C5a受体拮抗剂对小鼠脊髓损伤后后肢功能的影响及作用机制

Effect and mechanism of Complement 5a receptor antagonist on hindlimb function after spinal cord injury in mice

目的观察补体裂解因子C5a受体拮抗剂(C5a receptor antagonist,C5aRA)对小鼠脊髓损伤后炎症反应及功能变化的干预效果,探讨C5a在脊髓损伤后的作用机制。方法利用成年雌性C57BL/6J小鼠建立脊髓夹伤模型,在术前45 min经腹腔注入C5aRA AcF-[OP(D-Cha)WR](PMX53),同时建立甲基强的松龙(methylprednisolone,MP)注射组、生理盐水注射组和假手术组对照。应用ELISA、RT-PCR观察炎症因子TNF-α及IL-1β表达的变化,通过流式细胞方法检测小胶质细胞百分比的变化,并进行小鼠后肢的行为学评分(mouse scale for locomotion,BMS)。结果生理盐水组小鼠脊髓于伤后1 h即出现炎症因子的明显升高,TNF-α在1 h达峰值,IL-1β在12 h达峰值,后下降,到72 h两者均已接近基线值;小胶质细胞百分比在SCI后1 d即出现升高,3 d较明显,7 d达峰,14 d已下降,但仍高于正常水平。与生理盐水组相较,应用C5aRA特异性抑制C5a后,损伤早期的炎症因子表达水平及炎症细胞增殖均明显被抑制,BMS评分显著优于生理盐水组(P<0.05),但其对炎症反应的抑制作用略弱于MP组(P<0.05)。结论应用C5aRA特异性拮抗C5a可以抑制脊髓损伤后早期炎症因子TNF-α、IL-1β表达及炎症细胞小胶质细胞活化增殖,改善后肢的行为学评分,提示C5a通过增强脊髓损伤后早期炎症反应参与了脊髓损伤后的继发性损伤。

Objective To observe the effect of Complement 5a receptor antagonist（C5aRA） in the early inflammatory reaction and outcome of hindlimb function after spinal cord injury（SCI） in mice,and to investigate the role of complement C5a after spinal cord injury.Methods Totally 173 female C57BL/6J mice were randomly divided into sham operation,C5aRA group,methylprednisolone（MP） group,and normal saline group.Another 9 mice with no any treatment served as normal control.The mice of C5aRA group received an intraperitoneal injection of C5aRA peptides（1.0 mg/kg） in 45 min before,and 24 h after SCI establishment.MP（30 mg/kg） was given i.p.to the mice of this group followed by another 4 injections in every 6 h.Normal saline group received an injection of normal saline.Tumor necrosis factor alpha（TNF-α） and interleukin 1β（IL-1β） in injured spinal cord were measured by ELISA and RT-PCR.Microglia was examined by flow cytometry,and hindlimb locomotion was observed the by Mouse Scale for Locomotion（BMS）.Results TNF-α and IL-1β were increased greatly at 1 h following SCI,with the peak at 1 h and 12 h respectively,and nearly returned to the normal level at 72 h.The percentage of activated microglial cells was raised at 1 d after SCI,with obvious increase at 3 d and the peak at 7.At 14 d it was decreased but still higher than the normal level.The level of TNF-α,IL-1β and microglia percentage of the C5aRA group was obviously lower than that of the NS group（P〈0.05）,although significantly higher than the MP group（P〈0.05）.Conclusion C5aRA suppresses early inflammatory reaction and improves BMS score after SCI in mice.C5a may participate in the secondary damage process through up-regulating the early inflammation reaction after SCI.