RIN1 regulates ferroptosis and nociceptive perception via the Nrf2/HO-1 pathway in chronic constriction injury

Neuropathic pain (NP) has been a major focus of clinical research for decades. This study investigates the function of RAS- and RAB-interacting protein 1 (RIN1) in modulating NP and explore the involvement of the nuclear factor-2 erythroid factor-2 (Nrf2) and heme oxygenase 1 (HO-1) pathway in this context. A rat model of CCI was generated. The presence of mechanical and thermal hypersensitivity, as well as spontaneous pain behaviors, confirmed the successful modeling. Intrathecal injection of AAV9-shRNA targeting RIN1 attenuated nociception, reduced microglial activation in the L4-L6 spinal cord, and decreased the expression levels of c-Fos, GFAP, and IBA-1. Furthermore, the levels of NMDAR, PKC, Src, enzymes linked to neural hypersensitivity, was inhibited by RIN1 silencing. RIN1 was found to interact to Nrf2 protein, inhibiting its nuclear translocation and transcriptional activation. The RIN1 knockdown activated the Nrf2/HO-1 pathway, reducing oxidative stress and ROS levels in the spinal cord, while increasing the expression of Nrf2-target genes, including Nqo1, Gclc, and Gclm, which are key players in cellular antioxidant defense. Additionally, ferroptosis, characterized by mitochondrial damage and elevated Fe²⁺ levels, was reduced in RIN1 knockdown rats. Treatment with Nrf2 or HO-1 activators improved pain sensitivity and reduced inflammation, while inhibition of Nrf2 activity attenuated the protective effects of RIN1 silencing. In vitro, RIN1 silencing reduced activation LPS-treated of mouse BV2 microglial cells, leading to a decrease in the secretion of pro-inflammatory cytokines (IL-6, TNFα, and IL-1β), reduced microglial ferroptosis, and decreased the cytotoxicity of BV2 cells to co-cultured neurons. These effects were mediated by the Nrf2 pathway, as Nrf2 antagonism reversed the effects of RIN1 knockdown. These findings suggest that RIN1 plays a critical role in spinal cord hypersensitivity and pain perception by inhibiting the Nrf2/HO-1 pathway, influencing neuroinflammation and ferroptosis. Targeting RIN1 could provide a potential therapeutic strategy for managing NP and neuroinflammation.