Table_1_Macrophage polarization regulates intervertebral disc degeneration by modulating cell proliferation, inflammation mediator secretion, and extracellular matrix metabolism.docx

Macrophage infiltration and polarization have been increasingly observed in intervertebral disc (IVD) degeneration (IDD). However, their biological roles in IDD are still unrevealed. We harvested conditioned media (CM) derived from a spectrum of macrophages induced from THP-1 cells, and examined how they affect nucleus pulposus cells (NPCs) in vitro, by studying cell proliferation, extracellular matrix (ECM) synthesis, and pro-inflammation expression; and in vivo by injection CM in a rat IDD model. Then, high-throughput sequencing was used to detect differentially expressed genes (DEGs). Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks were used to further analysis. Higher CCR7+ (M1 marker) and CD206+ (M2 marker) cell counts were found in the degenerated human IVD tissues as compared with the control. Furthermore, the cell co-culture model showed M1CM attenuated NPC proliferation, downregulated the expression of ECM anabolic genes encoding aggrecan and collagen IIα1, upregulated the expression of ECM catabolic genes encoding MMP-13, and inflammation-related genes encoding IL-1β, IL-6, and IL-12, while M2CM showed contrasting trends. In IDD model, higher histological scores and lower disc height index were found following M1CM treatment, while M2CM exhibited opposite results. M1CM injection decreased ECM anabolic and increased ECM catabolic, as well as the upregulation of inflammation-related genes after 8 weeks treatment, while M2CM slowed down these trends. Finally, a total of 637 upregulated and 655 downregulated genes were detected in M1CM treated NPCs, and 975 upregulated genes and 930 downregulated genes in the M2CM groups. The top 30 GO terms were shown and the most significant KEGG pathway was cell cycle in both groups. Based on the PPI analysis, the five most significant hub genes were PLK1, KIF20A, RRM2, CDC20, and UBE2C in the M1CM groups and RRM2, CCNB1, CDC20, PLK1, and UBE2C in the M2CM groups. In conclusion, macrophage polarization exhibited diverse roles in IDD progression, with M1CM exacerbating cell proliferation suppression and IVD degeneration, while M2CM attenuated IDD development. These findings may facilitate the further elucidation of the role of macrophage polarization in IDD, and provide novel insights into the therapeutic potential of macrophages.

巨噬细胞浸润与极化在椎间盘（intervertebral disc, IVD）退变（intervertebral disc degeneration, IDD）中的作用日益受到关注，但其在IDD中的生物学功能仍未阐明。我们收集了由THP-1细胞诱导的不同表型巨噬细胞的条件培养基（conditioned media, CM），通过体外实验——检测细胞增殖、细胞外基质（extracellular matrix, ECM）合成以及促炎因子表达——探究其对髓核细胞（nucleus pulposus cells, NPCs）的影响；并通过在大鼠IDD模型中注射CM开展体内实验。随后，我们采用高通量测序检测差异表达基因（differentially expressed genes, DEGs），并通过基因本体论（Gene Ontology, GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）以及蛋白质-蛋白质相互作用（protein-protein interaction, PPI）网络进行进一步分析。相较于对照组织，退变的人IVD组织中CCR7+（M1型标志物）与CD206+（M2型标志物）的细胞数量更高。进一步的细胞共培养实验显示，M1型巨噬细胞条件培养基（M1CM）可抑制NPC增殖，下调编码聚集蛋白聚糖与Ⅱ型胶原α1的ECM合成相关基因的表达，上调编码MMP-13的ECM降解相关基因以及编码IL-1β、IL-6、IL-12的炎症相关基因的表达；而M2型巨噬细胞条件培养基（M2CM）则呈现相反的变化趋势。在IDD大鼠模型中，M1CM处理组的组织学评分更高、椎间盘高度指数更低，而M2CM处理组则表现出相反的结果。8周干预后，M1CM注射可下调ECM合成相关基因、上调ECM降解相关基因以及炎症相关基因的表达，而M2CM则可延缓这些变化。最终，在M1CM处理的NPC中共检测到637个上调基因与655个下调基因，在M2CM处理组中则检测到975个上调基因与930个下调基因。两组中排名前30的GO富集条目一致，且最显著的KEGG通路均为细胞周期。通过PPI分析，M1CM组中5个最重要的核心基因为PLK1、KIF20A、RRM2、CDC20与UBE2C；M2CM组则为RRM2、CCNB1、CDC20、PLK1与UBE2C。综上，巨噬细胞极化在IDD进展中发挥多样化作用：M1CM可加剧NPC增殖抑制与IVD退变，而M2CM则可延缓IDD的发展。本研究结果有助于进一步阐明巨噬细胞极化在IDD中的作用，并为巨噬细胞的治疗潜力提供新的研究思路。