Vitamin D3 Attenuates Neuropathic Pain via Suppression of Mitochondria-Associated Ferroptosis by Inhibiting PKCα/NOX4 Signaling Pathway

Aims: Neuropathic pain remains a significant unmet medical challenge due to its elusive mechanisms. Recent clinical observations suggest that vitamin D (VitD) holds promise in pain relief, yet its precise mechanism of action is still unclear. This study explores the therapeutical role and potential mechanism of VitD(3) in spared nerve injury (SNI)-induced neuropathic pain rat model. Methods: The analgesic effects and underlying mechanisms of VitD(3) were evaluated in SNI and naive rat models. Mechanical allodynia was assessed using the Von Frey test. Western blotting, immunofluorescence, biochemical assay, and transmission electron microscope (TEM) were employed to investigate the molecular and cellular effects of VitD(3). Results: Ferroptosis was observed in the spinal cord following SNI. Intrathecal administration of VitD(3), the active form of VitD, activated the vitamin D receptor (VDR), suppressed ferroptosis, and alleviated mechanical nociceptive behaviors. VitD(3) treatment preserved spinal GABAergic interneurons, and its neuroprotective effects were eliminated by the ferroptosis inducer RSL3. Additionally, VitD(3) mitigated aberrant mitochondrial morphology and oxidative metabolism in the spinal cord. Mechanistically, VitD(3) inhibited SNI-induced activation of spinal PKC alpha/NOX4 signaling. Inhibition of PKC alpha/NOX4 signaling alleviated mechanical pain hypersensitivity, accompanied by reduced ferroptosis and mitochondrial dysfunction in SNI rats. Conversely, activation of PKC alpha/NOX4 signaling in naive rats induced hyperalgesia, ferroptosis, loss of GABAergic interneurons, and mitochondrial dysfunction in the spinal cord, all of which were reversed by VitD(3) treatment. Conclusions: Our findings provide evidence that VitD(3) attenuates neuropathic pain by preserving spinal GABAergic interneurons through the suppression of mitochondria-associated ferroptosis mediated by PKC alpha/NOX4 signaling, probably via VDR activation. VitD, alone or in combination with existing analgesics, presents an innovative therapeutic avenue for neuropathic pain.