不同时机应用促红细胞生成素对感染所致新生大鼠脑损伤的保护作用

Protective effects of erythropoietin on infection induced neonatal rat brain injury using at different times

目的探讨促红细胞生成素（EPO）对感染致新生大鼠脑损伤保护作用的最佳应用时机及其相关机制。方法2日龄（P2）新生sD大鼠按随机数字表法分为4组，分别为对照组（A组）、脂多糖（LPS）感染组（B组）、早期EPO干预组（c组）、晚期EPO干预组（D组）。A、B、C组P2新生大鼠连续5d（132-P6）分别腹腔注射相应药物：等容积9g／L盐水＋等容积EPO空白对照品、0．6mg／kgLPS＋等容积EPO空白对照品、0，6rag／kgLPS＋5000IU／kgEPO；D组P2新生大鼠连续5d（1）2-P6）腹腔注射0．6rag／kgLPS，P7开始连续腹腔注射5000IU／kgEPO5d（即P7-P11）。A、B2组分别于P2（腹腔注射第1次药物后6h）、P7、P12，各随机数字表法抽取10只新生大鼠取脑，以矢状缝为标志分为左右半脑，右侧脑应用酶联免疫吸附法柃测脑组织EPO受体（EPOR）水平，左侧脑应用反转录-聚合酶链反应（RT—PCR）方法检测EPORmRNA水平；A、B、C3组于P7随机数字表法选取10只新生大鼠灌注取脑，余续养，4组均于P12灌注取脑，应用免疫组织化学方法检测髓鞘碱性蛋白（MBP）、胶质纤维酸性蛋白（GFAP）及EPOR的表达，采用HE染色观察各组大鼠脑组织的病理改变。结果1.HE染色示B组海马锥体细胞界限不清、层次紊乱，细胞数目减少，脑室扩张，周围白质有囊性软化区域形成；EPO干预组较B组病理改变减轻，早期干预组更明显。2．B组较A组EPOR蛋白及mRNA表达增加，随日龄的增加EPOR与EPORmRNA表达有下降趋势。3．B组MBP表达（107．46±3．65）较A组（146．78±3．13）明显减少（P＜0．05），EPO干预组较B组表达增加，且C组（126．25±4．42）较D组（117．35±3．42）增加更明屁（P＜0．05）。4．B组GFAP表达（P7、P12分别为141．46±11．92、149．48±13．59）较A组（P7、P12分别为120．63-4-13．32、119．74±12．48）增加（P＜0．05），P12时EPO干预组表达较B组降低，C组（134．59±12．19）与D组（137．27±13．87）差异无统计学意义（P＞0．05）。结论EPO对出生后感染所致的脑白质损伤有保护作用，且早期应用优于晚期，其机制可能与感染可使新生大鼠脑组织EPOR表达增加及EPOR表达随日龄增加而降低有关。

Objective To explore the influence of erythropoietin （EPO） on infection induced neonatal rat brain injury at different starting time and its related mechanism. Methods Postnatal day 2 （P2） newborn SD rats were randomly divided into 4 groups ： control group （ group A ） , lipopolysaccharide （ LPS ） group （ group B ） , the early EPO group （group C ）and the later EPO group（ group D）. Pups in group A, B and C were injected different drugs intraperitoneally （ group A for saline, group B for 0.6 mg/kg of LPS, and group C for 0.6 mg/kg of LPS and 5 000 U/kg of EPO） once a day for consecutive 5 days（ P2 - P6 ）. LPS in group D were injected 0.6 mg/kg of LPS intraperitoneally once a day for consecutive 5 days （ P2 - P6）, and with 5 000 U/kg of EPO once a day for consecutive 5 days （ P7 - P11 ）. Rats in each group were given different drugs starting at corresponding time by intraperitoneal injection for 5 consecutive days. Every 10 newborn rats in group A and B were selected randomly on P2 （6 h after intraperitoneal injection of drugs for the first time） , P7 and P12, the brains were divided into the left and the right hemispheres marked by sagittal suture, using enzyme-linked immunosorbent assay method to evaluate the erythropoietin receptor（EPOR） protein level with the right cerebral hemisphere and reverse transcription-polymerase chain reaction （RT-PCR） method was used to investigate EPOR mRNA level of the left cerebral hemisphere. Immunohistochemical method was adopted to evaluate the expression of myelin basic protein（MBP） ,glial fibrillary acidic protein（GFAP） and EPOR at specified time point,and HE dyeing for the pathological changes of brain damage in different groups. Results HE staining of the group A presented the normal structure in the neonatal rat brain. Reduced numbers of hippocampal pyramidal cells, expansion of the lateral ventricles and periventricular leukomalacia were found in group B. No leukomalacia or lateral ventricles＇s expansion in EPO administrated groups and it was more obvious in group C. The EPOR protein and mRNA of group B was in-creased compared with the group A. The EPOR protein and mRNA levels had a tendency to decline with the increase of age. The MBP expression of group B（ 107.46 ± 3.65 ） was significantly reduced compared with the group A（ 146.78 ±3.13） （P 〈0. 05 ）, and the expression of EPO groups increased in contrast to the group B, moreover, the group C （ 126. 25 ±4.42）increased more obviously than that of group D（ 117.35 ±3.42） （P 〈0.05）. The GFAP expression of group B（ 141.46 ±11.92 at P7 and 149.48 ±13.59 at P12） increased significantly than group A（ 120.63 ± 13.32 at P7 and 119.74 ±12.48 at P12） （P 〈0. 05 ）, the EPO group expressed lower than group B at the P12, and the group C （ 134. 59 ± 12.19 ） decreased than the group D （ 137.27 ± 13.87 ） （ P 〉0.05 ）. Conclusions EPO shows a protective effect on the cerebral white matter injury caused by postpartum infection,it is superior to administer EPO at early time than later time. The mechanism of the protective effect may be connected with the fact that the infection can induce the expression of brain EPOR and the EPOR expression level has a tendency to decline with the increase of age.