The Effects Of NADPH Oxidase Mediated NLRP1 Inflammasome Activation In The Aging Damage Of Primary Culture Hippocampal Neurous And The Regulation Of Ginsenoside Rg1

Brain aging is a phenomenon of gradual decline in the structure and morphology of the brain accompanied by the age,which leads to a lack of advanced brain function and cognitive impairment.The imbalance of oxidation-reduction equilibrium increase the production of reactive oxigen species?ROS?,promotes inflammatory reaction,and led to the damage of hippocampal neurons.Simultaneously,reactive oxygen species are the indispensable substance in the process of apoptosis and proliferation in cell.So,whether the oxidative stress damage of NADPH oxidase derived ROS and the promotion of inflammatory response has provoked interest of many scholars.The purpose of this study is to find new ideas and tragets for the prophylaxis and cure of brain aging recognition disfunction.Part OneObjectiveTo research the effect and mechanism of NADPH oxidase and NLRP1 inflammasome excitation in primary culture hippocampal neurons,and discuss the role of NADPH oxidase-NLRP1 inflammasome in hippocampal neurons aging damage.Methods1.Primary cultured hippocampal neurons cultured for 6 days?d?,9d,12d respectively.The DHE staining was used to observe the production of ROS in neurons.The?-GALC staining was used to observe neuronal aging.The Hoechst 33258 staining was employed to detect neuronal apoptosis.Immunofluorescence was used to observe the expression of MAP2.Western blot was employed to check the changes of NLRP1,ASC,caspase-1,IL-1?,NOX2,p47phox,p22phox and RAC1.ELISA was used to observe the release of IL-1?and IL-18 in supernatant.2.The primary cultured hippocampal neurons in 6 days were separated into 5 groups:control group,Lentivirus scramble control group,NLRP1-siRNA transfection groups and treated for another 6 days.The expression of NLRP1 was checked by Western blot.3.Primary cultured hippocampal neurons in 6 days were randomly separate into 4groups:control group,caspase-1 inhibitor group,scramble control group,NLRP1-siRNA?408?group and treated for 6 days.The?-GAL staining was used to observe neuronal aging.The neuronal apoptosis was employed by Hoechst 33258staining.Western blot was employed to observe the expression level of?-Gal and NLRP-1.ELISA was used to observe the release of IL-18 and IL-1?.Results1.Compared with 6d group,the DHE staining results showed that the reactive oxygen substances were significantly increased in 9d and 12d.The?-GAL staining results showed that the?-GAL in hippocampal neurons was significantly increased in 12d.The Hoechst 33258 staining results indicated that the hippocampal neurons apoptosis was significantly raised in 9d and 12d.Immunofluorescence results showed that the expression of MAP2 was significantly decreased in 12d.Western blot results revealed that the expression of NLRP-1,ASC,caspase-1,IL-1?,NOX2,p47phox,p22phox and RAC1 was significantly increased in 9d and 12d.ELISA results showed that the release of IL-18 and IL-1?was significantly increased in 12d.2.Compared with transfection for 3 days,the transfection for 6 days was significantly enhanced in hippocampal neurons.Western blot results showed that the expression of NLRP1 significantly reduced by NLRP1-siRNA?408?treatment for 6 days.3.Compared with normal group,the?-GAL staining results showed that caspase-1inhibitor and NLRP1-siRNA?408?treatment for 6 days significantly inhibited the hippocampal neurons aging.The Hoechst 33258 staining results showed that caspase-1inhibitor and NLRP1-siRNA?408?treatment for 6 days significantly reduced the hippocampal neurons apoptosis.Western blot results indicated that caspase-1 inhibitor and NLRP1-siRNA?408?treatment for 6 days significantly reduced the expression of NLRP-1 and?-Gal.ELISA results showed that caspase-1 inhibitor and NLRP1-siRNA?408?treatment for 6 days significantly reduced the release of IL-18 and IL-1?in supernatant.Part TwoObjectiveTo research the effect and machanism of ginsenoside Rg1 on the aging damage in primary hippocampal neurons induced by H₂O₂,and to provide support for the prophylaxis and cure of ginsenoside Rg1 in aging process.MethodsThe cultured primary hippocampal neurons in 7 days were divided into 6 groups:control group,H₂O₂?200?M?group,H₂O₂?200?M?+Tempol?100?M?group,H₂O₂?200?M?+Rg1?1?M?group,H₂O₂?200?M?+Rg1?5?M?group,H₂O₂?200?M?+Rg1?10?M?group.After 6 hours of the treatment of Tempol?100?M?and Rg1?1,5,10?M?,except the control group,the cells were treated with H₂O₂?200?M?for 18 hours.The DHE staining was used to observe the generation of ROS in hippocampal neurons.Western blot was employed to test the expression level of?-Gal,NLRP1,ASC,Caspase-1,NOX2,p22phox and p47phox.ELISA was used to observe the release of IL-1?and IL-18.ResultsThe expermental results showed that in the neuronal aging process,the activity of NADPH oxidase and the generation of reactive oxigen species in hippocampal neurons were signifiantly increased,and the NLRP1 inflammasomes were significantly activated,and leading to the aging and damage of hippocampal neurons.Caspase-1 inhibitor and down-regulation of NLRP1 could significantly reduce the aging related damage.Ginsenoside Rg1 and Tempol have anti-oxidative function,which could obviously reduce the expression of NADPH oxydase and the production of ROS and decrease NLRP1 inflammasomes.It suggest that the generation of ROS mediated by NADPH oxidase may participate in activating NLRP1 inflammasomes in hippocampal neuronal aging process,while Rg1 can inhibit the expression of NADPH oxidase and NLRP1inflammasomes,thus it has productive effect on the aging damage of hippocampal neurons.ConclusionThe expermental results showed that the neuronal aging process obviously increased the activity of NADPH oxidase and the generation of reactive oxigen species?ROS?in hippocampal neurons,activating NLRP1 inflammasomes,led to the senescence and damage of hippocampal neurons.Ginsenoside Rg1 and Tempol have anti-oxidative function,which can obviously reduce the production of ROS and NLRP1inflammasome expression.It suggest that NADPH oxidase mediated the generation of ROS may participate in activating NLRP1 inflammasome in hippocampal neuronal aging process,while ginsenoside Rg1 can inhibit the expression of NADPH oxidase and NLRP-1 inflammasomes,thus it has productive effect on the aging damage of hippocampal neurons.