目的:探讨吡拉西坦通过丝裂原活化蛋白激酶(MAPK)通路治疗大鼠脊髓损伤的机制。
方法:将54只健康的6周龄SD雌性大鼠分为假手术组,体重80~100g,脊髓损伤组和吡拉西坦组采用随机数字表法,每组18只大鼠。脊髓损伤组和吡拉西坦组采用打击乐器建立脊髓损伤模型,假手术组未损伤脊髓。吡拉西坦组按照5ml·kg-1标准经尾静脉注射吡拉西坦注射液,每天一次,连续3天;其他两组注射相同剂量的生理盐水,相同的频率和相同的持续时间。手术后1、3和7天处死大鼠,和巴索的变化,观察并比较了Beattie和Bresnahan(BBB)运动评分量表。采用酶联免疫吸附试验(ELISA)检测脊髓炎症因子,如白细胞介素-6(IL-6),白细胞介素-10(IL-10),白细胞介素-1β(白细胞介素-1β),坏死因子-α(IL-1β)和肿瘤坏死因子-α(TNF-α);HE染色观察脊髓损伤大鼠的形态学变化,免疫组化观察水通道蛋白4(AQP4)的表达水平。采用免疫印迹法(WB)观察大鼠脊髓损伤后MAPK信号通路的激活。
结果:假手术组1、3、7天BBB评分分别为21分。在脊髓损伤组中,得分为(1±1),(4±1)和(7±2);吡拉西坦组为(1±1),(5±1),(9±2),脊髓损伤组与假手术组比较差异有统计学意义(P<0.05)。HE染色显示假手术组未见异常。在脊髓损伤组中,术后1天出现脊髓组织出血和变性;术后3天出现片状坏死区,术后7天脊髓组织开始缓慢修复。在吡拉西坦组中,术后1天出血面积小于脊髓损伤组;术后3天,坏死面积减少,核消失范围减少;脊髓在手术后7天开始缓慢恢复。假手术组大鼠脊髓AQP4染色在造模后1、3、7天较弱,脊髓损伤组AQP4染色加深,面积增加,吡拉西坦组AQP4染色较脊髓损伤组轻,与假手术组相比,阳性细胞略有增加,染色稍深。在1、3和7天,脊髓损伤组IL-6、IL-10、IL-1β和TNF-α水平均高于假手术组和吡拉西坦组(P<0.05)。与脊髓损伤组相比,吡拉西坦组HE染色减少脊髓出血和坏死面积,免疫组化AQP4染色降低。WB结果表明,P-ERK,脊髓损伤组术后3天P-JNK和P-P38水平均高于假手术组和吡拉西坦组(P<0.05)。
结论:吡拉西坦不仅具有促进脊髓损伤后运动功能恢复的作用,而且在减少病变面积方面也显示出积极的治疗潜力,调节AQP4表达以减轻水肿,通过调节MAPK信号通路减轻炎症反应。
OBJECTIVE: To explore mechanism of
piracetam for the treatment of spinal cord injury in rats through mitogen-activated protein kinase (MAPK) pathway.
METHODS: Fifty-four healthy 6-week-old SD female rats with body weight of 80 to 100 g were divided into sham operation group, spinal cord injury group and
piracetam group by random number table method, with 18 rats in each group. Spinal cord injury model was established in spinal cord injury group and
piracetam group using percussion apparatus, while sham operation group did not damage spinal cord.
Piracetam group was injected with
piracetam injection through tail vein according to 5 ml·kg-1 standard, once a day for 3 days;the other two groups were injected with normal saline at the same dose, the same frequency and the same duration. The rats were sacrificed at 1, 3, and 7 days after surgery, and changes of Basso, Beattie and Bresnahan (BBB) locomotor rating scale was observed and compared. Enzyme-linked immunosorbent assay (ELISA) was used to detect spinal cord inflammatory factors, such as interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-1β (interleukin-1β), necrosis factor-α (IL-1β) and tumor necrosis factor-α (TNF-α);HE staining was used to observe morphological changes of rats with spinal cord injury, and immunohistochemistry was used to observe expression level of aquaporin 4 (AQP4). The activation of MAPK signaling pathway in spinal cord of rats after spinal cord injury was observed by western blotting (WB).
RESULTS: BBB scores of sham operation group on 1, 3 and 7 day were 21 points. In spinal cord injury group, the scores were (1±1), (4±1) and (7±2);
piracetam group was (1±1), (5±1), (9±2), respectively;the difference between spinal cord injury group and sham operation group was statistically significant (P<0.05). HE staining showed that no abnormality was found in sham operation group. In spinal cord injury group, bleeding and degeneration of spinal cord tissue appeared at 1 day after operation; flaky necrotic areas were appeared in spinal cord at 3 days after surgery, and spinal cord tissue began to slowly repair at 7 days after surgery. In piracetam group, the bleeding area was less than that of spinal cord injury group at 1 day after surgery;at 3 days after operation, the necrotic area was reduced and the range of nuclear disappearance was reduced; and the spinal cord began to recover slowly at 7 days after surgery. AQP4 staining of spinal cord of rats in sham operation group was weak at 1, 3 and 7 days after modeling, AQP4 staining was deepened and area increased in spinal cord injury group, AQP4 staining of piracetam group was lighter than that of spinal cord injury group, and the positive cells were slightly increased and the staining was slightly darker than that of sham operation group. At 1, 3 and 7 days, the level of IL-6, IL-10, IL-1β and TNF-α in spinal cord injury group were higher than those in sham operation group and piracetam group(P<0.05). Compared with spinal cord injury group, the area of spinal cord bleeding and necrosis were decreased by HE staining in piracetam group, and AQP4 staining was decreased by immunohistochemistry. WB results showed that P-ERK, P-JNK and P-P38 levels in spinal cord injury group at 3 days were higher than those in sham operation group and piracetam group(P<0.05).
CONCLUSIONS: Piracetam not only showed significant effect in promoting motor function recovery after spinal cord injury, but also showed positive therapeutic potential in reducing lesion area, regulating AQP4 expression to reduce edema, and reducing inflammatory response by regulating MAPK signaling pathway.