EGCG上调神经细胞自噬相关蛋白Beclin-1表达改善脊髓损伤后大鼠运动功能

Epigallocatechin gallate improves motor function via upregulation of Beclin-1 expression in rats with spinal cord injury

目的观察表没食子儿茶素没食子酸酯(EGCG)对脊髓损伤(SCI)大鼠神经元自噬和相关调控蛋白Beclin-1表达的影响,探讨其SCI保护机制。方法 SD大鼠90只,分为假手术组、SCI组、EGCG组(n=25)。钳夹法建立SCI模型,EGCG组和SCI组损伤后即刻和1h后分别腹腔注射EGCG (50 mg/kg)和等量生理盐水,三组大鼠于术后不同时间1d、3d、1周、2周、3周、4周(n=5),分别进行斜板试验和运动功能(BBB)评分实验,测定各组大鼠的运动功能;用结晶紫(CV)染色法和免疫组化方法,观察SCI后6h、12h、24h、3d和7d时间内,从损伤中心到头端0～5.00 mm空间范围内,EGCG对脊髓神经元存活、自噬以及相关调控蛋白Beclin-1表达的影响。结果 SCI组在各个时间点斜板角度均明显小于假手术组,EGCG组相比于SCI组,除1d和3 d组外,其余1周～4周各个时间点斜板角度明显增加。SCI后各个时间点,SCI组评分明显低于假手术组,EGCG组相比于SCI组,术后3d～4周各个时间点BBB评分明显增加;SCI后6 h～7d,在0～0.50mm空间范围内均未观察到存活的神经元;在1.00～3.50mm距离内, EGCG组和SCI组相比,存活的神经元数明显增加,4.00mm处达峰值。SCI后6h～7d,在1.05～4.55mm范围内,EGCG组和SCI组相比,Beclin-1阳性表达的神经元数明显增加,其中在3d,2.55mm,Beclin-1表达达高峰,7d时减少。结论 EGCG干预改善脊髓损伤大鼠运动功能与上调神经元自噬蛋白Beclin-1表达相关。

Objective To investigate the effect of epigallocatechin gallate(EGCG) on the neuronal autophagy and the expression of Beclin-1 in the spinal cord after SCI. Methods Ninety Sprague-Dawley rats were divided into sham, SCI(clamp method) and EGCG treated groups(injected with EGCG intraperitoneally, 50 mg/kg, n=25). All rats were subjected to inclined plane test and BBB test at different time points at 1, 3 days, and 1, 2, 3 and 4 weeks after operation. The expression of Beclin-1 in spinal cord was measured by crystal violet(CV) staining and immunohistochemistry at 6, 12, 24 hours, and 3 , 7 days post-injury in the space of 0-5.00 mm from the epicenter to the rostral. Results The angle of inclined plate was significantly decreased in SCI group than in sham, but increased in EGCG group than in SCI group at each time point during 1-4 weeks significantly except for 1 and 3 days. At each time point after SCI, BBB score was significantly lower in SCI than in sham, and significantly higher in EGCG group than in SCI group. In the distance of 1-3.50 mm, the number of survival neurons was significantly higher in EGCG group than in SCI group and peaked at 4.00 mm. The number of Beclin- 1-positive neurons increased significantly in EGCG group than in SCI group, peaked at 3 days and 2.55 mm, and decreased at 7 days. Conclusion EGCG improves the motor function, which is related to the up-regulation of autophagy protein Beclin-1 expression in rats with spinal cord injury.