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

PURPOSE: 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.

METHODS

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.

RESULTS

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.

CONCLUSION

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.