Abstract: Objective To investigate the therapeutic effects of D-pinitol (DP) on rats with cerebral ischemia-reperfusion injury (CI/RI). Methods Rats were randomly divided into 6 groups with 12 in each: sham group (sham), model group (CI/RI rat model established by the modified Longa suture method), low (L-DP), medium (M-DP), and high-dose DP group (H-DP) (gavaged with 10, 20, or 40 mg/kg DP, respectively) and H-DP+inhibitor group (inhibitor, gavaged with 40 mg/kg DP and intraperitoneally injected with 30 mg/kg Nrf2 inhibitor ML385). Zea Longa score were evaluated using a double-blind method, cerebral infarction volume was assessed by TTC staining, neuronal morphology was observed via hematoxylin-eosin (HE) and Nissl staining microcopy, apoptotic cells were labeled by TUNEL staining. The activity of SOD and the contents of GSH, MDA, TNF-α, IL-1β and IL-6 in brain tissues were detected by commercial kits, Fe²⁺ content in brain homogenates was measured by visible spectrophotometer. mRNA levels of heme oxygenase-1 (HO-1), NAD (P) H quinone dehydrogenase 1 (NQO1), and glutathione peroxidase 4(GPX4) were analyzed by RT-qPCR. Protein expression of nuclear Nrf2, Keap1, HO-1, NQO1, and GPX4 in brain tissues was detected by Western blot. Results Compared with sham group, the brain injury of rats in the model group showed significant brain injury, and the neurological function score, cerebral infarction volume and the proportion of TUNEL⁺ cells were increased (P＜0.05) while GSH content and SOD activity decreased (P＜0.05). The level of MDA, TNF-α, IL-1β, IL-6 and Fe²⁺ increased (P＜0.05). The protein level of GPX4, Nrf2 (nucleus), HO-1 and NQO-1 decreased (P＜0.05), while the protein level of Keap1 increased(P＜0.05). Compared with model group, the brain injury of rats in L-DP, M-DP and H-DP group was significantly alleviated. The neurological function score, cerebral infarction volume and the proportion of TUNEL⁺ cells were all decreased (P＜0.05). The GSH content and SOD activity increased (P＜0.05), while the levels of MDA, TNF-α, IL-1β, IL-6 and Fe²⁺ all decreased (P＜0.05). The protein levels of GPX4, Nrf2 (nucleus), HO-1 and NQO-1 increased (P＜0.05), while the protein level of Keap1 decreased (P＜0.05). Compared with H-DP group, the brain injury of rats in the inhibitor group was aggravated, and the neurological function score, cerebral infarction volume and the proportion of TUNEL⁺ cells were all increased (P＜0.05). The GSH content and SOD activity decreased (P＜0.05), while the level of MDA, TNF-α, IL-1β, IL-6 and Fe²⁺ all increased(P＜0.05). The protein level of GPX4, Nrf2 (nucleus), HO-1 and NQO-1 decreased (P＜0.05), while the protein level of Keap1 increased (P＜0.05). Conclusions D-pinitol alleviates brain injury in CI/RI rats through anti-oxidation, anti-inflammation, anti-apoptosis effects and inhibition of ferroptosis.

Key words: cerebral ischemia-reperfusion injury, D-pinitol, nuclear factor E2-related factor 2, antioxidant response element, oxidative stress, ferroptosis