Investigation of the mechanism by which sigma-1 receptor protein mediates the IRE1-XBP-1s pathway to alleviate cerebral ischemia-reperfusion injury

Objective: To investigate how pentazocine (PTZ) regulates the Inositol-Requiring Enzyme 1-X-box Binding Protein 1 Spliced(IRE1-XBP-1s) pathway via sigma-1 receptor (sigma-1R) to mitigate cerebral ischemia-reperfusion injury.

Methods: In vivo, 220-250 g rats were randomly assigned to control, Middle Cerebral Artery Occlusion/Reperfusion (MCAO/R), MCAO/R + PTZ, and MCAO/R + PTZ + BD1047 (sigma-1R inhibitor) groups. The MCAO model was established in all but the control group. PTZ group received 3 mg/kg PTZ daily for 3 consecutive days pre-MCAO/R; PTZ + BD1047 group received additional 3 mg/kg BD1047. Behavioral changes were assessed using Zea-longa neural function scores. Protein expressions of sigma-1R, IRE1, C/EBP Homologous Protein(CHOP), and XBP-1s were analyzed by Western blot; sigma-1R distribution was examined through immunohistochemistry. In vitro experiments involved human neuroblastoma cell(SH-SY5Y) divided into control, Oxygen-Glucose Deprivation/Reoxygenation (OGD/R), OGD/R + PTZ, and OGD/R + PTZ + BD1047 groups. Apart from untreated controls, cells at 70-80% confluency were subjected to 4 h hypoxia in glucose-free EBSS (37 °C, 5% CO₂, 95% N₂) followed by 18 h reoxygenation in complete medium. During reoxygenation: OGD/R group received no intervention, OGD/R + PTZ group received 10 μM PTZ, OGD/R + PTZ + BD1047 group received 10 μM PTZ plus 20 μM BD1047. Protein levels of sigma-1R, p-IRE1, XBP-1s, CHOP and cleaved Caspase-3 were measured; localization of sigma-1R was determined via immunofluorescence; Apoptosis rates were quantified using flow cytometry.

Results: In vivo experiments demonstrated that compared to the MCAO/R group, the MCAO/R + PTZ group exhibited significantly elevated neurological function scores, while the MCAO/R + PTZ + BD1047 group showed lower scores than the MCAO/R + PTZ group; concurrently, in vitro experiments revealed enhanced cell viability in the OGD/R + PTZ group. Both in vivo and in vitro analyses indicated that relative to the MCAO/R、OGD/R model group, MCAO/R + PTZ and OGD/R + PTZ treatment upregulated sigma-1R and IRE1 expression, downregulated p-IRE1, XBP-1s, the endoplasmic reticulum (ER) stress-related protein CHOP, and the apoptosis-related protein cleaved Caspase-3 (C-Cas-3), and reduced Apoptosis rates. However, BD1047 administration reversed these protective effects of PTZ.

Conclusion: PTZ activates sigma-1R to regulate the IRE1-XBP-1s pathway which inhibits endoplasmic reticulum stress and Apoptosis thereby alleviating cerebral ischemia-reperfusion injury.

Cell apoptosis; Endoplasmic reticulum stress; Ischemia–reperfusion injury; Pentazocine; Sigma-1R.