PRP-Exos通过TLR4/NF-κB通路对神经病理性疼痛的作用及其机制研究

Effects and mechanisms of PRP-Exos on neuropathic pain via the TLR4/NF-κB pathway

摘要:

目的 探讨富血小板血浆来源外泌体（PRP-Exos）通过TLR4/NF-κB信号通路对坐骨神经损伤（SNI）大鼠神经病理性疼痛的作用及其分子机制。方法 将30只雄性SD大鼠随机分为空白组、模型组和PRP-Exos组，每组10只。模型组和PRP-Exos组采用SNI法复制神经病理性疼痛模型。PRP-Exos组模型复制后第7天起鞘内注射200 mg/L PRP-Exos（0.5 mL/次，隔日1次，共10次），模型组注射等体积生理盐水。检测干预前后大鼠机械痛阈值（MWT）、热痛阈值（TWL）及坐骨神经运动神经传导速度（MNCV）；干预结束后，坐骨神经甲苯胺蓝染色观察病理形态，透射电镜观察并测量有髓神经纤维的轴突直径和髓鞘厚度，酶联免疫吸附试验测定脊髓组织肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平，Western blotting检测脊髓Toll样受体4（TLR4）、核因子-κB（NF-κB）、p65蛋白表达。结果 空白组、模型组和PRP-Exos组术前、模型复制后第6天、给药10次时MWT、TWL、MNCV水平比较，结果 ①不同时间点MWT和TWL水平比较，差异均有统计学意义（P <0.05）。②3组MWT、TWL、MNCV水平比较，差异均有统计学意义（P <0.05）；与空白组相比，模型组和PRP-Exos组模型复制后第6天、给药10次时MWT、TWL、MNCV水平均降低；与模型组相比，PRP-Exos组给药10次时MWT、TWL、MNCV水平均升高。③3组MWT、TWL、MNCV水平变化趋势比较，差异有统计学意义（P <0.05）。与空白组相比，模型组和PRP-Exos组轴突直径和髓鞘厚度均减小（P <0.05）；与模型组相比，PRP-Exos组轴突直径和髓鞘厚度均增大（P <0.05）。与空白组相比，模型组和PRP-Exos组TNF-α、IL-6水平和TLR4、NF-κB p65蛋白相对表达量均升高（P <0.05）；与模型组相比，PRP-Exos组TNF-α、IL-6水平和TLR4、NF-κB p65蛋白相对表达量均降低（P <0.05）。结论 PRP-Exos可通过抑制TLR4/NF-κB信号通路，减少脊髓炎症因子释放，改善SNI大鼠神经病理形态，从而缓解神经病理性疼痛。

Abstract:

Objective To investigate the therapeutic effects and molecular mechanisms of platelet-rich plasma-derived exosomes (PRP-Exos) on neuropathic pain in spared nerve injury (SNI) rats through the Toll-like receptor 4 (TLR4)/ nuclear factor-κB (NF-κB) signaling pathway.Methods Thirty male SD rats were randomly divided into the sham, SNI model, and PRP-Exos groups (each n = 10). Neuropathic pain models were established in the latter two groups using SNI. From day 7 post-modeling, the PRP-Exos group received intrathecal injections of 200 mg/L PRP-Exos (0.5 mL/dose, every other day for 10 doses), while the model group received saline. Before and after the intervention, the mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), and motor nerve conduction velocity (MNCV) were assessed. After the intervention, the pathological morphology of the sciatic nerve was assessed using toluidine blue staining. Transmission electron microscopy was performed to observe and measure the axonal diameter and myelin thickness of myelinated nerve fibers. Enzyme-linked immunosorbent assay (ELISA) was used to quantify tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in spinal cord tissues. Western blotting was conducted to determine the expression of TLR4, NF-κB, and p65 proteins in the spinal cord.Results The comparison of MWT, TWL, and MNCV at baseline, on day 6 after model establishment, and after 10 doses of treatment in the sham, SNI model, and PRP-Exos groups demonstrated that significant differences in MWT and TWL were observed across the different time points (P < 0.05) and that significant differences in MWT, TWL, and MNCV were observed among the three groups (P < 0.05). Compared with the sham group, both the SNI model and PRP-Exos groups showed decreased MWT, TWL, and MNCV on day 6 after modeling and after 10 doses of treatment. Compared with the SNI model group, the PRP-Exos group showed increased MWT, TWL, and MNCV after 10 doses of treatment. The change trends in MWT, TWL, and MNCV among the three groups were significantly different (P < 0.05). Compared with the sham group, both the SNI model and PRP-Exos groups exhibited reduced axonal diameter and myelin thickness (P < 0.05), while the PRP-Exos group showed increased axonal diameter and myelin thickness compared with the SNI model group (P < 0.05). Similarly, TNF-α and IL-6 levels, as well as spinal cord TLR4 and NF-κB p65 protein expression, were elevated in the SNI model and PRP-Exos groups compared with the blank group (P < 0.05), whereas these markers were reduced in the PRP-Exos group relative to the SNI model group (P < 0.05).Conclusion PRP-Exos can mitigate neuropathic pain in SNI rats by suppressing the TLR4/NF-κB pathway, thereby reducing the release of inflammatory factors in the spinal cord and improving neuropathological morphology.