瑞香素对脊髓损伤大鼠运动功能改善作用的TRL4/NF-κB信号通路机制研究

Effects of daphnetin on motor function of spinal cord injury based on the TRL4/NF-κB signaling pathway in rats

目的探讨瑞香素激活Toll样受体4/核转录因子kappa B(Toll like receptor 4/NF-κB,TRL4/NF-κB)信号通路对大鼠脊髓损伤(spinal cord injury,SCI)后神经再生及功能恢复的影响,为促进SCI的分子机制研究提供依据。方法建立脊髓损伤实验大鼠模型,并分别于术后1 d、8 d、14 d、21 d和30 d,采用Basso Beattie Bresnahan(BBB)运动功能评分和联合行为评分法(combine behavioral score,CBS)评分测试评价各组SCI大鼠治疗后的神经功能;采用相关试剂盒检测脊髓组织中超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性及丙二醛(maleicdialdehyde,MDA)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、白介素-1β(interleukiN-1β,IL-1β)、白介素-6(interleukin 6,IL-6)、白介素-10(interleukin 10,IL-10)的表达量变化;并采用蛋白质印迹法(Western blot)检测术后各组脊髓组织中对实验大鼠脊髓组织中BCL2-Associated X的蛋白质(BCL2-Associated X Protein,Bax)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Caspase-3、细胞色素C(cytochrome C,Cyt-c)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、酪氨酸激酶受体B(tyrosine kinase receptor B,TrkB)、磷酸化酪氨酸激酶受体B(phospho-tyrosine kinase receptor B,p-TrkB)、TOLL样受体4重组蛋白(recombinant toll like receptor 4,TRL4)和核因子-κB p65(nuclear factor-kappa b p65,NF-κB p65)水平的影响。结果瑞香素能够显著性改善实验大鼠BBB评分和CBS评分结果,且联合用药治疗组效果优于单一药物的疗效。此外,瑞香素能够改善实验大鼠脊髓组织中SOD、CAT、GSH-Px活性和MDA含量,能够降低脊髓组织中TNF-α、IL-1β、IL-6和IL-10含量;Western blot实验结果表明瑞香素对脊髓损伤诱发的凋亡相关蛋白异常表达具有显著的调节作用,并能够显著性调节BDNF/TrkB信号通路和TRL4/NF-κB信号通路。结论瑞香素改善脊髓损伤的作用机制与是激活TRL4/NF-κB信号密切相关。

Objective To investigate the effect of daphnetin on the TRL4/NF-κB signaling pathway involved in nerve regeneration and functional recovery after spinal cord injury(SCI)in rats.This study aimed to provide a basis for study of the molecular mechanism of SCI.Methods A laminectomy was performed at the T9 level and the exposed dorsal surface of the spinal cord was subjected to a contusion injury as established a spinal cord injury model.On the 1st,8th,14th,21st,and 30th days after the operation,the Basso Beattie Bresnahan motor function score and combine behavioral score were used to evaluate neurological function of rats with SCI in each group.The activity of SOD,CAT,and GSH PX enzymes,and MDA,TNF-α,IL-1β,IL-6,and IL-10 expression were detected by kits.Western blotting was used to determine protein levels of Bax,Bcl-2,caspase-3,Cyt-C,BDNF,TrkB,and p-TrkB in the spinal cord tissue of experimental rats.Results The BBB and CBS scores were significantly improved by daphnetin(P<0.05),and the effect of the high daphnetin group was better than that in the low daphnetin group.Additionally,daphnetin improved the activity of SOD,CAT,and GSH PX and MDA levels in the spinal cord of experimental rats(P<0.05),and reduced TNF-α,IL-1β,IL-6,and IL-10 levels in the spinal cord(P<0.05).Western blot showed that daphnetin significantly regulated abnormal expression of apoptosis-related proteins and the BDNF/TrkB signaling pathway.Conclusions Daphnetin may play a role in spinal cord injury by activating TRL4/NF-κB signaling.