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中华神经创伤外科电子杂志

中华神经创伤外科电子杂志 ›› 2024, Vol. 10 ›› Issue (02) : 65 -70. doi: 10.3877/cma.j.issn.2095-9141.2024.02.001

基础研究

法舒地尔对大鼠急性脊髓损伤后神经细胞凋亡和BCL-2蛋白表达水平的影响
李景德1, 张保艳2, 卢培刚3,†(), 李博3   
  1. 1. 250031　济南，济南市第四人民医院神经外科
    2. 250031　济南，山东第一医科大学第三附属医院神经内科
    3. 250031　济南，解放军联勤保障部队第九六〇医院神经外科
  • 收稿日期:2023-08-07 出版日期:2024-04-15
  • 通信作者: 卢培刚

Effect of Fasudil on neuronal apoptosis and expression of BCL-2 protein after acute spinal cord injury in rats

Jingde Li1, Baoyan Zhang2, Peigang Lu3,†(), Bo Li3   

  1. 1. Department of Neurosurgery, the Fourth People's Hospital of Ji'nan, Ji'nan 250031, China
    2. Department of Neurology, the Third Affiliated Hospital of Shandong First Medical University, Ji'nan 250031, China
    3. Department of Neurosurgery, the 960th Hospital of the Joint Logistics Support Force of the People's Liberation Army, Ji'nan 250031, China
  • Received:2023-08-07 Published:2024-04-15
  • Corresponding author: Peigang Lu
  • Supported by:
    National Natural Science Foundation of China General Project(81471214)
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李景德, 张保艳, 卢培刚, 李博. 法舒地尔对大鼠急性脊髓损伤后神经细胞凋亡和BCL-2蛋白表达水平的影响[J]. 中华神经创伤外科电子杂志, 2024, 10(02): 65-70.

Jingde Li, Baoyan Zhang, Peigang Lu, Bo Li. Effect of Fasudil on neuronal apoptosis and expression of BCL-2 protein after acute spinal cord injury in rats[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2024, 10(02): 65-70.

目的

探讨法舒地尔通过抑制Rho激酶活性对急性脊髓损伤（SCI）后神经细胞凋亡和BCL-2蛋白表达水平的影响。

方法

选取清洁级健康成年雌性Wistar大鼠90只，随机分为假手术组、SCI对照组和SCI治疗组，每组30只。SCI对照组和SCI治疗组均选取大鼠脊髓T10节段为损伤节段，剪断脊髓的后3/4深度损伤；假手术组只行椎板切除术，不进行脊髓的损伤。3组分别于造模后6 h和1、3、7、14 d各取6只大鼠，采用开放场地试验（BBB）运动功能评分法对大鼠的后肢运动功能进行评价；采用TUNEL法检测大鼠SCI局部的神经细胞凋亡；采用免疫组织化学法检测BCL-2蛋白的表达水平。

结果

大鼠SCI模型制备后，假手术组大鼠双后肢活动正常，各时间点的BBB评分均为21分；SCI对照组和SCI治疗组在SCI后6 h和1 d的BBB评分均为0分，而后逐渐增高。SCI治疗组SCI后3、7、14 d的BBB评分均高于SCI对照组，差异均有统计学意义（P<0.05）。SCI后6 h及1、3、7、14 d，SCI治疗组的TUNEL阳性细胞数少于SCI对照组，BCL-2蛋白阳性细胞数多于SCI对照组，差异均有统计学意义（P<0.05）。

结论

SCI后早期应用Rho激酶抑制剂法舒地尔可以抑制神经细胞凋亡，促进BCL-2蛋白的表达，有助于脊髓神经功能恢复。

关键词: 急性脊髓损伤, 法舒地尔, BCL-2, 细胞凋亡
Objective

To explore the effects of the drug Fasudil on neuronal apoptosis and BCL-2 protein expression levels after spinal cord injury (SCI) by inhibiting Rho-kinase activity.

Methods

Ninety clean and healthy adult female Wistar rats were selected and randomly divided into sham surgery group, SCI control group, and SCI treatment group, with 30 rats in each group. Both SCI control group and SCI treatment group selected the T10 segment of the rat spinal cord as the injury segment, and cut the posterior 3/4 depth of the SCI; The sham surgery group only underwent laminectomy without SCI. Six rats were selected from 3 groups at 6 h and 1, 3, 7, and 14 d after modeling, and their hind limb motor function was evaluated using the Basso, Beattie and Bresnehan open field locomotor test (BBB) motor function scoring method; The TUNEL method was used to detect neuronal apoptosis in the local area of SCI in rats; Immunohistochemical method was used to detect the expression level of BCL-2 protein.

Results

After the preparation of the SCI model in rats, the sham surgery group showed normal activity of both hind limbs and a BBB score of 21 points at each time point; The BBB scores of both SCI control group and SCI treatment group were 0 points at 6 h and 1 d after SCI, and gradually increased thereafter. The BBB scores of SCI treatment group at 3, 7, and 14 d after SCI were higher than those of SCI control group, and the differences were statistically significant (P<0.05). At 6 h, 1, 3, 7, and 14 d after SCI, the number of TUNEL positive cells in SCI treatment group was lower than that in SCI control group, and the number of BCL-2 protein positive cells was higher than that in SCI control group, with statistical significance (P<0.05).

Conclusion

Early application of the Rho-kinase inhibitor Fasudil after SCI inhibits neuronal apoptosis and promotes BCL-2 protein expression, which contributes to the recovery of spinal cord neurological function.

Key words: Acute spinal cord injury, Fasudil, BCL-2, Cell apoptosis
表1 3组大鼠SCI后不同时间点BBB评分比较（分，±s）
Tab.1 Comparison of BBB scores at different time periods after SCI in three groups of rats (score, Mean±SD)
组别 只数 6 h（n=6） 1 d（n=6） 3 d（n=6） 7 d（n=6） 14 d（n=6）
假手术组 30 21 21 21 21 21
SCI对照组 30 0 0 0a 1.42±0.20a 3.92±0.58a
SCI治疗组 30 0 0 1.33±0.41ab 3.17±0.52ab 8.17±0.61ab
F值       50.333 89.142 146.337
P值       <0.001 <0.001 <0.001
图1 2组大鼠急性SCI后神经细胞凋亡染色图（TUNEL染色，×100）A~E：SCI对照组（A：SCI后6 h；B：SCI后1 d；C：SCI后3 d；D：SCI后7 d；E：SCI后14 d）；F~J：SCI治疗组（F：SCI后6 h；G：SCI后1 d；H：SCI后3 d；I：SCI后7 d；J：SCI后14 d）
Fig.1 Staining plots of neuronal apoptosis after acute SCI in two groups of rats (TUNEL staining, ×100)
表2 2组大鼠SCI后各时间点TUNEL阳性细胞数比较（±s）
Tab.2 Comparison of TUNEL positive cells at different time points after SCI in two groups of rats (Mean±SD)
组别 只数 6 h（n=6） 1 d（n=6） 3 d（n=6） 7 d（n=6） 14 d（n=6）
SCI治疗组 30 2.17±0.75 56.50±9.40 96.33±12.32 54.0±13.33 44.17±7.78
SCI对照组 30 4.16±1.60 82.17±7.36 182.67±16.98 79.17±12.32 58.50±12.44
t值   6.168 11.780 22.540 7.595 5.349
P值   <0.001 <0.001 <0.001 <0.001 <0.001
图2 2组大鼠急性SCI后BCL-2免疫组织化学染色图（EnVision，×200）A~E：SCI对照组（A：SCI后6 h；B：SCI后1 d；C：SCI后3 d；D：SCI后7 d；E：SCI后14 d）；F~J：SCI治疗组（F：SCI后6 h；G：SCI后1 d；H：SCI后3 d；I：SCI后7 d；J：SCI后14 d）
Fig.2 Immunohistochemical staining of BCL-2 after acute SCI in two groups of rats (EnVision, ×200)
表3 2组大鼠SCI后各时间点BCL-2蛋白阳性细胞数比较（±s）
Tab.3 Comparison of BCL-2 protein positive cells count at different time points after SCI between two groups of rats (Mean±SD)
组别 只数 6 h（n=6） 1 d（n=6） 3 d（n=6） 7 d（n=6） 14 d（n=6）
SCI治疗组 30 13.33±1.37 26.17±1.94 38.17±1.47 33.0±2.45 25.50±1.38
SCI对照组 30 6.50±1.05 17.17±1.60 22.83±1.17 27.67±2.25 20.33±1.63
t值   21.670 19.601 44.722 8.776 13.260
P值   <0.001 <0.001 <0.001 <0.001 <0.001
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