Effects Of Lycium Barbarum Polysaccharides On Keap1-Nrf2/HO-1 Pathway After Cerebral Ischemic Injury

ObjectTo investigate whether LBP exerts beneficial effects mediated by theKeap1-Nrf2/HO-1 signaling pathway after cerebral ischemic injury in vivo and in vitro,We used the model of middle cerebral artery occlusion（MCAO）in mice and oxygen/glucose deprivation（OGD）in PC12 cell.Methods1.In present experiments,the mice were subjected to middle cerebral artery occlusion 2h,reperfusion 22h to preparation model.（1）The change of Regional cortical blood flow（r CBF）in the ischemic cortex of mice was detected by laser speckle imager;（2）The total power of ischemic cortex in mice was recorded by EEG;（3）The Nissl staining and FJB staining were used to observe the ischemia brain tissue histopathology;（4）The protein levels of Nrf2,Keap-1,HO-1,LC3-Ⅱand Beclin-1 in the ischemic brain tissue were detected by Western blotting.2.The cell injury model was established us ing PC12 cells with oxygen/glucose deprivation-reoxygenation.（1）MTT assay was used to detect the cell viability;（2）The intracellular ROS levels was assayed by Fluorescence spectrophotometer;（3）The laser scanning confocal microscopy was used to evaluate Ca²⁺content and mitochondrial membrane potential by Fluo-3fluorescent probe and JC-1;（4）The protein levels of Nrf2,Keap-1,HO-1,LC3-Ⅱand Beclin-1 in PC12 cells were detected by Western blotting.Results1.LBP Exerts Neuroprotection Effects on Focal Cere bral Ischemia in MiceCompared with the model group,（1）the LBP group（40 mg/kg）significantl y reduced the score of neurological deficit（p<0.05）;（2）LBP group significantl y increased the r CBF（p<0.05）.（3）LBP group could increase the total power of cerebral cortex（p<0.05）;（4）The LBP group（40mg/kg）prevented neuronal cell death.Compared with LBP group（40mg/kg）,Nrf2 inhibitor（1mg/kg）group reduced neurological deficits,r CBF,cerebral cortex total power and aggravated neuronal necrosis in ischemic brain tiss ue.2.LBP Exerts Protection Effects on oxygen/glucose deprivation in PC12 cellsCompared with OGD group,In the LBP（40μg/ml）group,the cell viabilit y and mitochondrial membrane potential of PC12 cells were significantly increased（p<0.05）;the level of intracellular free calcium and ROS were significantly decreased（p<0.05）（40μg/ml）.Nrf2 inhibitor（4μg/ml）group can partiall y eliminate the effects of LBP on cell viability,mitochondrial membrane potential,ROS levels,calcium content.3.Effect of LBP on Keap1-Nrf2/HO-1 Signaling Pathway After Focal Cerebral Ischemia in MiceCompared with model group,LBP（40mg/kg）group could increase the protein levels of Nrf2 and HO-1 in ischemic brain tissue significantly,but such rise in levels was attenuated by Nrf2 inhibitor（1mg/kg）in the LBP+Bru group relative to that in the LBP group.Compared with model group,LBP（40mg/kg）group could significantly reduce the expression of Keap-1,LC3-Ⅱand Beclin-1protein in ischemic brain tissue（p<0.01）.In contrast,Nrf2 inhibitor（1mg/kg）group increased the expression of Keap-1,LC3-Ⅱand Beclin-1 protein in ischemic brain tissue（p<0.05）.4.Effect of LBP on Keap1-Nrf2/HO-1 Signaling Pathway After Oxygen/Glucose Deprivation in PC12 cellsCompared with OGDgroup,LBP（40μg/ml）treated group could increase the protein levels of Nrf2 and HO-1 significantly in PC12 cells.In contrast,Nrf2inhibitor group decreased the expression of Nrf2 and HO-1 protein（p<0.05）.Compared with OGD group,LBP（40μg/ml）group could significantly reduce the expression of Keap-1,LC3-Ⅱand Beclin-1 protein in PC12 cells（p<0.01）.In contrast,Nrf2 inhibitor group increased the expression of Keap-1,LC3-Ⅱand Beclin-1 protein（p<0.05）.Conclusion1.The present study demonstrated that LBP ex erted neuroprotective effects after cerebral ischemic injury.2.LBP exerted protective effects after cerebral ischemic injury the underl ying mechanisms may include activating the Nrf2/HO-1 pathway.