重组人促红细胞生成素在早产儿脑白质损伤模型鼠中调控血管内皮生长因子受体2的机制

Mechanisms of vascular endothelial growth factor receptor 2 expression regulation by erythropoietin in a premature rat model of periventricular white matter damage

目的探讨在早产儿脑白质损伤模型鼠脑血管生成中，重组人促红细胞生成素（recombinant human erythropoietin, rh-EPO）调控血管内皮生长因子受体2（ vascularendothelial growth factor receptor 2, VEGFR2） 表达的可能机制。方法选取3日龄Sprague Dawley仔鼠，采用随机数余数分组法分为假手术组、缺血缺氧组、缺血缺氧＋促红细胞生成素（erythropoietin，EPO）组、缺血缺氧＋EPO受体（ erythropoietin receptor, EPOR ）拈抗剂组、缺血缺氧＋EPO＋EPOR拮抗剂组。假手术组仔鼠仅游离右侧颈总动脉。缺血缺氧组仔鼠结扎右侧颈总动脉，并吸人氮氧混合气体2h。缺血缺氧＋EPO组、缺血缺氧＋EPO＋EPOR拮抗剂组结扎后4h腹腔内给予rh—EPO5U／g。缺血缺氧＋EPOR拈抗剂组、缺血缺氧＋EPO＋EPOR拮抗剂组，结扎后脑室内给予EPOR拮抗剂5μl，其余组均给予等量生理盐水。采用蛋白质印迹技术检测分组处理完成后60和90min脑组织中EPOR和磷酸化EPOR（phosphorylated—EPOR，p-EPOR）、总细胞外信号调节激酶（ extracellular regulated protein kinases, ERK ）和磷酸化ERK（phosphorylated—ERK，p-ERK）及分组处理完成后2、4d脑组织中的VEGFR2表达情况。多组间数据比较采用单因素方差分析，组间两两比较采用SNK检验。结果分组处理完成后60和90min，仔鼠脑组织EPOR蛋白表达在假手术组呈较低水平（分别为1．095±0．182和0．751±0．136）；在缺血缺氧组（1．717±0．206和1．416±0．242）、缺血缺氧＋EPO组（2．557±0．222和2．111±0．159）和缺血缺氧＋EPO＋EPOR拮抗剂组（1．547±0．170和1．452±0．250）表达增加，高于假手术组；在缺血缺氧＋EPO组高于缺血缺氧组和缺血缺氧＋EPO＋EPOR拮抗剂组；在缺血缺氧＋EPOR拈抗剂组（1．088±0．160和1．020±0．174）低于缺血缺氧组；差异均有统计学意义（F值分别为30．154和20．265，P值均〈0．05）。p-EPOR和p-ERK，以及分组处理完成后2和4dVEGFR2在各组的表达差异与EPOR一致，差异均有统计学意义（P值均〈0．05）；此外，分组处理完成后4d，VEGFR2表达在缺血缺氧＋EPO＋EPOR拈抗剂组低于缺血缺氧组（1．053±0．118与1．439±0．074，F=54．248，P〈0．05）。分组处理完成后60和90min，各组仔鼠脑组织ERK表达差异均无统计学意义（F值分别为1．117和0．734，P值均〉O．05）。结论在早产儿脑白质损伤模型鼠，EPO与EPOR作用后影响下游ERK信号通路，增加脑组织VEGFR2的表达。

Objective To explore the mechanisms of vascular endothelial growth factor receptor 2 （VEGFR2） expression regulated by recombinant human erythropoietin （rh-EPO） in a premature rat model of periventricular white matter damage. Methods Sprague-Dawley rats aged three days were randomly divided into five groups： sham group without hypoxia-ischemia （HI）, HI group （HI with saline administration）, HI＋erythropoietin （EPO） group, HI＋erythropoietin receptor （EPOR） antagonist group and HI＋EPO＋EPOR antagonist group. Rat pups were either subjected to permanent ligation of the right common carotid artery and 6% 02＋94% N2 for two hours （HI） or sham operated and exposed to normal air （sham）. After the operation, rats in the HI＋EPOR antagonist and HI＋EPO＋EPOR antagonist groups received a single intraventricular injection of EPOR antagonist （5 μ l）. Four hours after the operation, rats in the HI＋EPO and HI＋EPO＋EPOR antagonist groups received a single intraperitoneal injection of rh-EPO （5 U/g）, Western-blot was performed to detect EPOR, phosphorylated EPOR （p-EPOR）, extracellular regulated protein kinases （ERK） and phosphorylated ERK （p-ERK） at 60 and 90 minutes after the models were established successfully, and also used to analyze the expression of VECFR2 on day 2 and 4. Analysis of variance and SNK test were used as statistical methods. Results At 60 and 90 minutes after model establishment, the expression of EPOR protein in rat brain tissues was increased in HI （1.717±0.206 and 1.416±0.242）, HI＋EPO （2.557±0.222 and 2.111±0.159） and HI＋EPO＋EPOR antagonist （1.547±0.170 and 1.452±0.250） groups as compared with that in sham group （1.095±0.182 and 0.751±0.136）, that in HI＋EPO group was higher than that in HI and HI＋EPO＋EPOR antagonist groups, and that in HI＋EPOR antagonist group （1.088 ±0.160 and 1.020± 0.174） was lower than that in HI group. All differences were statistically significant （F=30.154 and 20.265, both P〈0.05）. The expressions of p-EPOR, p-ERK and VEGFR2 in the five groups were consistent with the expression of EPOR, and the differences were also statistically significant （all P〈0.05）. In addition, the expression of VEGFR2 in HI＋EPO＋EPOR antagonist group was lower than that in HI group on day 4 （1.053 ± 0.118 vs 1.439 ±0.074, F=54.248, P〈0.05）. No statistically significant difference in ERK expression was found among all groups at 60 or 90 minutes after modeling （F=1.117 and 0.734, both P〉0.05）. Conclusions ERK signaling pathways will be affected by EPO binding to EPOR. As a result, VEGFR2 expression was increased leading to enhanced angiogenesis in a premature rat model of periventricular white matter damage.