Ferroptosis Regulated by 5-HT3a Receptor via Calcium/Calmodulin Signaling Contributes to Neuropathic Pain in Brachial Plexus Avulsion Rat Model

Abstract

Abstract Neuropathic pain is a prevalent complication following brachial plexus avulsion (BPA). Ferroptosis has been implicated in various nervous system disorders. However, the association between ferroptosis and neuropathic pain induced by BPA remains unclear. This study aimed to investigate the role of ferroptosis in BPA-induced neuropathic pain. A rat model of neuropathic pain was established via BPA induction. Pain thresholds of rats were measured after BPA surgery and intraperitoneal injection of Fer-1. On day 14 post-surgery, SDH (spinal dorsal horn) samples were collected for western blotting, biochemical analysis and immunohistochemistry to analyze the expression and distribution of ferroptosis-related markers. The relationships among 5-HT3a receptor, calcium/calmodulin (CaM) pathway and ferroptosis were assessed via western blotting, biochemical analysis and lipid peroxidation assays, including iron and calcium content, ROS, GPX4, ACSL and CaM expression. BPA-induced neuropathic pain was associated with iron accumulation, increased lipid peroxidation, and dysregulated expression of ACSL4 and GPX4, and changes in transferrin receptor (TFR), divalent metal transporter 1 (DMT1), and ferroportin-1 (FPN1). Intraperitoneal administration of Fer-1 reversed all these alterations and mitigated mechanical and cold hypersensitivity. Inhibition of the 5-HT3a receptor reduced ferroptosis. Furthermore, the 5-HT3a receptor can regulate the calcium/calmodulin pathway via L-type calcium channels (LTCCs), and blocking LTCCs with nifedipine also alleviated ferroptosis in the SDH of BPA rats. Taken together, in rats with brachial plexus avulsion, the development of neuropathic pain involves ferroptosis, which is regulated by the 5-HT3a receptor through the L-type calcium channels and the calcium/calmodulin signaling pathway in the spinal dorsal horn.