脊髓损伤组织匀浆通过甲酰基肽受体2促进神经元突起的生长

Spinal cord homogenate promotes neurite outgrowth through activation of formyl peptide receptor 2 after spinal cord injury

背景:前期研究观察到神经干细胞新分化的神经元表达甲酰基肽受体2,并证实甲酰基肽受体2能促进神经干/祖细胞迁移,诱导向神经元分化。脊髓损伤组织中存在甲酰基肽受体2配体,然而不同的配体与甲酰基肽受体2结合可能导致不同、甚至相反的生物学效应。目的:探讨脊髓损伤产生的配体与甲酰基肽受体2作用后对神经元突起生长的影响。方法:采用酶消化法提取胎鼠大脑皮质神经元;制备SD大鼠脊髓损伤模型,提取损伤脊髓组织匀浆。①实验分组1:观察甲酰基肽受体2激活对神经元突起的影响,分组如下:对照组、甲酰基肽受体2阻断剂组(即添加WRW4)、脊髓匀浆组、脊髓匀浆+WRW4组;②实验分组2:观察甲酰基肽受体2激活后AKT和ERK信号通路阻断对神经元突起的影响,分组如下:对照组、AKT和ERK信号通路阻断剂组(即添加Ly294002+PD98059)、脊髓匀浆组、脊髓匀浆+Ly294002+PD98059组。神经元细胞贴壁24 h后,按上述分组处理7 d,免疫荧光染色共聚焦显微镜观察脊髓匀浆激活甲酰基肽受体2对神经元突起的影响;按上述分组处理30 min,Western blotting检测磷酸化蛋白水平;按上述分组处理24 h,Western blotting检测F-actin水平,观察在甲酰基肽受体2特异性阻断剂WRW4存在的情况下,对MAPK和PI3K/Akt通路中关键蛋白磷酸化的影响。结果与结论:①脊髓损伤组织匀浆液能够使神经元突起长度、初级分枝数、分枝节点数显著增加,这种增加效应大部分被甲酰基肽受体2受体特异性阻断剂WRW4阻断;②脊髓损伤组织匀浆液能够使神经元中ERK1/2和Akt的磷酸化增加,这种效应能够被WRW4阻断;③Akt信号通路阻断剂Ly294002和Erk信号通路阻断剂PD98059能够阻断脊髓匀浆的促进作用;④脊髓损伤组织匀浆能够使F-actin表达量明显增加,这种效应能够被甲酰基肽受体2特异性阻断剂WRW4所阻断;⑤这些实验结果说明,脊髓匀浆液能够通过激活甲酰基肽受体2促进神经元突起生长,这种作用机制可能与ERK1/2和Akt的磷酸化增加相关。

BACKGROUND:Previous studies have observed the expression of formyl peptide receptor 2 in newly differentiated neurons from neural stem cells and confirmed that formyl peptide receptor 2 can promote the migration of neural stem/progenitor cells and induce them to differentiate into neurons.Formyl peptide receptor 2 ligands are present in damaged spinal cord tissues,but the binding of different ligands with FPR2 may lead to different and even opposite biological effects.OBJECTIVE:To investigate the neurite outgrowth after the binding of the ligands produced following spinal cord injury with formyl peptide receptor 2.METHODS:The fetal rat cerebral cortical neurons were extracted by enzymatic digestion.Spinal cord injury models were established in Sprague-Dawley rats,and the injured spinal cord homogenate was extracted.(1)Experiment 1:To observe the effect of the activation of formyl peptide receptor 2 on neurite outgrowth,the cells were divided into control group,formyl peptide receptor 2 blocker group(addition of WRW4),spinal cord homogenate group,spinal cord homogenate+WRW4 group.(2)Experiment 2:To observe the effect of blockade of AKT and ERK signaling pathways on neurite outgrowth after activation of formyl peptide receptor 2,the cells were divided into control group,AKT and ERK signaling pathway blocker group(addition of Ly294002+PD98059),spinal cord homogenate group,spinal cord homogenate+Ly294002+PD98059 group.After 24 hours of culture,adherent neurons were treated with above-mentioned regimens for 7 days.Immunofluorescence staining with confocal microscope detection was used to observe the effect of spinal cord homogenate on neurite outgrowth via the activation of formyl peptide receptor 2.The cells were treated by the above-mentioned regimens for 30 minutes and phosphorylated protein levels were detected by western blot.The cells were treated with the above-mentioned regimens for 24 hours,and western blot assay was used to detect F-actin levels and observe the phosphorylation of key proteins in MAPK and PI3K/Akt pathways with the presence of formyl peptide receptor 2 specific blocker WRW4.RESULTS AND CONCLUSION:WRW4 could eliminate the effects of injured spinal cord homogenates on neurite outgrowth,including neurite length,the number of primary neurites,and the number of branch points.Spinal cord homogenate increased the phosphorylation of ERK1/2 and Akt in neurons,whereas this effect could be blocked by WRW4.Ly294002 and PD98059 could also eliminate the effects of homogenates on the neurite outgrowth.Spinal cord homogenate significantly increased the expression of F-actin in neurons,but this effect was blocked by WRW4.These results suggest that spinal cord homogenates can expedite neurite outgrowth by activating formyl peptide receptor 2,which may be related to the increased phosphorylation of ERK1/2 and Akt.