The CLIC1-Nrf2/HO-1 pathway mediates multiple modes of neuronal cell death in cerebral ischemia–reperfusion injury: an MRI-based study in rats

To establish a rat model of cerebral ischemia–reperfusion injury (CIRI) to investigate how chloride intracellular channel protein 1 (CLIC1) exacerbates neuronal damage and to assess the neuroprotective effect of its inhibition. The potential of quantitative MRI parameters (MP2RAGE T 1 and R2*) as non-invasive biomarkers was also explored. Thirty-two SD rats were randomized into four groups: Sham, MCAO/R, NC + MCAO/R, and CLIC1-GS. Following MCAO/R modeling and intracerebroventricular lentivirus injection, multi-parametric MRI (MP2RAGE T 1 and R2*) was performed on day 7. Hippocampal tissues were then analyzed via histology (H&E, IHC, TUNEL, IF) and molecular assays (PCR, WB, ELISA, flow cytometry). CLIC1 was associated with exacerbation of CIRI through multi-pathway mechanisms, including inhibition of Nrf2 nuclear translocation and the Nrf2/HO-1 pathway, reducing antioxidant enzymes (SOD, CAT, GSH-Px) and elevating LDH and ROS levels; promoting apoptosis by altering the Bcl-2/Bax ratio; activating the NLRP3/caspase-1/GSDMD pathway and the release of inflammatory cytokines (IL-1β, IL-18), inducing pyroptosis and triggering ferroptosis, with increased 4-HNE staining, upregulated PTGS2, and downregulated SLC7A11. CLIC1 silencing was associated with amelioration of these pathological alterations, ameliorated brain damage, and significantly reduced the elevated T 1 and R2* values in the ipsilateral hippocampus post-injury. T 1 values showed strong correlations with oxidative, apoptotic, and pyroptotic markers, while R2* values showed robust correlations with ferroptosis markers. Our findings suggest that CLIC1 contributes to CIRI exacerbation, likely by inhibiting Nrf2 nuclear translocation and the Nrf2/HO-1 pathway, thereby modulating oxidative stress, apoptosis, pyroptosis, and ferroptosis. CLIC1 silencing may be neuroprotective. MP2RAGE T 1 and R2* values show promise as non-invasive quantitative biomarkers for assessing CIRI severity and treatment response.