IL-32通过激活FAT4介导的Hippo/YAP信号通路加重人髓核细胞的代谢紊乱

在炎症微环境中细胞外基质（ECM）代谢障碍在椎间盘退行性疾病（IDD）的发病机制中起关键作用。已报道白细胞介素-32（IL-32）参与多种炎症性疾病的进展；然而，目前尚不清楚它是否参与髓核（NP）细胞的基质代谢。因此，本研究旨在探讨IL-32在炎症微环境中调控ECM代谢的机制。采用RNA测序（RNA-seq）鉴定炎症微环境中NP细胞的异常表达基因。通过Western印迹、实时定量PCR、免疫组化和免疫荧光分析，检测人类NP组织或用肿瘤坏死因子-α（TNF-α）处理的NP细胞中IL-32及代谢标志物的表达。在体内模型中，将过表达IL-32的腺相关病毒（AAV）注射到大鼠尾椎间盘中，以评估其对IDD的影响。通过免疫沉淀和质谱技术鉴定与IL-32相互作用的蛋白质。使用过表达IL-32的慢病毒或敲除FAT4、YAP抑制剂Verteporfin（VP）处理人类NP细胞，探讨IL-32的发病机制。在人类NP组织中验证Hippo/YAP信号通路的活性。临床样本显示，退行性NP组织中IL-32表达显著上调。此外，TNF-α诱导的退行性NP细胞中IL-32表达也显著升高。IL-32过表达诱导大鼠模型中IDD的进展。在机制层面，炎症微环境中IL-32的升高增强其与FAT4及哺乳动物无菌20样激酶1/2（MST1/2）蛋白的相互作用，促使MST1/2磷酸化，并激活Hippo/YAP信号通路，导致NP细胞的基质代谢紊乱。我们的结果表明，IL-32通过FAT4/MST/YAP轴介导炎症微环境中NP细胞的基质代谢障碍，为IDD的精准治疗提供理论基础。

Extracellular matrix (ECM) metabolism disorders in the inflammatory microenvironment play a key role in the pathogenesis of intervertebral disc degeneration (IDD). Interleukin-32 (IL-32) has been reported to be involved in the progression of various inflammatory diseases; however, it remains unclear whether it participates in the matrix metabolism of nucleus pulposus (NP) cells. Therefore, this study aimed to investigate the mechanism of IL-32 on regulating the ECM metabolism in the inflammatory microenvironment. RNA-seq was used to identify aberrantly expressed genes in NP cells in the inflammatory microenvironment. Western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence analysis were performed to measure the expression of IL-32 and metabolic markers in human NP tissues or NP cells treated with or without tumor necrosis factor-α (TNF-α). In vivo, an adeno-associated virus overexpressing IL-32 was injected into the caudal intervertebral discs of rats to assess its effect on IDD. Proteins interacting with IL-32 were identified via immunoprecipitation and mass spectrometry. Lentivirus overexpressing IL-32 or knocking down Fat atypical cadherin 4 (FAT4), yes-associated protein (YAP) inhibitor-Verteporfin (VP) were used to treat human NP cells, to explore the pathogenesis of IL-32. Hippo/YAP signaling activity was verified in human NP tissues. IL-32 expression was significantly upregulated in degenerative NP tissues, as indicated in the clinical samples. Furthermore, IL-32 was remarkably overexpressed in TNF-α-induced degenerative NP cells. IL-32 overexpression induced IDD progression in the rat model. Mechanistically, the elevation of IL-32 in the inflammatory microenvironment enhanced its interactions with FAT4 and mammalian sterile 20-like kinase1/2 (MST1/2) proteins, prompting MST1/2 phosphorylation, and activating the Hippo/YAP signaling pathway, causing matrix metabolism disorder in NP cells. Our results suggest that IL-32 mediates matrix metabolism disorders in NP cells in the inflammatory micro-environment via the FAT4/MST/YAP axis, providing a theoretical basis for the precise treatment of IDD.