TNF-α与IL-1β上调ADAMTS-1表达加速椎间盘退变的分子机制研究

Chronic low back pain affects a lot of people in their lives. The primary cause of low back pain is degeneration of the intervertebral discs. Intervertebral disc degeneration (IVDD) is linked to loss of extracellular matrix (ECM), particularly the early loss of aggrecan. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family that cleave aggrecan in vitro are likely to play a role in aggrecan degradation and subsequent disk degeneration as in the pathogenesis of osteoarthritis. According to our previous studies, ADAMTS-1 is expressed at a very low level in tissues of the normal intervertebral disk (in adult rat and human). ADAMTS-1 expression is significantly elevated in degenerative disk. It has been reported that during disk degeneration, in addition to infiltrating immune cells, resident nucleus pulposus(NP) and annulus fibrosus(AF) cells produce high levels of the cytokines TNF-α and IL-1β. We found that there was a dose-dependent increase in ADAMTS-1 mRNA expression by the cytokines treatment. Both cytokines significantly increased the ADAMTS-1 promoter activity. We also found that cytokines modulated ADAMTS-1 expression by NF-κB signaling in rat NP cells. Surprisingly, despite the importance of these aggrecanases in the pathogenesis of osteoarthritis and disk disease, only few studies have investigated regulation of ADAMTS-1 transcription in NP cells, and none have used promoter analysis. Above all, these observations beg the question of how TNF-α and IL-1β control the expression of ADAMTS-1 and what is their relative contribution in NP cells in terms of aggrecan degradation. According to our previous study, we hypothesis that TNF-α and IL-1β associated with ADAMTS-1 expression in degenerative disk through NF-κB pathway. Targeting these enzymes may be a possible future therapeutic strategy for the prevention of intervertebral disc degeneration and its associated morbidity.

慢性下腰痛是临床极为常见的疾病，椎间盘退变（IVDD）被认为是引起下腰痛的重要原因之一。研究发现聚集蛋白聚糖减少引起髓核细胞外基质(ECM)合成与分解代谢的失衡是IVDD的重要机制，含I型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS)是一种新发现的可降解ECM聚集蛋白聚糖的蛋白酶，研究已经证明ADAMTS家族在骨性关节炎的发病过程中起到重要作用，我们的预实验发现，ADAMTS-1在正常大鼠和人的椎间盘中有微量表达，在退变的椎间盘中表达明显升高；TNF-α与IL-1β可上调ADAMTS-1的表达，可增加ADAMTS-1启动子活性，并可激活NF-κB传导通路。在上述预实验的基础上，我们提出假设：TNF-α与IL-1β通过激活NF-κB信号通路上调ADAMTS-1表达加速IVDD的发生。该研究为延缓IVDD进展提供可能的分子生物学依据，为腰痛患者的早期治疗提供新的选择。

本项目针对椎间盘退变的发病机制进行研究，重点关注ADAMTS-1在椎间盘退变中的作用及调控机制，通过组织层面检测发现ADAMTS-1在椎间盘退变时表达显著上调，提示ADAMTS-1可能在椎间盘退变时发挥重要作用，为此采用促炎因子构建髓核细胞退变模型，在细胞层面验证ADAMTS-1的表达变化，并构建ADAMTS-1干扰腺病毒探索该基因对细胞退变的调控作用，发现干扰ADAMTS-1表达后，促炎因子诱导的髓核细胞外基质aggrecan降解被显著缓解，此外，动物实验进一步证实干扰ADAMTS-1可有效延缓促炎因子诱导的椎间盘退变进程。综上结果说明ADAMTS-1在椎间盘退变时发挥促进作用。而关于ADAMTS-1受促炎因子调控表达的调控机制，通过检查相关通路激酶磷酸化水平并构建相关位点干扰慢病毒，发现NF-KB通路在促炎因子诱导ADAMTS-1表达过程中发挥重要作用，明确了ADAMTS-1在退变椎间盘内表达调控的上游机制。课题组在本项目实施中共发表SCI论著5篇，已接收尚未见刊1篇，为椎间盘退变的诊疗带来新的思路。