促红细胞生成素通过降低血小板反应蛋白-1和转化生长因子-β的表达对实验性脊髓损伤的保护作用

Protection of erythropoietin on experimental spinal cord injury by reducing the expression of thrombospondin-1 and transforming growth factor-beta.

作者信息

Fang Xiang-qian, Fang Mei, Fan Shun-wu, Gu Chuan-long

机构信息

Department of Orthopaedics, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, China.

出版信息

Chin Med J (Engl). 2009 Jul 20;122(14):1631-5.

DOI:

PMID:19719963

Abstract

BACKGROUND

Erythropoietin (EPO) functions as a tissue-protective cytokine in addition to its crucial hormonal role in red cell production and neuron protection. This study aimed to determine the neuron protective effect of erythropoietin on experimental rats enduring spinal cord injury (SCI) by assessing thrombospondin-1 (TSP-1) level and transforming growth factor-beta (TGF-beta) in the development of a rat model of SCI.

METHODS

Sixty Sprague-Dawley rats were randomly assigned to three groups: sham operation control group, SCI group and EPO treatment group. By using a weight-drop contusion SCI model, the rats in the SCI group and EPO treatment group were sacrificed at 24 hours and 7 days subsequently. The Basso, Beattie, and Bresnahan (BBB) scores were examined for locomotor function. Pathological changes were observed after HE staining. The expressions of thrombospondin-2 (TSP-1) and TGF-beta were determined by immunohistochemical staining and Western blotting.

RESULTS

Slighter locomotor dysfunction was discovered and it was recovered abruptly as higher BBB scores were found in the EPO treatment group than in the SCI group (P < 0.01). Pathologically, progressive disruption of the dorsal white matter and regeneration of a few neurons were also observed in SCI rats. TSP-1 and TGF-beta expression increased at 24 hours and 7 days after SCI in the injured segment, and it was higher in the SCI group than in the EPO treatment group. Spinal cord samples from the animals demonstrated a TSP-1 optical density of 112.2 +/- 6.8 and TSP-1 positive cells of 5.7 +/- 1.3 respectively. After injury, the TSP-1 optical density and cell number increased to 287.2 +/- 14.3/mm(2) and 23.2 +/- 2.6/mm(2) at 24 hours and to 232.1 +/- 13.2/mm(2) and 15.2 +/- 2.3/mm(2) at 7 days respectively. When EPO treated rats compared with the SCI rats, the TSP-1 optical density and cell number decreased to 213.1 +/- 11.6/mm(2) and 11.9 +/- 1.6/mm(2) at 24 hours and to 189.9 +/- 10.5/mm(2) and 9.3 +/- 1.5/mm(2) at 7 days, respectively (P < 0.01). In the SCI rats, the TGF-beta optical density and positive neuron number were 291.4 +/- 15.2/mm(2) and 28.8 +/- 4.9/mm(2) at 24 hours and 259.1 +/- 12.3/mm(2) and 23.9 +/- 4.1/mm(2) at 7 days respectively. They decreased in the EPO treated rats to 222.8 +/- 11.9/mm(2) and 13.7 +/- 2.1/mm(2) at 24 hours and to 196.5 +/- 9.7/mm(2) and 8.7 +/- 2.2/mm(2) at 7 days (P < 0.01).

CONCLUSIONS

Increased expression of TSP-1 and TGF-beta can be found in the injured segment of the spinal cord at 24 hours and 7 days after injury. EPO treatment can effectively prevent pathological alterations from severe spinal cord injury by reduced expression of TSP-1 and TGF-beta.

摘要

背景

促红细胞生成素（EPO）除了在红细胞生成和神经元保护中发挥关键的激素作用外，还作为一种组织保护细胞因子发挥作用。本研究旨在通过评估脊髓损伤（SCI）大鼠模型发育过程中血小板反应蛋白-1（TSP-1）水平和转化生长因子-β（TGF-β），确定促红细胞生成素对实验性脊髓损伤大鼠的神经元保护作用。

方法

将60只Sprague-Dawley大鼠随机分为三组：假手术对照组、SCI组和EPO治疗组。采用重物坠落挫伤性SCI模型，SCI组和EPO治疗组的大鼠分别在24小时和7天后处死。检测Basso、Beattie和Bresnahan（BBB）评分以评估运动功能。苏木精-伊红（HE）染色后观察病理变化。通过免疫组织化学染色和蛋白质印迹法测定血小板反应蛋白-2（TSP-1）和TGF-β的表达。

结果

发现EPO治疗组的运动功能障碍较轻，且随着BBB评分高于SCI组而迅速恢复（P<0.01）。病理上，SCI大鼠还观察到背侧白质的进行性破坏和少数神经元的再生。SCI后24小时和7天，损伤节段TSP-1和TGF-β表达增加，且SCI组高于EPO治疗组。动物的脊髓样本显示TSP-1光密度分别为112.2±6.8，TSP-1阳性细胞为5.7±1.3。损伤后，TSP-1光密度和细胞数在24小时分别增加至287.2±14.3/mm²和23.2±2.6/mm²，在7天分别增加至232.1±13.2/mm²和15.2±2.3/mm²。与SCI大鼠相比，EPO治疗的大鼠在24小时时TSP-1光密度和细胞数分别降至213.1±11.6/mm²和11.9±1.6/mm²，在7天时分别降至189.9±10.5/mm²和9.3±1.5/mm²（P<0.01）。在SCI大鼠中，TGF-β光密度和阳性神经元数在24小时分别为291.4±15.2/mm²和28.8±4.9/mm²，在7天分别为259.1±12.3/mm²和23.9±4.1/mm²。在EPO治疗的大鼠中，它们在24小时降至222.8±11.9/mm²和13.7±2.1/mm²，在7天降至196.5±9.7/mm²和8.7±2.2/mm²（P<0.01）。