吡拉西坦通过MAPK通路治疗大鼠脊髓损伤的疗效观察与机制研究
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作者Author单位AddressE-Mail
董博 DONG Bo 西安交通大学第二附属医院骨科, 陕西 西安 710048 Department of Orthopaedics, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710048, Shaanxi, China  
李越 LI Yue 陕西中医药大学第一临床医学院, 陕西 咸阳 712000 The First Clinical College of Medicine, Shaanxi University of Traditional Chinese Medicine, Xianyang 712000, Shaanxi, China  
李迎春 LI Ying-chun 陕西中医药大学第一临床医学院, 陕西 咸阳 712000 The First Clinical College of Medicine, Shaanxi University of Traditional Chinese Medicine, Xianyang 712000, Shaanxi, China  
王桐 WANG Tong 陕西中医药大学第一临床医学院, 陕西 咸阳 712000 The First Clinical College of Medicine, Shaanxi University of Traditional Chinese Medicine, Xianyang 712000, Shaanxi, China  
梁壮 LIANG Zhuang 陕西中医药大学第一临床医学院, 陕西 咸阳 712000 The First Clinical College of Medicine, Shaanxi University of Traditional Chinese Medicine, Xianyang 712000, Shaanxi, China  
贺西京 HE Xi-jing 西安交通大学第二附属医院骨科, 陕西 西安 710048 Department of Orthopaedics, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710048, Shaanxi, China xijing_h@vip.tom.com 
期刊信息:《中国骨伤》2024年,第37卷,第6期,第591-598页
DOI:10.12200/j.issn.1003-0034.20230540
基金项目:陕西省重点研发计划项目"两链"融合重点专项(编号:2021LLRH-08);陕西省教育厅青年创新团队计划(编号:22JP018);秦都区2022年科技计划项目(编号:QK2022sf04)
中文摘要:

目的: 探讨吡拉西坦通过丝裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)通路治疗脊髓损伤大鼠的作用机制。

方法: 将54只体重为80~100 g的6周龄SD雌性健康大鼠采用随机数字表法分为假手术组、脊髓损伤组、吡拉西坦组,每组18只。脊髓损伤组、吡拉西坦组使用打击器建立脊髓损伤模型,假手术组不损伤脊髓。吡拉西坦组按照5 ml·kg-1标准予尾静脉注射吡拉西坦,连续干预3 d,每日1次;其他2组注射等剂量、等次数、等时长的生理盐水。分别于术后1、3、7 d处死大鼠并取材,观察并比较大鼠脊髓损伤行为学评分(Basso,Beattie and Bresnahan locomotor rating scale,BBB)的变化,使用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测脊髓炎症因子白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)、白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平变化,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(aquaporin 4,AQP4)表达水平,蛋白质免疫印迹法(western blotting,WB)观察脊髓损伤后MAPK信号通路在大鼠脊髓中的激活状态。

结果: 假手术组1、3、7 d的BBB评分均为21分;脊髓损伤组分别为(1±1)、(4±1)、(7±2)分;吡拉西坦组分别为(1±1)、(5±1)、(9±2)分;脊髓损伤组与假手术组比较,差异有统计学意义(P<0.05);吡拉西坦组与脊髓损伤比较,差异有统计学意义(P<0.05)。HE染色结果显示,假手术组形态未见异常。脊髓损伤组在术后1 d时,脊髓组织出现出血、变性;术后3 d,脊髓组织内出现片状坏死区;术后7 d,脊髓组织开始缓慢修复。吡拉西坦组术后1 d,脊髓损伤出血面积较脊髓损伤组减小;术后3 d,坏死区域较脊髓损伤组减少,细胞核消失范围较脊髓损伤组缩小;术后7 d,脊髓开始缓慢恢复。假手术组大鼠造模后1、3、7 d脊髓组织中AQP4染色较淡;脊髓损伤组AQP4染色加深,面积增大;吡拉西坦组AQP4染色较脊髓损伤组变淡,较假手术组阳性细胞略增多,染色微深。造模第1、3、7天,脊髓损伤组IL-6,IL-10,IL-1β和TNF-α水平高于手术组和吡拉西坦组(P<0.05)。与脊髓损伤组比,吡拉西坦组HE染色脊髓出血及坏死区域面积减小,免疫组化显示AQP4染色变淡。WB结果显示,造模第3天,脊髓损伤组p-ERK,p-JNK和p-p38水平高于假手术组和吡拉西坦组(P<0.05)。

结论: 吡拉西坦不仅在促进脊髓损伤后的运动功能恢复方面表现出显著效果,而且在减少病变面积、调节AQP4表达以减轻水肿,以及通过调控MAPK信号通路降低炎症反应方面均显示出积极的治疗潜力。
【关键词】吡拉西坦  脊髓损伤  MAPK信号通路
 
Therapeutic effect and mechanism of piracetam for the treatment of spinal cord injury in rats through MAPK pathway
ABSTRACT  

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).

Conclusion 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.
KEY WORDS  Piracetam  Spinal cord injury  MAPK signaling pathway
 
引用本文,请按以下格式著录参考文献:
中文格式:董博,李越,李迎春,王桐,梁壮,贺西京.吡拉西坦通过MAPK通路治疗大鼠脊髓损伤的疗效观察与机制研究[J].中国骨伤,2024,37(6):591~598
英文格式:DONG Bo,LI Yue,LI Ying-chun,WANG Tong,LIANG Zhuang,HE Xi-jing.Therapeutic effect and mechanism of piracetam for the treatment of spinal cord injury in rats through MAPK pathway[J].zhongguo gu shang / China J Orthop Trauma ,2024,37(6):591~598
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