表没食子儿茶素没食子酸酯对大鼠脊髓损伤后神经功能恢复的影响

The therapeutic effect of epigallocatechin gallate on the neurological recovery after spinal cord injury in rat

中文关键词:  脊髓损伤  表没食子儿茶素没食子酸酯  神经营养因子  凋亡  大鼠

英文关键词:Spinal cord injury  Epigallocatechin gallate  Neurotrophic factor  Apoptosis  Rat

中文摘要:

  【摘要】 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。

英文摘要:

  【Abstract】 Objectives: To investigate the therapeutic effects and underlying mechanism of epigallocatechin gallate(EGCG) administered by subarachnoid injection in spinal cord injury(SCI) in rats. Methods: 40 adult Sprague-Dawley rats were randomly divided into four groups with 10 in each group as follows: sham group(group A), laminectomy only; control group(group B), after SCI, subarachnoid injection of same volume of control solution; 10mg/kg EGCG-treated group(group C), after SCI, 10mg/kg EGCG was given by subarachnoid injection; 20mg/kg EGCG-treated group(group D), after SCI, 20mg/kg EGCG was given by subarachnoid injection. SCI was induced by using the modified weight-drop method(10g×4cm) at T10 level. EGCG(10 or 20mg/kg) was administered by subarachnoid injection at lumbar level 4 immediately after SCI. At 1d, 3d, 1w, 2w, 3w and 4w of post-operation, the locomotor functional recovery was assessed by using open-field locomotor tests and inclined-plane tests. At 4 weeks of post-operation, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemical and Western blot analyses were performed to observe the expressions of: the brain-derived neurotrophic factor(BDNF), glial cell line-derived neurotrophic factor(GDNF), B cell CLL/lymphoma-2(Bcl-2) and Bcl-2-associated X protein(Bax). Results: In group A, the BBB score and inclined plate test angle did not change much after operation. While at 1d, 3d, 1w, 2w, 3w and 4w after operation, the BBB scores and inclined pate test angles of group B, C and D were significantly lower than those in group A(P＜0.05). At 1d and 3d after operation, the BBB scores and inclined-plated angles of group B, C and D did not show significantly difference(P＜0.05). At 1w, 2w, 3w and 4w after operation, the BBB scores and inclined plated test angles in group C and D were significantly higher than those in group B(P＜0.05). There were no differences between group C and D in the BBB scores and inclined plated test angles(P＞0.05). At 4w of post-operation, the LFB staining in group B, C and D shows less myelin distribution compared with group A(P＜0.05). Group C and D have more myelin distribution compared with group B(P＜0.05). Further, in the center of the injured cord, there was more myelin in group D than group C(P＜0.05). Immunohistochemistry result showed that the positive expressions of BDNF, GDNF, Bcl-2 and Bax in group A were weaker than those in group B, C and D. The positive expression of BDNF, GDNF and Bcl-2 in group C and D were higher than those in the group B, while, the expression of Bax was weaker. Western blot results showed that expressions of BDNF, GDNF, Bcl-2 and Bax in group B, C and D were much higher than those in the group A(P＜0.05). The expressions of BDNF and GDNF in group C and D were higher than group B(P＜0.05). There was no difference between group C and D. The expression of Bcl-2 in group C and group D was higher than group B(P＜0.05). The expressions of Bax in group C and D was lower than that in group B(P＜0.05). Moreover, the expression of Bax in group D was lower than that in group C(P＜0.05). Conclusions: EGCG administered by subarachnoid injection can significantly improve locomotor recovery, and this neuroprotective effect may be related to the up-regulation of BDNF and GDNF, and the inhibition of apoptosis-related proteins.