程序性细胞死亡配体-1通过调节T细胞免疫在脊髓损伤小鼠模型中发挥神经保护作用

Programmed cell death ligand-1 exerts neuroprotective effects in a mouse model of spinal cord injury by modulating T cell immunity

目的探究程序性细胞死亡配体-1(programmed cell death ligand 1,PD-L1)通过调节T细胞免疫以及PI3K/Akt/mTOR信号通路在脊髓损伤(SCI)小鼠模型中发挥保护作用。方法采用C57BL/6小鼠建立SCI模型,分为假手术组(Sham)、SCI组、SCI+PD-L1抗体组(SCI+Ab)和SCI+PD-L1蛋白组(SCI+PRO)。C57BL/6小鼠和PD-L1基因敲除小鼠进行SCI造模,分为假手术组(Sham WT)、PD-L1敲除假手术组(Sham PD-L1 KO)、SCI模型组(SCI WT)、PD-L1敲除SCI模型组(SCI PD-L1 KO)。应用Western blotting和qRT-PCR检测SCI后不同时间点脊髓组织中PD-L1的表达;通过Basso小鼠运动量表(BMS)评估小鼠运动功能;利用qRT-PCR和流式细胞术分析SCI后炎症因子水平和T细胞亚群的变化;Western blotting检测PI3K/Akt/mTOR信号通路活化情况。结果小鼠经SCI造模后脊髓组织中PD-L1表达上调,于第7天达峰值。与SCI PD-L1 KO组相比,SCI WT组的小鼠在SCI后7 d、14 d和28 d时BMS评分均显著升高(P<0.05),造模后第7天炎症因子IL-1α、IL-2、IFN-γ和TNF-α水平显著降低(P<0.05);与SCI+PBS组相比,SCI+PRO组Foxp3水平显著升高,Th1和Th17细胞水平显著降低,Th2和Treg细胞水平显著升高(P<0.05);与SCI PD-L1 KO组相比,SCI WT组小鼠PI3K/Akt/mTOR信号通路磷酸化程度显著升高(P<0.05);与SCI+PBS组和SCI+Ab组相比,SCI+PRO组PI3K/Akt/mTOR信号通路磷酸化程度显著降低(P<0.05)。结论PD-L1通过调节Th1、Th17、Th2和Treg细胞平衡,并抑制PI3K/Akt/mTOR信号通路,从而减轻SCI后的炎症反应,发挥神经保护作用。PD-L1有望成为治疗SCI的新靶点。

Objective The aim of this study was to investigate the protective role of programmed cell death ligand-1(PD-L1)in a mouse model of spinal cord injury(SCI)by regulating T cell immunity and the PI3K/Akt/mTOR signaling pathway.Methods C57BL/6 mice used to establish SCI models were divided into the sham operation group(Sham),SCI group,SCI+PD-L1 antibody group(SCI+Ab),and SCI+PD-L1 protein group(SCI+PRO).c57BL/6 mice and PD-L1 knockout mice were used for SCI mapping,and they were divided into the sham operation group(Sham WT),PD-L1 knockout sham operation group(Sham PD-L1 KO),SCI model group(SCI WT),and PD-L1 knockout SCI model group(SCI PD-L1 KO).Western blotting and qRT-PCR were applied to detect the expression of PD-L1 in spinal cord tissues at different time points after SCI;mouse motor function was assessed by the Basso Mouse Motor Scale(BMS);changes in the levels of inflammatory factors and T-cell subpopulations after SCI were analyzed using qRT-PCR and flow cytometry;and Western blotting was used to detect changes in the PI3K/Akt/mTOR signaling pathway activation.Results PD-L1 expression was upregulated in spinal cord tissues of mice subjected to SCI palliation,peaking on day 7.Compared with the SCI PD-L1 KO group,mice in the SCI WT group had significantly higher BMS scores at 7,14,and 28 days after SCI(P<0.05),and the levels of inflammatory factors IL-1α,IL-2,IFN-γand TNF-αwere significantly lower on day 7 after palpation(P<0.05).Compared with the SCI+PBS group,mice in the SCI+PRO group had significantly higher Foxp3 levels and significantly lower Th1 and Th17 levels.Foxp3 levels were significantly higher,but Th1 and Th17 cell levels were significantly lower,and Th2 and Treg cell levels were significantly higher(P<0.05).The phosphorylation of the PI3K/Akt/mTOR signaling pathway was significantly higher in the SCI WT group mice than the SCI PD-L1 KO group ones(P<0.05).In contrast,the phosphorylation of PI3K/Akt/mTOR signaling pathway was significantly lower in the SCI+PRO group than in the SCI+PBS group and the SCI+Ab group(P<0.05).Conclusion PD-L1 plays a neuroprotective role by regulating the balance of Th1,Th17,Th2 and Treg cells and inhibiting the PI3K/Akt/mTOR signaling pathway,thereby reducing the inflammatory response after SCI.PD-L1 is expected to be a new target for the treatment of SCI.