Secretome and Pathway Analysis of Stress Induced Disc Degeneration In Vitro

ABSTRACT Background Intervertebral disc degeneration (IDD) involves complex inflammatory and oxidative stress pathways within nucleus pulposus (NP) cells, leading to catabolic remodeling and loss of disc function. Traditional single‐analyte assays capture only a small part of these coordinated networks. Leveraging the multiplex capability of Luminex, this study uses targeted immunoassays to simultaneously quantify 73 extracellular proteins and 21 intracellular signaling molecules in primary human NP cells stimulated with IL‐1β, Pam2CSK4, or hydrogen peroxide, enabling comparison of shared and stimulus‐specific responses. Methods Human NP cells isolated from patients undergoing spine surgery were plated in monolayer or encapsulated in alginate and stimulated for 1 week with either: 100 pg/mL or 1 ng/mL Interleukin (IL)‐1β, or the Toll‐Like Receptor (TLR) 2/6 agonist Pam2CSK4 at 100 ng/mL, or 50 μM Hydrogen Peroxide (H 2 O 2 ). The role of the stimuli was investigated using mRNA expression analysis and Luminex multiplex immunoassays. Results TLR2/6 agonist and IL‐1β stimulation significantly up‐regulated IL‐6, IL‐8, IL‐1β, and MMP‐3 mRNA expression, while H 2 O 2 failed to induce any changes. The secretome showed that IL‐8, IL‐6, MMP‐7, Intercellular Adhesion Molecule 1 (ICAM‐1), and Growth‐Regulated Oncogene‐α (GRO‐α) were the most responsive proteins across treatments and culture conditions. Intracellularly, phosphorylation of Jun Proto‐Oncogene AP‐1 Transcription Factor Subunit (cJUN), Nuclear Factor Kappa B (NF‐kB), and Inhibitor of Nuclear Factor Kappa B Alpha (IKBA) was observed following IL‐1β and Pam2CSK4 stimulation. Conclusions Across inflammatory and innate immune activation and under both monolayer and 3D culture, a concise five‐marker panel, namely IL‐6, IL‐8, GRO‐α (CXCL‐1), ICAM‐1, and MMP‐7 captured NP cell responses with high consistency, while phosphoproteomics demonstrated convergence on NF‐κB/AP‐1 signaling. These markers are well established within disc‐degeneration pathways (inflammation, innate immunity, and ECM remodeling); thus, they offer practical, reusable readouts for comparative pharmacology, assay qualification, and early biomarker development in translational spine research.