| Objective: Chronic cerebral ischemia is caused by the long-term cerebral hypoperfusion, which is clinically one of the common brain injuries, and is a common pathological process that usually occurs in conditions such as Alzheimer's disease and vascular dementia, both of which are characterized by cognitive impairment. Oxidative stress plays an important role in nerve cell damage and cognitive dysfunction induced by chronic cerebral ischemia, but the PI3K/AKT/Nrf-2/HO-1 signaling pathway plays an important role in inhibiting oxidative stress. This study aims to observe the antioxidation of Curcumin on chronic cerebral ischemia model in rats and PC12 ischemic cell models and investigate the molecular mechanism of Curcumin inducing HO-1 and Nrf-2, which is PI3K/AKT/Nrf-2/HO-1 signaling pathway related factors as the research target.Methods: The chronic cerebral ischemia was produced in male Sprague-Dawley rats by permanent occlusion of bilateral common carotid arteries (2VO). Animals were randomly divided into 5 groups: normal control group, sham-operated group, 2VO+DMSO group, 2VO+Curcumin 100mg/kg group, 2VO+Curcumin50mg/kg group. After surgery, all animals were injected intraperitoneally with DMSO solution of Curcumin or a same volume of normal DMSO. Each group was injected once daily for four consecutive weeks. The spatial learning and memory ability was tested by Morris water maze after administration for 28d. After the completion of the behavioral testing, rats were sacrificed. Hippocampus was used to spectrophotometrically determine the level of MDA and the activity of SOD. The pathological changes in the hippocampus CA1 area were observed with hematoxylin and eosin (HE) staining and Nissl staining. Western Blot was used to detect the levels of PI3K,AKT,Nrf-2 and HO-1 protein. Nrf-2 and HO-1 protein were located by immunohistochemistry.The PC12 ischemic cell models were cultured in vitro and treated with different concentrations of Curcumin ( 0,1.25,5.0,20μmol/L) for 24h. The fluorescent probe DCFH-DA and fluorescent spectrophotometer were performed to detect the levels of the active oxygen, to observe the protective effect of Curcumin on PC12 cells.PC12 ischemic cell models were divided into 9 groups: control group (0μmol/L Curcumin); empty plasmid group; Curcumin group (5μM) ; Curcumin+ LY294002 group (5μM); Curcumin+ LY294002+ Nrf-2 siRNA group; LY294002+ Nrf-2 siRNA group; LY294002 group; Nrf-2 siRNA group; Curcumin+ Nrf-2 siRNA group. Nrf-2 siRNA or empty plasmid was transiently transfected in PC12 cells by LipofectaminTM2000, and then the cells were treated for 24h. The protein expression of PI3K,AKT,Nrf-2 and HO-1 were detected by Western Blot.Results: The results displayed that Curcumin significantly improved the spatial learning and memory and attenuated pathological change in the hippocampus Cornu Ammonis layer 1 (CA1) area. The level of MDA decreased, but the activity of SOD increased, and the changes were in a dose-dependent manner (p<0.05). In the hippocampus the levels of PI3K,AKT,Nrf-2 and HO-1 protein significantly increased after treated with Curcumin. Immunohistochemistry results not only confirmed the above changes, but also displayed that Nrf-2 had translocated into the nuclear gradually with the increased dosage of Curcumin.In vitro the levels of the active oxygen decreased in PC12 cells after treated with Curcumin, and the changes were in a dose-dependent manner (p<0.05). Western Blot datas showed that the protein levels of PI3K,AKT,Nrf-2 and HO-1 increased in Curcumin group, whereas, the protein expression of PI3K,AKT,Nrf-2 and HO-1 decreased or vanished after treated with Nrf-2 siRNA or LY294002.Conclusion: Curcumin has obviously neuroprotective effect on brain injury induced by chronic cerebral ischemia and can inhibit oxidative stress induced by ischemia. It is the main performance that improving the spatial learning and memory, attenuating pathological change, decreasing the level of MDA, increasing the activity of SOD and inducing HO-1 protein expression. Our data demonstrated that the neuroprotective effect of Curcumin involved in activating PI3K/AKT/Nrf-2/HO-1 signaling pathway, and then promoting Nrf-2 transfer to the nuclear, increasing the protein levels of downstream product HO-1, and inhibiting oxidative stress induced by ischemia.
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