The Effect Of Astaxanthin On Neurodegenerative Respones In The Glia And Neurons Induced By Aβ_1-42

OBJECTIVE:Based on the documented,stablly established the cellular models of the oxidative-stress responses in the primary mixed glia,which were derived from 2-3d Sprague-Dawley rats,induced by oligomeric or fibrillar Aβ1-42 or Lipopolysaccharide(LPS)or BV-2 activation by LPS in our previous studies,to establish a couple of cellular models of oxidative-stress responses,neuroinflammatory responses or apoptosis of activated microglia,mixed glia,as well as neurons by LPS or Aβ1-42 or brain tissue induced by LPS,and to explore the effect of Astaxanthin on oxidative-stress responses with these models.Furthermore,we test the time-and concentration-dependent relations to the effect of Astaxanthin on modulating oxidative-stress reponses and apoptosis in the cellular or tissue models.METHODS:With the well-known and documented methods in the published academic papers and our previous studies,Aβ1-42 at 10μM or LPS at 100ng/mL were used to induce primary glia or neurons derived from 2-3d neonatal pups of SD rats or fresh cerebral cortex tissue derived from the brains of Rhesus Macaque to produce the oxidative-stress or neuroinflammatory responses or apoptosis in reestablished glia model,our neuronal cellular model or novel monkey cerebral cortex tissue models.Nitric oxide(NO),inducible nitric oxide synthase(iNOS)、reactive oxygen species(ROS)were chosen as oxidative-stress biomarkers,Caspase-3 and Bax as apoptosis proteins,and cyclooxygenase-2(COX-2)as neuroinflammatory biomarkers of neurodegenerative toxicity either in the primary glia and BV-2 or neurons or monkey brain tissue mediated by oAβ1-42 or fAβ1-42 or LPS.Meanwhile,NO,iNOS,ROS,Caspase-3 or Bax were chosen as the targeting endpoints of Astaxanthin(AST)in the different experiments.AST was used from the concentration range of 11.7 to 200μg/mL in the initial screening assays.12.5、25 and 50μg/mL of AST were seleted to the experiments to test the concentration-dependent relations in a series of further studies after screening assays.Time course of 12h,16h/24h or 36h was applied to test time-dependent experiments.The methods of cell culture,Griess assay,Western blotting analysis,immunofluorecence staining,NBT assay and microscopy were employed in the related experiments.The first generation of primary glia cells or neuronal cultures were derived from the brain cortex of 2-3d neonatal pups of SD rats or the fresh monkey cortex tissue was derived from the brains of Rhesus monkeys(N=3).The primary mixed glia grew to confluence after seeding for 10-15 days,and then they were split to the secondary passage and then kept growing for additional 10 days until confluency.The tertiary of the glial cells were used in the experiments.The first generation of neuronal cultures cannot be split after initially seeding into the culture dishes and they were be used for experiments after growing for 25-30d.The fresh cortex tissue from the monkey brains were dissected into the similar size to that of a hemisphere of 2-3d SD rats,and were directly used for the novel-model experiments.Each experiment was repeated for three times at least.SPSS 20.0 of Statistic software was used to analyse the data from the experiments.T test was used to analyse the data generated from two-two comparison while ANOVA was used to analyse the data generated from one-way tail,multi-factor comparison.P<0.05 or below were with significant differences.RESULTS:1.In screening assay,NO was up-regulated in the conditioned medium after using 100ng/mL of LPS to incubate with BV-2 cells for 16h,which indicated that the oxidative-stress model of BV-2 successfully reestablished.Also,11.7、17.56、26.33、39.26、59.25、88.89、133.3、200μg/mL of Astaxanthin were treated with BV-2 induced by 100ng/mL of LPS for 16h,and then NO was down-regulated by Astaxanthin.According to this experiment,12.5、25 or 50μg/mL of Astaxanthin were suggested for the following experiments.2.In the established oxidative-stress models,NO was up-regulated in the conditioned medium in all experiments with the primary glia,neurons or monkey brain cortex induced by the stimuli when conparing to that in control,and the difference between two groups showed significance(P<0.05 to P<0.001),and Caspase-3 and Bax up-regulated in the neurons,iNOS and COX-2 were up-regulated in the glia cells after using 100ng/mL of LPS or 10μM of oAβ1-42 or fAβ1-42 to incubate with the glia cells,neurons or cortex tissue at the different time points.The results suggested that all the oxidative-stress models be successfully established.However,the upregulated value of NO in the conditioned medium was slightly lower in Aβ groups than that in LPS groups.3.In the application of the models with oxidative-stress damage or inflammatory responses,we found out that Astaxanthin down-regulated the targeting molecules and endpoints including NO、iNOS、ROS、COX-2、Caspase-3 or Bax in the cells or tissue induced by 100μM of oAβ1-42 or fAβ1-424.In this study,NO was obviously down-regulated in the primarny neurons induced by LPS after using p38MAPK and JNK inhibitors,which suggested that p38MAPK and JNK signaling pathways be involved in the stimulation by LPS.CONCLUSION:1.In this study,the two cellular models of oxidative-stress damage were successfully reestablished.(1)The model of microglia cell line BV-2 induced by LPS;(2)The model of primary mixed glia or microglia induced either by LPS or by Aβ1-42.The two models showed oxidative-stress reponse.2.Two novel models of oxidative-stress damage were successfully established and following below:(1)A model of the primary neurons derived from 2-3d SD rats induced by oAβ1-42 or fAβ1-42 showed neurodegenerative responses including oxidative-stress reponse and apoptosis in which were with the up-regulation of NO,Caspase-3 or Bax.(2)A model of the primary mixed glia or brain cortex tissue,which were derived from Rhesus monkeys rats,induced by LPS and showed neurodegenerative responses including oxidative-stress reponse and apoptosis in which were with the up-regulation of NO,Caspase-3 or Bax.3.We have confirmed that Astaxanthin could down-modulate the neurodegenerative responses in the glia and neuronal cells induced by oAβ1-42 and fAβ-42.It demonstrated Astaxanthin was with anti-oxidative stress,anti-apoptosis or anti-inflammtory effects in this study.Also,the results indicated that MAPK signaling pathway was involved in the mechanisms of Alzheimer’ s disease.