Mechanism of Netrin-1 in electroacupuncture-mediated inhibition of nerve fiber ingrowth in degenerative intervertebral discs.

Lumbar intervertebral disc degeneration (IVDD) is characterized by abnormal innervation and neurogenic inflammation, contributing to chronic discogenic pain. Electroacupuncture (EA) alleviates IVDD-related pain, yet its underlying mechanisms remain incompletely understood. This study aimed to investigate the role of the axonal guidance factor Netrin-1 in EA-mediated inhibition of myelinated nerve fiber ingrowth into degenerative intervertebral discs and to elucidate the downstream signaling pathways involved. A rabbit axial compression IVDD model was established. Animals were divided into sham, model, EA, model+OV-Netrin-1, EA+sh-Netrin-1, EA+μ-opioid antagonist (β-FNA), and EA+δ-opioid antagonist (NTI) groups. Tarlov scoring, MRI, HE, AB-PAS, immunohistochemistry, TEM, Western blot, RT-qPCR, Co-IP, ELISA and flow cytometry were used to evaluate disc histology, nerve ingrowth, Netrin-1 expression in annulus fibrosus (AF) and dorsal root ganglion (DRG), endogenous opioid peptides, receptor interactions, cAMP/cGMP levels and intracellular calcium concentration. EA improved Tarlov scores, restored disc structure and reduced pathological nerve ingrowth in degenerative discs. EA downregulated Netrin-1 in AF but upregulated Netrin-1 in L2 DRG. DRG Netrin-1 overexpression mimicked EA's anti-neoinnervation effect, whereas Netrin-1 knockdown abolished EA efficacy. EA elevated spinal POMC/M-ENK; blocking μ/δ opioid receptors reversed EA-mediated Netrin-1 upregulation in DRG. Mechanistically, EA promoted UNC5B-DCC complex formation, reduced cAMP, increased cGMP and lowered intracellular Ca2+ via Netrin-1 receptors to inhibit myelinated axon sprouting. EA upregulates DRG Netrin-1 by activating endogenous opioid peptide signaling. Netrin-1 subsequently modulates UNC5B/DCC downstream cAMP-cGMP-calcium pathways to suppress abnormal myelinated nerve fiber ingrowth, thereby alleviating discogenic pain and delaying IVDD progression.