髓核细胞基于YAP/TAZ表达变化的P2Y2受体激活致椎间盘退变的机制研究

Low back pain has become a major public health problem worldwide, with a main cause of intervertebral discs degeneration (IDD). The mechanisms of IDD remain unclear. Un-physiologic mechanical loading (UPL) is generally thought to play a very crucial role in the process of IDD. Our previous studies and other literatures show that: 1) Activation of P2Y2Rs by ATP takes part in process of chronic pain under pathological conditions. 2) We found that P2Y2Rs were not only expressed on NP cells of human degenerated IVD but also co-expressed with YAP and TAZ. Furthermore, the number of profiles of P2Y2Rs expression was significantly increased on NP cells of human degenerated IVD compared with control group. 3) We also found the protein expression of P2Y2Rs and YAP/TAZ was increased with the increase of IVD degeneration. 4) Activations of YAP/TAZ play an important role in proliferation, apoptosis and proinflammatory cytokines releasing. 5) As IDD proceeds, proinflammatory cytokines play the multiple critical roles. So we provide a hypothesis about activation of P2Y2Rs under UPL could active YAP/TAZ to regulate TNF，IL-1β, IL-6 in the process of IDD. The present study was designed to verify our hypothesis from levels of in vivo, organization and cells by using human IDD and establishing compressive loading on rat tail model, which are with animal behavioral test, siRNA or AS-ODN treatment and molecular biology methods to explore regulation of IVD P2Y2 receptors activation on proinflammatory cytokines in the process of IDD and underlying signaling pathway. This study aimed to explore novel targets and provide experimental evidence for treating IDD.

下腰痛是全球主要健康问题，椎间盘退变(IDD)是主要原因，非生理负重(UPL)是IDD重要因素，但确切机制未阐明。国际及我们前期研究结果表明：①大量ATP释放，激活P2Y2受体参与疼痛发病过程；②人IDD时NP细胞P2Y2受体表达增加，且与YAP和TAZ 共表达；③髓核组织P2Y2受体, YAP/TAZ随IVD退变程度增加蛋白表达量增加；④YAP/TAZ激活在细胞增殖凋亡、促炎因子释放中发挥重要作用；⑤促炎因子在IDD中发挥关键作用。我们推测UPL下IVD释放大量ATP，激活NP细胞P2Y2受体，激活YAP/TAZ，调控TNF，IL-1β和IL-6，启动并维持IDD。本项目拟对人退变IVD及机械加压大鼠尾部IVD退变模型，采用动物行为学，分子生物学，干扰治疗等方法从“整体-组织-细胞”层面观察P2Y2受体激活对促炎因子致IVD退变的调控及其信号通路，为临床治疗IDD提供新靶点及理论依据。

下腰痛是全球主要健康问题，椎间盘退变(IDD)是主要原因，但确切机制未阐明。本项目对人退变IVD及机械加压大鼠尾部IVD退变模型的髓核组织采用动物行为学，分子生物学，干扰治疗等方法从“整体-组织-细胞”层面观察P2Y2受体激活对促炎因子致IVD退变的调控及其信号转导通路。研究发现，①在髓核组织中，P2Y2受体和YAP，P2Y2受体和TAZ共表达；且P2Y2受体、YAP、TAZ随人退变椎间盘退变程度增加蛋白表达增加；与正常椎间盘髓核组织比较，轻度退变椎间盘髓核组织表达IL-1 beta、IL-6和TNF-alpha。②培养的人退变椎间盘髓核细胞，P2Y2受体激活YAP、TAZ表达增加，细胞外基质降解增加，IL-1 beta、IL-6和TNF alpha含量增加。③下调大鼠尾部椎间盘模型中髓核组织P2Y2受体，大鼠椎间盘髓核组织YAP、TAZ蛋白表达下降，尾部机械疼痛、细胞凋亡和细胞外基质降解减轻。血清IL-1 beta、IL-6和TNF alpha含量降低。通过本研究明确了椎间盘退变过程中，髓核组织或细胞中的P2Y2受体激活，可通过YAP/TAZ两个信号分子活化，以及炎症因子释放增加，推进椎间盘退变发病过程。且可能起到了关键作用，由于本研究在人退变椎间盘髓核组织和大鼠椎间盘退变模型中观察到与假说比较一致的结果，因此，它们可能作为开发延缓椎间盘退变疾病药物的新靶点应用于临床。