Carbamylated erythropoietin promotes vascular microcirculation following cerebral infarction

doi:10.3969/j.issn.2095-4344.2017.16.013

Abstract

Abstract:

BACKGROUND: Carbamylated erythropoietin (CEPO) cannot only remarkably promote the prognosis of cerebral infraction, but also improve the microcirculation.
OBJECTIVE: To explore the underlying mechanism of CEPO promoting the microcirculation following cerebral infraction.
METHODS: 150 Wistar rats were selected, and 120 rats were used for establishing the models of cerebral infarction, followed by allotted into four groups. The model rats were treated with 500, 1 000 and 2 000 u/kg CEPO as experimental groups, and those received no treatment as model group. The other 30 rats were as controls. Vascular endothelial cells were isolated and cultured in vitro, and the cell proliferation was detected by cell counting kit-8 assay. The expression levels of proliferation-related genes (Ki67 and p16) and vascular endothelial growth factor (VEGF) were detected using western blot assay. After selective silencing of VEGF through RNA interference, all above indicators were detected again.
RESULTS AND CONCLUSION: Cell counting kit-8 assay results showed that the proliferation ability of vascular endothelial cells was increased with CEPO concentration increasing. Western blot assay results showed a significant upregulation of Ki67, p16 and VEGF. After shRNA-VEFG interference, these indicators had no positive correlation with the increased concentration of CEPO. Our findings indicate that CEPO can improve the proliferation of vascular endothelial cells in an animal model of cerebral infarction via upregulating the VEGF expression.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Cerebral Infraction, Microcirculation, Vascular Endothelial Growth Factors, Tissue Engineering

CLC Number:

R318

Zhang Qi-shun, Chen Yong, Wang Zhao-hui, Wu Chun-fang, Zhao Jun. Carbamylated erythropoietin promotes vascular microcirculation following cerebral infarction[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(16): 2534-2539.

Figures/Tables 2

2.1 实验动物数量分析实验选用大鼠150只，分为3组，实验过程中涉及所有大鼠均无任何脱失，全部进入结果分析。 2.2 C促红细胞生成素可明显促进脑梗死大鼠脑微循环灌注结果显示，相比于空白对照组，脑梗死大鼠的所有检测指标均显著上调；而相比于脑梗死组，在经C促红细胞生成素处理后的脑梗死大鼠中，所有检测指标均明显下调(P < 0.05)，提示C促红细胞生成素可明显改善脑梗死大鼠脑脉微循环的功能。然而，与血管损伤相关因子的表达变化显著高于与血栓形成相关因子的变化幅度：前列环素 (3.7±1.9)倍、内皮素1(15.0±2.1)倍、血管紧张素Ⅱ(12.3±1.2)倍、vWF(5.7±1.9)倍。同时在C促红细胞生成素不同浓度处理大鼠，与对照及脑梗死动物模型相比，差异均有显著性意义，其中以浓度为1 000 U/kg差异最为明显(图1)。提示，C促红细胞生成素明显改善脑梗死大鼠脑脉微循环的功能，可能与脑梗死循环内皮修复相关。 2.3 C促红细胞生成素可明显促进脑梗死大鼠脑微循环内皮细胞体外增殖采用CCK8体外细胞活力检测实验，结果显示，相比于空白对照组，脑梗死大鼠脑微循环内皮细胞的数量显著降低[(0.3±1.2)倍，P < 0.01]；而C促红细胞生成素却可明显提升脑梗死大鼠脑微循环内皮细胞数量[(1.9±0.8)倍，P < 0.01](图2)，其中以浓度为1 000 U/kg差异最为明显。为进一步确定C促红细胞生成素是否可通过促进脑梗死大鼠脑微循环内皮细胞体外增殖且，且抑制凋亡，采用realtimePCR实验检测了与增殖及凋亡相关基因的表达，结果显示，相比于未经C促红细胞生成素处理的脑梗死大鼠，与增殖相关基因表达均明显上调[Ki67 (9.0±1.1)倍，P < 0.01]及p16[(6.0±1.2)倍，P < 0.01]；而同时与凋亡相关基因的表达均显著下调Bad[(0.1±0.6) 倍，P < 0.01]及Bax[(0.08±0.07)倍，P < 0.01]。提示，C促红细胞生成素可通过促进脑梗死大鼠脑微循环内皮细胞体外增殖，以改善脑梗死大鼠脑微循环的功能。 2.4 C促红细胞生成素通过上调血管内皮生长因子表达以促进脑梗死大鼠脑微循环内皮细胞体外增殖通过实时定量PCR(qPCR)和蛋白免疫印记(western blot)实验分别从mRNA和蛋白水平两方面，检测了脑微循环内皮细胞内血管内皮生长因子和血管紧张素1的表达(图3A-C)。结果显示，相比于脑梗死组，在经C促红细胞生成素处理后的脑梗死大鼠中，血管内皮生长因子的表达水平显著上调[(9.0±1.5)倍， P < 0.01]，而血管紧张素1表达未见显著变化，其中以浓度为1 000 U/kg差异最为明显，提示血管紧张素1可能在C促红细胞生成素促进脑微循环内皮细胞增殖过程中并未起主导作用。进一步，采用RNA干扰技术，选取C促红细胞生成素差异最为明显浓度1 000 U/kg为处理剂量，特异性降低脑微循环内皮细胞内血管内皮生长因子蛋白的转录。结果显示，经RNA干扰后，C促红细胞生成素促进脑微循环内皮细胞体外增殖能力显著降低[(0.6±0.2)倍，P < 0.01](图3D-E)，提示，C促红细胞生成素可通过上调血管内皮生长因子表达以促进脑梗死大鼠脑微循环内皮细胞体外增殖，以改善脑梗死大鼠脑微循环的功能。"

References

[1] Gurav AN. Management of diabolical diabetes mellitus and periodontitis nexus: Are we doing enough? World J Diabetes. 2016;7(4):50-66.

[2] Balc?o?lu AS, Müderriso?lu H. Diabetes and cardiac autonomic neuropathy: Clinical manifestations, cardiovascular consequences, diagnosis and treatment. World J Diabetes.2015;6(1):80-91.

[3] 侯兰,吕娟, 陈莉娜.促红细胞生成素的多器官保护作用研究进展[J].中国药房,2014(17):1626-1628

[4] Schöffel N,Börger JA,Quarcoo D,et al.[Erythropoietin - state of science]. Sportverletz Sportschaden.2008;22(4):201-206.

[5] Xu K,George I,Klotz S,et al.Erythropoietin derivate improves left ventricular systolic performance and attenuates left ventricular remodeling in rats with myocardial infarct-induced heart failure. J Cardiovasc Pharmacol.2010;56(5):506-512.

[6] Sanchis-Gomar F, Garcia-Gimenez JL, Pareja-Galeano H,et al.Erythropoietin and the heart: physiological effects and the therapeutic perspective.Int J Cardiol.2014;171(2):116-125.

[7] Ogunshola OO, Bogdanova AY. Epo and non-hematopoietic cells: what do we know? Methods Mol Biol. 2013;982:13-41.

[8] He H,Qiao X,Wu S.Carbamylated erythropoietin attenuates cardiomyopathy via PI3K/Akt activation in rats with diabetic cardiomyopathy. Exp Ther Med. 2013;6(2):567-573.

[9] Xu X,Cao Z,Cao B,et al.Carbamylated erythropoietin protects the myocardium from acute ischemia/reperfusion injury through a PI3K/Akt-dependent mechanism. Surgery. 2009;146(3):506-514.

[10] Dashkevich A,Hagl C,Beyersdorf F,et al. VEGF Pathways in the Lymphatics of Healthy and Diseased Heart. Microcirculation.2016;23(1):5-14.

[11] Cubranic A,Redzovic A,Dobrila-Dintinjana R,et al. Mystery Story about Erythropoietin (Epo) and Erythropoietin Receptor (EpoR) are Disguised? Hepatogastroenterology. 2015;62 (139):585-589.

[12] Bjornstad P, Truong U, Dorosz JL, et al.Cardiopulmonary Dysfunction and Adiponectin in Adolescents With Type 2 Diabetes.J Am Heart Assoc. 2016;5(3):e002804.

[13] Meredith IT,Tanguay JF,Kereiakes DJ,et al.Diabetes Mellitus and Prevention of Late Myocardial Infarction After Coronary Stenting in the Randomized Dual Antiplatelet Therapy Study. Circulation.2016;133(18):1772-1782.

[14] Heinonen I,Sorop O,de Beer VJ,et al.What can we learn about treating heart failure from the heart's response to acute exercise? Focus on the coronary microcirculation. J Appl Physiol (1985). 2015;119(8):934-943.

[15] Fiordaliso F,Chimenti S,Staszewsky L,et al.A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. Proc Natl Acad Sci U S A. 2005;102(6):2046-2051.