镁金属内植物介导巨噬细胞NF-κB/TNF-α通路治疗腰突症神经根炎性病变的效果及机制研究

The inflammatory reaction in the nerve roots resulting from nucleus pulposus of intervertebral disc is the main cause of postoperative refractory lumbar disc herniations (LDH) neuropathy. Macrophage polarization to M1 phenotype and the release of Tumor necrosis factor-α(TNF-α) are critical in the process of neuroinflammation, and new biomaterials with the potential to modulate the local concentration of TNF-α combining with spinal surgery are desired in the treatment of inflammatory neuropathy. In our previous experiment, magnesium (Mg) materials were confirmed to promote sciatic neuropathy and inhibit the release of TNF-α from macrophage. Thererfore, we put forward the hypothesis that Mg could modulate macrophage polarization and expression of TNF-α via the NF-κB/TNF-α signaling, and improve the inflammatory neuropathy caused by herniated lumbar discs. In our project, we will firstly investigated the effect of Mg extraction on macrophage polarization and cytokines release in vitro. Secondly, Mg materials will be implant in the spine of autologous nucleus pulposus induced rats LDH model, and the degradation of Mg will be observed with its effects on recovery of sciatic function and neuroinflammation in the associated nerve roots. Furthermore, the contribution of NF-κB/TNF-α signaling blockage to the treatment of inflammatory neuropathy by Mg materials will be studied both in vitro and in vivo. Our project will elucidate the influence and mechanism of Mg on inflammatory reaction, and provide effective treatment for inflammatory neuropathy caused by LHD .

椎间盘髓核外漏引起的神经根炎症反应是术后难治性腰椎间盘突出神经根症状的主要诱因，炎症早期M1型巨噬细胞释放TNF-α对神经根炎性病变至关重要，研制新型骨科生物材料结合手术治疗控制TNF-α局部浓度是改善神经根症状的理想方案。本课题组前期研究发现镁金属脊柱植入物可改善腰突症动物坐骨神经功能，同时镁金属材料可抑制巨噬细胞释放TNF-α。由此得出镁金属材料介导NF-κB/TNF-α通路调控巨噬细胞极化及TNF-α释放，缓解腰突症神经根炎性病变的机制假设。基于此，本课题在细胞实验中研究镁金属浸提液对巨噬细胞M1型极化及细胞因子释放等细胞功能的作用；在自体髓核诱导的大鼠腰突症模型中观察镁金属降解对坐骨神经功能恢复及神经根炎症反应的影响；从体外和体内深入探讨阻断NF-κB/TNF-α通路对镁金属治疗神经根炎症反应的影响。本研究将阐明镁金属调控炎症反应的分子机制，为腰突症神经根炎性病变提供新的治疗方案。

腰椎间盘突导致的腰腿神经放射性疼痛，是成人慢性疼痛的首要病因，终身患病率高达40%。而椎间盘髓核外漏引起的神经根炎症反应是术后难治性腰椎间盘突出神经根症状的主要诱因，研制新型骨科生物材料结合手术治疗控制局部炎症是改善神经根症状的理想方案。本课题组工艺改善了高纯镁材料的力学强度和降解性能，制备出了降解均匀，生物相容性良好，细胞毒性小的高纯镁材料，并在此基础上进一步设计并且制备了镁金属椎体融合器。在体外检测中，分别在RAW 264.7细胞系诱导出的M1/M2巨噬细胞模型和小鼠原代巨噬细胞BMDM诱导出的M1/M2巨噬细胞模型中，采用流式细胞技术、免疫荧光技术、RTPCR、Western Blot以及Elisa证明了高纯镁浸提液可以抑制M1型巨噬细胞极化以及IL-6和TNF-a等炎症因子释放，同时促进M2型巨噬细胞极化以及抗炎因子Arg1和TGF-b的表达。之后采用了RNA-seq测序以及KEGG通路富集分析法，发现PI3K-AKT通路表达介导了高纯镁浸提液介导M1/M2巨噬细胞极化分型的分子信号通路。进一步构建Akt1和Akt2的siRNA序列并进行细胞内转染，证实了AKT1/2蛋白的表达差异介导了高纯镁巨噬细胞调控M2型巨噬细胞极化分型。在体内试验中，我们将高纯镁棒材植入了自体髓核组织诱导的腰突症大鼠模型中，发现了高纯镁材料体内降解均匀，周围骨长入良好，并在降解同时可提高坐骨神经术后功能恢复，抑制背根神经节处的炎症因子表达，组化以及免疫荧光验证了高纯镁材料抑制M1型巨噬细胞促进了M2型巨噬细胞的转归。我们进一步在前交叉韧带重建的新西兰兔模型以及大鼠的牵张成骨的大段骨缺损模型中验证了高纯镁材料对巨噬细胞极化分型的调控作用。制备了镁金属椎体融合器并进行了大型动物的体内实验，为了进一步探究镁材料的高纯镁锥体植入物的临床应用前景打下基础。