Necrostatin-1通过抑制细胞凋亡及M1型小胶质/巨噬细胞极化促进小鼠脊髓损伤后运动功能恢复

Necrostatin-1 promotes locomotor recovery after spinal cord injury through inhibiting apoptosis and M1 polarization of microglia/macrophage in mice

目的研究坏死抑制蛋白1(necrostatin-1)对小鼠脊髓损伤(SCI)的保护作用,并探讨该作用与细胞凋亡及M1型小胶质/巨噬细胞介导的促炎性免疫的相关性。方法将雄性C57BL/6小鼠随机分为空白对照组、necrostatin-1对照组、脊髓损伤(SCI)模型组、SCI后necrostatin-1治疗组,每组20只。各组小鼠在造模后1、3、5、7、10、14 d,进行旷场试验BMS评分及标准化尾高指数测定,以评价小鼠SCI后的运动功能恢复;于损伤后1、3、7、14 d,TUNEL染色观察脊髓组织的细胞凋亡情况;损伤后14 d,采用Western blot法及免疫组织化学染色检测脊髓组织中M1型小胶质/巨噬细胞标志物诱导型一氧化氮合酶(iNOS)的水平,采用实时定量PCR检测脊髓组织中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-18、IL-4、IL-5及IL-10的mRNA水平。结果Necrostatin-1可明显促进小鼠SCI后运动功能恢复,减少SCI区周围细胞凋亡;降低M1型小胶质/巨噬细胞标志物iNOS的蛋白表达及iNOS阳性小胶质/巨噬细胞数量,下调促炎细胞因子TNF-α、IL-18及IL-1β的转录水平,同时促进抑炎性细胞因子IL-4、IL-5及IL-10的转录。结论Necrostatin-1可通过抑制细胞凋亡及抑制炎性免疫反应显著改善小鼠SCI后运动功能的恢复。

Objective To investigate the effect of necrostatin-1 on locomotor recovery after spinal cord injury(SCI)in mice,and to explore the role of apoptosis and M1 type-microglia/macrophage-mediated pro-inflammation in the protective effect.Methods Male C57 BL/6 mice were randomly divided into four groups:control group,necrostatin-1 group,SCI model group,necrostatin-1-treated group after SCI,with20 mice in each.For SCI model group,mice were anesthetized with10 g/L pentobarbital sodium with a dose of8 mL/kg.After skin disinfection,T8 laminectomy was performed under operating microscope,and the T8 spinal cord was clearly revealed.The injury model was established with a device designed by our own with the parameter at0.2 mm-width for20 seconds.Manual urination was performed once a day.For necrostatin-1-treated group after SCI,7.8 mg/kg of necrostatin-1 was intravenously administrated at the1,2,and3 days after SCI.For necrostatin-1 group,necrostatin-1 was intravenously injected for three days.Basso Mouse Scale(BMS)score and standardized rump-height index were used to evaluate locomotor function at1-,3-,5-,7-,10-and14-day after injury.To observe cell apoptosis in injured cord,TUNEL staining was performed at1-,3-,7-,and14-day after injury.Western blot and immunohistochemical staining were performed to detect the expression of inducible nitric oxide synthase(iNOS),a classical marker of M1 type microglia/macrophage.Real time quantitative PCR was used to detect mRNA levels of TNF-α,interleukin-1β(IL-1β),IL-18,IL-4,IL-5,and IL-10.Results Necrostatin-1 significantly promoted the locomotor recovery in mice after SCI,reduced cell apoptosis around the SCI area;decreased the protein expression of M1 type microglia/macrophage marker iNOS and the number of iNOS-positive microglia/macrophage,and down-regulated the transcription levels of pro-inflammatory cytokines TNF-α,IL-18,and IL-1β,while promoting the transcription of anti-inflammatory cytokines IL-4,IL-5,and IL-10.Conclusion Necrostatin-1 significantly promotes locomotor function recovery after SCI in mice by reducing the number of apoptotic cells and inhibiting M1 microglia/macrophages-mediated pro-inflammatory factors.