Effect Of Astaxanthin On Oxidative Stress And Inflammatory Response In Primary Microglia Mediated By LPS

Objective:Chronic oxidative stress and neuroinflammatory responses have been long lasting in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease(AD).Nitric Oxide(NO),Inducible Nitric Oxide Synthase(i NOS),Cyclooxygenase-2(COX-2)and Interleukin-6(IL-6)were up-regulated in those diseases,which were the tested endpoints of the activated microglia and the main targets of screening anti-oxida-nt or anti-inflammatory drugs.Astaxanthin(AST),a strongest oxidant,was tested if it was with the efficacy of modulating the activated microglia that were isolated from2-3d pups of Sprague-Dawley(SD)rats or oxidative stress and inflammatory responses on the Kunming mice induced by LPS.The aim of this study was to understand the relationships bet-ween the oxidative stress and neuroinflammation in microglial activation and three the sub-pathways of MAPK signaling pathway.Methods:The mixed glial cells were derived from the cortex of the brains of 2-3d pups,SPF-grade Sprague Dawley(SD)rats.The primary microglia were isolated by Shake-off on an orbit shaker when the cultures grew to complete confluence.The primary mixed glia were with astrocytes and microglia.To confirm them,immunofluorescence staining was used to mixed cultures or microglial cultures grown on the round slides in 12-well plates.GFAP antibody was labeled with green fluorescence while CD11b antibody labeled with red.GFAP was the specific antigen of astrocytes while CD11b antibody was for identifying microglia.The morphology was recored by a microscope and a computer.Counting DAPI for total numbers of the cells and counting the red for microglia while counting green for astrocytes by microscopy.Thus,the purity of microglia was counted by 5 views each slide under a microscope,and the purity of microglia was calculated by getting the the average of the five numbers.To get the primary microglia activated with oxidative stress and neuroinflammatory responses,the cells were treated with 100ng/ml of LPS for 12h,24h or 36h.The supernatant of the microglia seeded with the density at 1.25 x 10~5in 24-well plates was obtained for measure of NO by Griess Assay after incubation with LPS for 12h,24h or 36h.Meanwhile,the cell lysates were harvested and tested for i NOS,COX-2 or IL-6through Western Blotting Analysis.In vivo,to get oxidative stress and inflammation,1mg/kg of LPS was injected to Kunming mice via,and the serum was collected from each mouse and stored at 4℃until use.There were 3 mice in each group in which were control,LPS or AST.The animals were pretreated with AST at 50mg/kg for 3days before giving LPS injection.Along with LPS,AST was remained to be administered until the experiments were finished at 24h,36h or 60h.Results:Rat astrocytes and microglia in the mixed primary cultures were confirmed respectively via immunofluorescence staining with specific GFAP or CD11b antibodies.DAPI in blue for the nuclei of the cells.The purity of primary microglia was at 98.6%at average.NO,i NOS,COX-2 or IL-6 were upregulated in the activated primary microglia mediated by LPS for 12h,24h or 36h.To compare with control,the upregulation of each endpoint in LPS group,the difference was significant(P<0.05),which suggested that the model of oxidative stress and neuroinflammatory response of primary microglia was successfully established.The microglia with AST treatment and LPS co-incubated for 12h,24h or 36h,the endpoints of NO,i NOS,COX-2 or IL-6were diferently downregulated with the time points.Furthermore,AST also down-regulated oxidative stress response in animal experiments which showed that NO in serum was upregulated in LPS alone while it was downregulated in AST and LPS co-incubation for 24h,36h or 60h.However,there were no significant differences.Conclusion:LPS mediated oxidative stress and inflammatory responses in rat primary microglia,and led Kunming mice be with oxidative stress state.The cellular or animal oxidative-stress or inflammatory models induced by LPS were successfully established.AST could downregulate the oxidative stress and/or inflammatory responses in the primary microglia or NO in serum in Kunming mice.Also,MAPK pathway involved in the oxidative stress and inflamma-tory responses.AST working mechanisms were not completely clear in this study,and it needed to be explored in the future experiments.AST could be a potentially useful candidate drug in clinics.