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目的:研究经蛛网膜下腔给予表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)对大鼠脊髓损伤(spinal cord injury,SCI)后神经功能恢复的影响及其作用机制。方法:成年雌性SD大鼠40只,随机分为4组,每组10只,假手术组(A组)仅切除椎板;对照组(B组)SCI后,蛛网膜下腔注射同体积载体溶液;10mg/kg EGCG治疗组(C组)SCI后经蛛网膜下腔注射EGCG 10mg/kg;20mg/kg EGCG治疗组(D组)SCI后经蛛网膜下腔注射EGCG 20mg/kg。改良Allen法(40g·cm)制作T10节段SCI模型,L4水平蛛网膜下腔注射EGCG或载体溶液。术前、术后1d、术后3d及术后1、2、3、4周进行盲法BBB评分、斜板试验;术后4周时处死大鼠,病理学检查(Luxol fast blue染色)观察脊髓损伤部位残余髓鞘情况;免疫组化及Western blot法检测胶质细胞源性营养因子(GDNF)、脑源性神经营养因子(BDNF)、Bcl-2和Bax的表达水平。结果:A组术前及术后各时间点BBB评分均为21分,斜板试验角度无明显变化;术后各时间点B、C组和D组BBB评分及斜板试验角度均小于A组(P<0.05);术后1d、3d时,B、C组和D组BBB评分以及斜板试验角度无统计学差异(P>0.05);术后1、2、3、4周时,C、D组的BBB评分及斜板试验角度均大于B组(P<0.05),C组的BBB评分及斜板试验角度与D组比较均无统计学差异(P>0.05)。术后4周时,B、C、D组大鼠脊髓损伤部位的髓鞘残余面积均小于A组(P<0.05),C、D组明显大于B组(P<0.05),在损伤脊髓中心D组明显大于C组(P<0.05)。术后4周时免疫组化检查,B、C、D组的BDNF、GDNF、Bcl-2和Bax的阳性表达强于A组,C、D组的BDNF、GDNF和Bcl-2的阳性表达强于B组,Bax的阳性表达弱于B组。术后4周时Western blot法检测,B、C、D组的BDNF、GDNF、Bcl-2和Bax的表达高于A组(P<0.05);C、D组的BDNF和GDNF表达明显高于B组(P<0.05),C组与D组无统计学差异(P>0.05);C、D组的Bcl-2表达明显高于B组(P<0.05),C组与D组比较无统计学差异(P>0.05);C组和D组的Bax表达明显低于B组(P<0.05),D组明显低于C组(P<0.05)。结论:EGCG可有效促进大鼠SCI后的神经功能恢复,其机理可能与髓鞘的丢失减少、神经营养因子BDNF和GDNF的表达上调及细胞凋亡被抑制等有关。
Objective: To investigate the effect and mechanism of epigallocatechin gallate (EGCG) administered to the subarachnoid on neurological recovery after spinal cord injury (SCI) in rats. Methods: Forty adult female Sprague-Dawley rats were randomly divided into 4 groups (10 rats in each group). The sham-operated group (group A) only received laminectomy. In the control group (group B) Solution; 10mg / kg EGCG treatment group (group C) after SCI subarachnoid injection of EGCG 10mg / kg; 20mg / kg EGCG treatment group (D group) SCI after subarachnoid injection of EGCG 20mg / kg. T10 segmental SCI model was made by modified Allen’s method (40 g · cm), and EGCG or vehicle solution was injected into L4 horizontal subarachnoid. Blind BBB score and oblique plate test were performed before operation, 1 day after operation, 3 days after operation and 1, 2, 3, 4 weeks after operation. Rats were sacrificed at 4 weeks after operation, and pathological examination (Luxol fast blue staining) The remnant myelin in spinal cord injury area was detected by immunohistochemistry and Western blot. The expressions of GDNF, BDNF, Bcl-2 and Bax were detected by immunohistochemistry and Western blot. Results: The score of BBB was 21 points in group A before and after operation, and there was no significant change in the angle of oblique plate test. The BBB score and the angle of oblique plate in groups B, C and D at all time points were lower than those in group A (P <0.05). At 1 day and 3 days after operation, there was no significant difference in the BBB scores of the groups B, C and D as well as the angle of the inclined plate (P> 0.05) , BBB score of D group and angle of inclined plate test were all greater than those of B group (P <0.05). There was no significant difference between BBB score of C group and oblique plate test (P> 0.05). At 4 weeks after operation, the remnants of myelin in spinal cord injury in groups B, C and D were significantly smaller than those in group A (P <0.05), while those in groups C and D were significantly greater than those in group B (P <0.05) D group was significantly larger than C group (P <0.05). The positive expression of BDNF, GDNF, Bcl-2 and Bax in groups B, C and D was stronger than that in group A at 4 weeks after operation, and positive expression of BDNF, GDNF and Bcl-2 in groups C and D was stronger In group B, the positive expression of Bax was weaker than that in group B. The expression of BDNF, GDNF, Bcl-2 and Bax in group B, C and D were higher than that in group A at 4 weeks after operation (P <0.05). The expression of BDNF and GDNF in group C and D were significantly higher than that of group A (P <0.05). There was no significant difference between group C and group D (P> 0.05). The expression of Bcl-2 in group C and D was significantly higher than that in group B (P <0.05) (P> 0.05). The expression of Bax in group C and group D was significantly lower than that in group B (P <0.05), and in group D was significantly lower than that in group C (P <0.05). Conclusion: EGCG can effectively promote the recovery of neural function after SCI in rats. The mechanism may be related to the decrease of myelin loss, the upregulation of BDNF and GDNF, and the inhibition of apoptosis.