丙戊酸钠联合甲强龙对大鼠脊髓损伤的影响及其机制

目的 探讨丙戊酸钠(VAP)联合甲强龙(MP)在大鼠脊髓损伤(SCI)后神经功能恢复中的作用及其机制。方法 选取8～10周龄雄性健康SD大鼠60只,按照数字表法随机分为假手术组、SCI组、VAP组、MP组和VAP+MP组,每组12只;各组大鼠再按照数字表法随机分为A组、B组2个亚组,每组6只。假手术组仅暴露脊髓、不做SCI处理,其余4组均采用改良Allen法进行大鼠SCI模型制备。假手术组、SCI组术后30 min、6 h、8 h和24 h腹腔注入生理盐水(剂量为30 mg/kg),24 h后每天腹腔注入相同剂量生理盐水,持续28 d;VAP组:分别于术后于30 min、6 h和24 h腹腔注入生理盐水(30 mg/kg),术后8 h腹腔注入VAP(30 mg/kg),24 h后每天腹腔注入相同剂量VAP,持续28 d;MP组:分别于术后30 min、6 h和24 h腹腔注入MP(30 mg/kg),术后8 h腹腔注入生理盐水(30 mg/kg),24 h后每天腹腔注入相同剂量生理盐水,持续28 d;VAP+MP组:分别于术后30 min、6 h和24 h腹腔注入MP(30 mg/kg),术后8 h腹腔注入VAP(30 mg/kg),24 h后每天腹腔注入相同剂量VAP,持续28 d。选取各组A亚组大鼠:术后第1、3、7、14、28天,分别采用脊髓损伤行为学运动功能(BBB)评分标准和改良Rivlin斜板试验评价SCI后各组大鼠肢体运动功能的恢复情况。选取各组B亚组大鼠:术后第7天手术切取SCI区域脊髓组织,HE染色观察各组大鼠脊髓组织形态变化,Nissl染色观察脊髓运动神经元情况并计算凋亡运动神经元数目,免疫组织化学染色半定量分析肿瘤坏死因子(TNF-α)、白细胞介素-1β(IL-1β)蛋白的相对表达情况,Western blot法检测凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关的X因子(Bax)、caspase-3的相对表达情况。结果 (1)组内比较:与术前相比,假手术组术后第1、3、7、14、28天BBB评分和Rivlin斜板试验结果差异均无统计学意义(P值均＞0.05);SCI组、VAP组、MP组、VAP +MP组术后不同时间点BBB评分和Rivlin斜板试验结果均明显降低,但随着时间延长,BBB评分和Rivlin斜板试验结果逐渐升高,差异均有统计学意义(P值均＜0.05)。组间比较:SCI组、VAP组、MP组、VAP+MP组SCI后BBB评分和Rivlin斜板试验结果均显著低于假手术组,VAP组、MP组、VAP+MP组的BBB评分和Rivlin斜板试验结果均高于SCI组,VAP+MP组BBB评分和Rivlin斜板试验结果均高于VAP组、MP组,差异均有统计学意义(P值均＜0.05)。(2)假手术及造模后第7天,HE染色观察组织学特征:假手术组脊髓组织形态正常;SCI组脊髓组织可见脊髓灰质出现大量炎性细胞因子浸润,细胞出血、水肿, 运动神经元坏死、凋亡明显;VAP组、MP组、VAP+MP组较SCI组大鼠脊髓组织可见出血水肿显著减轻,炎性细胞因子浸润显著减少,运动神经元溶解、凋亡减轻,其中VAP+MP组效果更加显著。SCI组、VAP组、MP组、VAP+MP组脊髓空洞面积均明显大于假手术组,VAP组、MP组、VAP+MP组脊髓空洞面积均明显小于SCI组,VAP+MP组脊髓空洞面积均小于VAP组和MP组,差异均有统计学意义(P值均＜0.05)。(3)假手术及造模后第7天,Nissl染色观察脊髓运动神经元数目:SCI组、VAP组、MP组、VAP+MP组大鼠脊髓组织正常运动神经元数目明显少于假手术组,VAP组、MP组、VAP+MP组正常运动神经元数目均高于SCI组,VAP+MP组正常运动神经元数目均高于VAP组、MP组,差异均有统计学意义(P值均＜0.05)。(4)假手术及造模后第7天,免疫组织化学检测TNF-α、IL-1β表达情况:SCI组、VAP组、MP组、VAP+M组TNF-α、IL-1β表达明显高于假手术组,VAP组、MP组、VAP+MP组TNF-α、IL-1β表达水平显著低于SCI组,VAP+MP组TNF-α、IL-1β表达明显低于VAP组和MP组,差异均有统计学意义 (P值均＜0.05)。(5)假手术及造模后第7天,Western blot法测试大鼠脊髓组织中凋亡蛋白表达情况:与假手术组比较,SCI组、VAP组、MP组、VAP+MP组凋亡相关蛋白Bcl-2、Bax和caspase-3的相对表达量明显增加;与SCI组比较,VAP组、MP组、VAP+MP组Bcl-2的相对表达量明显增加,Bax、caspase-3的相对表达量明显减少;VAP+MP组Bcl-2的相对表达量明显高于VAP组、MP组,Bax、caspase-3的相对表达量明显低于VAP组、MP组:差异均有统计学意义 (P值均＜0.05)。结论 VAP联合MP能够显著改善大鼠SCI后神经运动功能,其机制可能与抑制局部炎性反应、促进抗凋亡蛋白Bcl-2的表达和减少凋亡蛋白Bax、caspase-3的表达有关。

Experimental study on the effects of valproate sodium combined with methylprednisolone on spinal cord injury in rats

Objective This study aims to explore the role and potential mechanism of sodium valproate (VAP) combined with methylprednisolone (MP) in the recovery of nerve functions after spinal cord injury (SCI) in rats. Methods Sixty healthy male SD rats aged 8-10 weeks were selected and randomly divided into sham operation, SCI, VAP, MP, and VAP+MP groups via the number table method, and each group had 12 rats. The rats in each group were further randomly divided into two subgroups, group A and B, with six rats in each group. In the sham operation group, only the spinal cord was exposed, and no SCI treatment was performed. The four other groups were subjected to the modified Allen method to prepare rat SCI models. In the sham operation and SCI groups, 30 mg/kg normal saline was injected into the abdominal cavity 30 min, 6 h, 8 h, and 24 h after the operation, and the same dose of normal saline was injected into the abdominal cavity every day after 24 h for 28 days. In the VAP group, 30 mg/kg normal saline was injected into the abdominal cavity 30 min, 6 h, and 24 h after the operation, 30 mg/kg VAP was injected into the abdominal cavity 8 h after operation, and the same dose of VAP was injected into the abdominal cavity every day after 24 h for 28 days. In the MP group, 30 mg/kg MP was injected into the abdominal cavity 30 min, 6 h, and 24 h after the operation, 30 mg/kg normal saline was injected into the abdominal cavity 8 h after the operation, and the same dose of normal saline was injected into the abdominal cavity every day after 24 h for 28 days. In the VAP+MP group, 30 mg/kg MP was injected into the abdominal cavity 30 min, 6 h, and 24 h after the operation, 30 mg/kg VAP was injected into the abdominal cavity 8 h after the operation, and the same dose of VAP was injected into the abdominal cavity every day after 24 h for 28 days. Each group of rats in subgroup A was selected 1, 3, 7, 14, and 28 days after the operation. The motor function basso-beattie-bresnahan (BBB) score standard of SCI and the modified Rivlin inclined plate test were used to evaluate the limbs motor function recovery of rats in each group after SCI. The rats in subgroup B were selected, and their spinal cord tissue was surgically cut from the SCI area 7 days after the operation. The obtained tissues were stained with HE to observe their morphological changes. They were also stained with Nissl to examine the spinal cord motor neurons and calculate the number of apoptotic neurons. Histochemical staining was performed to analyze the protein expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) semiquantitatively. Western blot was performed to detect the relative expression of apoptosis-related proteins, namely, B lymphoma-2 (Bcl-2), Bcl-2-related factor X (Bax), and caspase-3. Results (1) In intragroup comparison, the BBB score and Rivlin inclined plate test results at 1, 3, 7, 28 days after the operation in the sham operation group did not significantly differ from those recorded before injury (all P values >0.05). SCI, VAP, and MP groups. The BBB scores and the Rivlin inclined plate test results at different time points after injury in the VAP+MP group significantly decreased. As time passed, the BBB score and the Rivlin inclined plate test results gradually increased, and the differences were statistically significant (all P values＜0.05). The BBB scores and the Rivlin oblique plate test results of the rats at different time points in each group: The BBB scores and the Rivlin inclined plate test results of SCI, VAP, MP and VAP+MP groups were significantly lower than those of the sham operation group. The BBB scores and the Rivlin inclined plate test results of the VAP, MP, and VAP+MP groups were higher than those of the SCI group. The BBB scores and the Rivlin inclined plate test results of the VAP+MP group were significantly higher than those of the VAP and MP groups (all P values＜0.05). (2) HE staining revealed that the morphological characteristics of the spinal cord in the sham operation group were normal. In the spinal cord tissue of the SCI group, a large degree of inflammatory cytokine infiltration in the spinal cord grey matter, cell hemorrhage, edema, and obvious neuronal necrosis and apoptosis were observed. The rats in the VAP, MP, and VAP+MP groups had significantly reduced hemorrhage, edema, inflammatory cytokine infiltration, neuronal dissolution, and apoptosis compared with those in the SCI group. The VAP+MP group had a more significant effect than the other groups. The syringomyelia area of the SCI, VAP, MP, and VAP+MP groups was significantly larger than that of the sham operation group. The syringomyelia area of the VAP, MP, and VAP+MP groups was significantly smaller than that of the SCI group. The syringomyelia area of VAP+MP group was significantly smaller than that of the VAP and MP groups (all P values＜0.05). (3) Nissl staining indicated that the number of normal motor neurons in the SCI, VAP, MP, and VAP+MP groups was significantly less than that in the sham operation group, the number of normal motor neurons in VAP, MP and VAP+MP groups was higher than that in SCI group, and the number of normal motor in VAP+MP group was significantly higher than that in the VAP and MP groups (all P values＜0.05). (4) Immunohistochemical detection showed that the expression levels of TNF-α and IL-1β in the SCI, VAP, MP, and VAP+MP groups were significantly higher than those in the sham operation, VAP, and MP groups. The expression levels of TNF-α and IL-1β in the VAP+MP group were significantly lower than those in the SCI group. The expression levels of TNF-α and IL-1β in the VAP+MP group were significantly lower than those in the VAP and MP groups (all P values<0.05). (5) Western blot indicated that the expression levels of Bcl-2, Bax, and caspase-3 in the SCI, VAP, MP, and VAP+MP groups compared with those in the sham operation group were significantly increased. The relative expression of Bcl-2 in the VAP, MP, and VAP+MP groups significantly increased compared with that in the SCI group, and the relative expression levels of Bax and caspase-3 significantly decreased compared with that in the SCI group. The relative expression of Bcl-2 in the VAP+MP group was significantly higher than that in the VAP and MP groupS. The relative expression levels of Bax and caspase-3 in the VAP+MP group were significantly lower than those in the VAP and MP groups. The differences were statistically significant (all P values＜0.05). Conclusions VAP combined with MP can significantly improve the neuromotor function of rats after SCI. The mechanism might be related to inhibiting local inflammatory responses, promoting the inhibition of Bcl-2 production, and reducing the expression of Bax and caspase-3.