晚期糖基化终产物AGEs介导炎症小体NLRP3的表观遗传学改变在椎间盘退变中的作用及其机制研究

The inflammation, which secretes cytokines mainly composed of proinflammatory IL-1β in nucleus pulposus(NP) cells, is a distinguishing characteristic of the intervertebral disc degeneration(IDD). The inflammasome NLRP3 is the crucial protein complex regulating maturation and secretion of IL-1β. In recent years, it has been highly valued by the international academic community. Both genetic and environmental factors contribute to the regulation of inflammasome NLRP3. However, limited studies have explored the epigenetic modifications of inflammasome NLRP3 in IDD. Our previous studies have found that the NP tissue metabolite, advanced glycation end products(AGEs), is involved in activating inflammasome NLRP3 in NP cells. On the basis of previous studies, our research team will further investigate the epigenetics changes of NLRP3 component proteins in AGEs-induced NP cells degeneration by using immunoprecipitation and microarray technique. We will explore whether these changes promote the expression and activation of NLRP3, and then cause the degeneration of NP cells. By using specific intervention means, aberrant epigenetic modifications of the inflammasome NLRP3 components are rescued to identify the key regulatory mechanisim of IDD. We will also investigate the feasibility of alleviating and reversing IDD, which will lay a foundation for clinical diagnosis and molecular targeted therapy of IDD.

髓核细胞爆发以分泌IL-1β为主的炎症反应是椎间盘退行性变的主要特征，炎症小体NLRP3是调控IL-1β成熟和分泌的关键蛋白复合体，近年来受到国际学术界高度重视。炎症小体NLRP3的调控是基因与环境因素共同作用的复杂性过程，而表观遗传改变对其表达和激活的影响尚不清楚，在椎间盘退行性变领域更未见报道。申请者前期研究发现，髓核组织代谢积聚的晚期糖基化终产物AGEs参与了炎症小体NLRP3的激活。本课题拟在以往研究基础上，采用免疫沉淀加芯片技术，深入研究炎症小体NLRP3各组成蛋白在AGEs诱导髓核细胞退变过程中的表观遗传改变，并明确上述改变是否会促进炎症小体NLRP3表达和激活，继而导致髓核细胞发生退变表型。进一步运用特异性干预手段，改变炎症小体NLRP3表观遗传修饰状态，以期明确椎间盘退行性变的关键调控机理，探讨缓解或者逆转退变的可行性，为椎间盘退行性疾病的诊断和靶向治疗奠定基础。

椎间盘退变是引起下腰痛的重要原因，髓核细胞退变、炎性反应、细胞外基质降解重塑是诱发椎间盘退变的三要素。NLRP3炎症小体组装激活促使无活性的前体形式Pro-IL-1β向活性形式IL-1β转变，可促使髓核细胞合成与降解细胞外基质失衡，且AGEs沉积是椎间盘退变的重要危险因素。因此，本课题拟进一步解析AGEs相关椎间盘退变中NLRP3炎性小体组装激活的关键机制。我们发现AGEs沉积与椎间盘退变呈显著正相关，且AGEs干扰髓核细胞内关键蛋白去乙酰化酶功能进而促使线粒体损伤，是介导NLRP3炎性小体组装激活的关键机制。靶向调控去乙酰化酶功能对AGEs诱导髓核细胞死亡及椎间盘退变具有显著治疗效果。证实了AGEs诱导NLRP3炎性小体组装激活过程中Ca2+水平紊乱的关键机制--内质网应激，并发现BMSC外泌体可显著缓解AGEs退变模型中髓核细胞内质网应激，是椎间盘退变的有效治疗手段。发现了酸离子敏感通道在髓核细胞NLRP3炎性小体组装激活过程中的关键作用，以酸离子敏感通道为调控靶点可减缓髓核细胞炎性反应、细胞外基质降解及椎间盘退变进展。明确了线粒体DNA的时空网络在髓核细胞NLRP3炎性小体组装激活过程中的关键信号通路，并进一步探索了靶向修饰的工程化外泌体在缓解髓核细胞NLRP3炎性小体组装激活过程中的关键作用，对缓解炎性椎间盘退变具有显著治疗效果。该项目的顺利实施不仅明确了AGEs相关椎间盘退变过程中NLRP3炎症小体组装激活的关键机制，也为相关治疗靶点提供了重要干预策略，为探索椎间盘损伤修复策略提供了重要理论依据。