Diosmetin Alleviated Cerebral Ischemia/Reperfusion Injury Through SIRT1/Nrf2 Signaling Pathway In Vivo And In Vitro

Background Ischemic stroke is a leading cause of death and long-term disability worldwide and imposing a heavy burden on family and society.However,recanalization of ischemic brain tissue could lead to cerebral ischemia reperfusion injur（CI/RI）,which exacerbate neuronal injury.Objective This study was designed to explore the neuroprotective effect and underlying mechanism of diosmetin（Dios）on cerebral ischemia/reperfusion injury in vivo and in vitro.It is expected to provide a theoretical basis for the clinical application and new drug development of Dios in the prevention and treatment of ischemic stroke and other ischemi c encephalopathy.1.Protective effect and underlying mechanism of Dios on cerebral I/R injury in ratsMethods（1）60 healthy SPF male SD rats were randomly divied into 5 groups:sham,I/R,I/R+Dios（40mg/kg）,I/R+Dios（80mg/kg）,and I/R+Dios（80mg/kg）+EX527.The rats in groups of I/R+Dios（40mg/kg）,I/R+Dios（80mg/kg）,and I/R+Dios（80mg/kg）+EX527 were gavaged with corresponding Dios respectively,once a day for 7 days.The rats in sham and I/R groups were gavaged with the same amount of physiological saline.After 1h of the last intragastric administration,the right middle cerebral artery occlusion（MCAO）model was established.After 1.5h of occlusion,the monofilament was gently removed to restore blood flow and allow 24h reperfusion.（2）The neurological function of all groups of rats was observed and scored.（3）The cerebral infarct volume was observed by TTC staining.（4）HE and Nissl staining were used to observe cell morphological alterations and the number of Nissl bodies in the right cortical area of rats.（5）The activity of SOD、CAT and the content of MDA and GSH were detected by biochemical method.（6）The proteins expression of SIRT1、T-Nrf2、N-Nrf2、NQO1、HO-1 were measured by western blot.（7）All the experimental data were analyzed using SPSS 20.0 statistical software,and graph generations were performed in Graph Pad Prism 6.0.Results Pretreatment with Dios significantly reduced cerebral I/R rat neurological deficit,cerebral infarct volume,the destruction of cell morphology and neuronal damage.Meanwhile,Dios evidently increased activity of SOD and CAT,increased GSH content,decreased MDA level.And Dios increased the proteins expression of SIRT1、T-Nrf2、N-Nrf2、NQO1 and HO-1.However,the protective effects of Dios were blocked by SIRT1 inhibitor EX527.2.The Protective effect and underlying mechanism of Dios on OGD/R-induce d PC12Methods（1）Screening of drug concentrations:PC12 cells were divided into 10 groups:Control group,OGD/R group,OGD/R+DMSO group,Dios-treated groups（OGD/R+Dios5n M,10n M,20n M,40n M,80n M,160n M）,si RNA-NC group.To simulate cerebral I/R in vitro,the cells were cultured using a glucose-free medium under a 93%N₂,5%CO₂and 2%O₂atmosphere at 37℃for 2h.Then,the cells were cultured with normal serum-contained normal medium for 24h.Cell viability was detected by MTT,and two concentrations of Dios were selected for subsequent experiments.（2）LDH released was detected by the LDH cytotoxicity assay kit.（3）The activity of SOD、CAT and the content of MDA and GSH were detected by biochemical method.（4）ROS level was measured by the fluorescence method using DCFH-DA.（5）The proteins expression of SIRT1、T-Nrf2、N-Nrf2、NQO1、HO-1 were measured by Western blot.（6）All the experimental data were analyzed using SPSS 20.0statistical software,and graph generations were performed in Graph Pad Prism 6.0Results The results showed that Dios significantly increased cell viability,decreased LDH release and ROS level in OGD/R-induced PC12 cell.Consistent with in vivo experiments,Dios increased the activity of SOD and CAT,increased GSH content,decreased the content of MDA.Furthermore,Dios increased the proteins expression of SIRT1、T-Nrf2、N-Nrf2、NQO1 and HO-1.However,the protective effect of Dios on OGD/R induced PC12was partially eliminated after silencing SIRT1 by si RNA transfection.Conclusion Dios could attenuate CI/RI by inhibiting oxidative stress,and the underlying mechanism might be partially achieved by activating SIRT1/Nrf2 signaling pathway.