Abstract：
Nucleus pulposus (NP) cell pyroptosis is crucial for intervertebral disc degeneration (IDD). However, the precise mechanisms underlying pyroptosis in IDD remain elusive. Therefore, this study aimed to investigate how dickkopf-1 (DKK1) influences NP cell pyroptosis and delineate the regulatory mechanisms of IDD. Behavioral tests and histological examinations were conducted in rat IDD models to assess the effect of DKK1 on the structure and function of intervertebral discs. Detected pyroptosis levels using Hoechst 33,342/propidium iodide (PI) double staining, and determined pyroptosis-related protein expression via western blotting. The cellular mechanisms of DKK1 in pyroptosis were explored in interleukin (IL)-1β-induced NP cells transfected with or without DKK1 overexpression plasmids (oe-DKK1). In addition, IL-1β-treated NP cells transfected with sh-EZH2 and/or sh-DKK1 were utilized to clarify the interplay between the enhancer of zeste homologue 2 (EZH2) and DKK1 in pyroptosis. Additionally, the epigenetic regulation of DKK1 by EZH2 was explored in NP cells treated with the EZH2 inhibitors GSK126/DZNep. DKK1 expression decreased in IDD rats. Transfection with oe-DKK1 reduced pro-inflammatory factors and extracellular matrix markers in IDD rats. In IL-1β-induced NP cells, DKK1 overexpression suppressed pyroptosis and inhibited the NLRP3 and NAIP/NLRC4 inflammasome activation. EZH2 knockdown increased DKK1 expression and reduced pyroptosis-related proteins. Conversely, DKK1 downregulation reversed the inhibitory effects of EZH2 knockdown on pyroptosis. Furthermore, EZH2 suppressed DKK1 expression via H3K27 methylation at the DKK1 promoter. EZH2 negatively regulates DKK1 expression via H3K27me3 methylation, promoting NP cell pyroptosis in IDD patients. This regulatory effect involves the activation of NLRP3 and NAIP/NLRC4 inflammasomes.
摘要：
髓核（NP）细胞退变对椎间盘退变（IDD）至关重要。然而,IDD中焦凋亡的确切机制仍然难以捉摸。因此,本研究旨在探讨dickkopf-1（DKK1）如何影响NP细胞焦亡并描述IDD的调控机制。在大鼠IDD模型中进行行为测试和组织学检查，以评估DKK1对椎间盘结构和功能的影响。使用Hoechst33,342/碘化丙啶（PI）双重染色检测焦亡水平，并通过蛋白质印迹法测定焦亡相关蛋白的表达。在转染或不转染DKK1过表达质粒（oe-DKK1）的白介素（IL）-1β诱导的NP细胞中探索了DKK1在焦亡中的细胞机制。此外,用sh-EZH2和/或sh-DKK1转染的IL-1β处理的NP细胞用于阐明zeste同源物2（EZH2）的增强子与DKK1之间的相互作用。此外,在用EZH2抑制剂GSK126/DZNep处理的NP细胞中探索了EZH2对DKK1的表观遗传调控。IDD年夜鼠DKK1表达下降。转染oe-DKK1可降低IDD大鼠的促炎因子和细胞外基质标志物。在IL-1β诱导的NP细胞中，DKK1过表达抑制了焦凋亡并抑制了NLRP3和NAIP/NLRC4炎性体的活化。EZH2敲低增加了DKK1的表达并减少了焦亡相关蛋白。相反，DKK1下调逆转了EZH2敲低对焦凋亡的抑制作用。此外，EZH2通过DKK1启动子处的H3K27甲基化抑制DKK1表达。EZH2通过H3K27me3甲基化负调控DKK1的表达，在IDD患者中促进NP细胞焦亡。这种调节作用涉及NLRP3和NAIP/NLRC4炎性体的激活。
