氨基胍对大鼠急性脊髓损伤后脊髓水肿的作用机制研究

EFFECT OF AMINOGUANIDINE ON SPINAL CORD EDEMA OF ACUTE SPINAL CORD INJURY IN RATS

目的氨基胍(aminoguanidine,AG)能显著减轻脑外伤及中风动物模型脑水肿,提高神经功能恢复程度。探讨AG对大鼠急性脊髓损伤(spinal cord injury,SCI)后脊髓水肿的作用及相关机制。方法取成年雄性SD大鼠150只(体重230～255 g),分为对照组(A组,25只)、假损伤组(B组,25只)、SCI后未治疗组(C组,25只)和SCI后AG治疗组(75只);AG治疗组按给药剂量分为AG 75 mg/kg组(D组,25只)、AG 150 mg/kg组(E组,25只)和AG300 mg/kg组(F组,25只)。A组未行任何处理,B组仅行椎板切除术但不治疗;C、D、E、F组制备静压型大鼠SCI模型后,C组腹腔注射5%DMSO,D、E、F组腹腔注射相应剂量AG。于造模后0、12、24、48 h用干湿重法检测受损脊髓组织含水量以筛选最佳剂量,进一步用伊文思兰(Evans blue,EB)法评测血-脊髓屏障功能,用RT-PCR检测水通道蛋白4(aquaporins 4,AQP4)mRNA表达,Western blot和免疫组织化学染色检测AQP4蛋白表达。结果脊髓组织含水量检测示,E组在造膜后12、24、48 h对SCI后脊髓组织水肿有明显抑制作用(P<0.05),选择该剂量组用于后续实验。造模后12、24、48 h,E组EB含量明显低于C组(P<0.05),降低血-脊髓屏障通透性。RT-PCR检测结果示造模后12、24、48 h,B、E组AQP4 mRNA表达明显低于C组;Western blot检测示造模后24、48 h,B、E组AQP4蛋白表达明显低于C组;免疫组织化学染色示造模后48 h,B、E组AQP4蛋白表达明显低于C组,差异均有统计学意义(P<0.05);但各指标各时间点B、E组间比较,差异均无统计学意义(P>0.05)。结论急性SCI后大鼠经150 mg/kg AG治疗后,能降低AQP4表达,改善脊髓水肿,减轻损伤。

Objective Aminoguanidine （AG） can reduce brain edema and increase the recovery of neuron functions in surgical brain injury and stroke. To investigate the effect of AG on spinal cord injury （SCI） in rats and its mechanism. Methods A total of 150 adult male Sprague Dawley rats （weighing, 230-255 g） were divided into control group （group A, 25 rats without treatment）, the sham-operated group （group B, 25 rats undergoing laminectomy）, SCI group （group C, 25 SCI rats with injection of 5% DMSO）, SCI ＋ AG groups （groups D, E, and F, 25 SCI rats and AG injection of 75, 150, and 300 mg/kg, respectively）. The optimal dosage of AG was screened by dry-wet weight method with the percentage of water content at 0, 12, 24, and 48 hours after injury. The blood-spinal cord barriar permeability was further detected by Evans blue （EB） method, aquaporins 4 （AQP4） mRNA expression by RT-PCR, AQP4 protein expression by immunohistochemistry and Western blot. Results AG injection at dosage of 150 mg/kg can significantly reduce edema of spinal cords at 12, 24, and 48 hours after SCI （P 〈 0.05）, so 150 mg/kg was the optimal dosage. The EB content in group E was significantly lower than that in group C at 12, 24, and 48 hours after SCI, and the permeability of blood-spinal cord barrier was significantly decreased compared with group C （P 〈 0.05）. The AQP4 mRNA expressions in groups B and E were significantly lower than that in group C at 12, 24, and 48 hours after SCI （P 〈 0.05）. AQP4 protein expressions in groups B and E were significantly lower than that in group C at 24 and 48 hours after SCI （P 〈 0.05） by Western blot. Immunohistochemical staining revealed that AQP4 protein expression in group C was significantly higher than that in groups B and E （P 〈 0.05） at 48 hours after SCI, but no significant difference was found between group B and group E （P 〉 0.05）. Conclusion AG injection at dosage of 150 mg/kg can induce spinal cord edema and injury in rats, which could be correlated with the down-regulation of AQP4 expression.