Front Neurosci，2017，11：402. https：//doi.

org/10.3389/fnins.2017.00402.

[24] DU R，TENG J F，WANG Y，et al. Clinical study of

butylphthalide combined with Xue Shuan Tong on

serum inflammatory factors and prognosis effect of

patients with cerebral infarction[J]. Pak J Pharm Sci，

2015，28（5 Suppl）：1823-1827.

[25] HU J，WEN Q，WU Y，et al. The effect of

butylphthalide on the brain edema，blood-brain

barrier of rats after focal cerebral infarction and the

expression of Rho A[J]. Cell Biochem Biophys，2014，

69（2）：363-368.

[26] ZHAO Y J，NAI Y，MA Q S，et al. Dl-3-nbutylphthalide

protects the blood brain barrier

of cerebral infarction by activating the Nrf-2/

HO-1 signaling pathway in mice[J]. Eur Rev Med

Pharmacol Sci，2018，22（7）：2109-2118.

[27] TANG S C，LUO C J，ZHANG K H，et al. Effects

of dl-3-n-butylphthalide on serum VEGF and bFGF

levels in acute cerebral infarction[J]. Eur Rev Med

Pharmacol Sci，2017，21（19）：4431-4436.

[28] LI W，WEI D，XIE X，et al. Dl-3-n-butylphthalide

regulates the Ang-1/Ang-2/Tie-2 signaling axis to

promote neovascularization in chronic cerebral

hypoperfusion[J/OL]. Biomed Pharmacother，

2019，113：108757. https：//doi.org/ 10.1016/

j.biopha.2019.108757.