The Effects Of NOX2-NLRP1 Signaling Pathway In Brain Aging Injury Of Mice And The Regulation Of Ginsenoside Rg1

Brain aging refers to the phenomenon that the learning and memory function of the brain tissue declines with the increase of age after the body matures,and its tissue morphology changes.Oxidative stress and neuroinflammation are closely related to the development of brain aging and related diseases.NADPH oxidase 2(NOX2)is a major source of reactive oxygen species(ROS)production in the brain.Increased production of ROS is the main pathway for activation of inflammasomes.Activation of inflammasome of NLRP1 regulates the maturation and secretion of cytokine precursors pro-IL-1β and pro-IL-18,leading to inflammation in the body damage.Ginsenoside Rg1 has anti-aging and anti-oxidation effects.This study investigated the effects of NOX2-NLRP1 signaling pathway on brain aging damage in mice and the regulation of Rg1 on NOX2-mediated activation NLRP1 inflammasome and brain aging injury in SAMP8 mice.This study may find new targets and ideas for prevention and treatment of brain aging injury.Part One The effects of NOX2-NLRP1 signaling pathway in brain aging injury of natural aging mice ObjectiveTo research the effect and mechanism of NOX2-mediated NLRP1 inflammasome activation in mice brain aging injury,and the correlation between NOX2-NLRP1 signaling pathway and brain aging injury in natural aging mice.MethodsMale 6-month-old KM mice were randomly divided into 6-month-old youth group,16-month-old group,20-month-old group,and 24-month-old group.Each group of mice was routinely raised and fed freely.The mice in each group were weighed and observed monthly,and the changes of body weight were recorded.The changes of exercise and inquiry behavior of mice at 6 months,16 months,20 months and 24 months were observed in the open field experiment.The changes of learning and memory in mice at6 months,16 months,20 months and 24 months were measured by using Morris water maze.The pathological changes of neurons in the cortex and hippocampal CA1 and CA3 regions were observed by HE staining.DHE fluorescence was used to detect the production of ROS in mouse cortex and hippocampal CA1 and CA3 regions.β-galactosidase staining was used to detect the brain aging in each group.And the expressions of NOX2,p22 phox,p47phox,NLRP-1,ASC,caspase-1 and IL-1β were detected by Western Blot.ResultsBody weight measurements showed a gradual increase in body weight from 6months to 24 months of age.The results of open field experiments showed that the activity and exploration behavior decreased significantly as the mice growing older.Morris water maze experiments showed that compared with 6M mice,the 16 th,20th,and 24 M mice showed significant memory loss.The results of DHE showed that compared with 6-month-old mice,16-month-old ROS production increased,but the difference was not significant,while in 20-month-old and 24-month-old mice,the ROS production was significantly increased in brain cortical neurons,and the age group is particularly evident.The results of β-galactosidase staining showed that the expression of β-galactosidase in the cortex and CA1 and CA3 areas of the 6-month-old mice was less,compared with 6-month group,the expression of β-galactosidase was significantly increased in the 20-month-old and 24 months old.The results of HE staining showedthat the neurons in the hippocampus of the 6 and 16-month-old group were uniformly stained,no abnormal changes,clear nuclear outline,abundant cytoplasm in cortex and hippocampal neurons.While in 20-month-old and 24-month-old mice,there were obvious neuronal damage in cortex and hippocampus,especially in 24-month-old mice.The Western Blot results showed that,compared to the 6 month-old mice,the expressions of NOX2,p22 phox,p47phox,NLRP1,ASC,caspase-1 and IL-1β were significantly increased in 20-month-old and 24-month-old mice.Part Two The protective effect of ginsenoside Rg1 on brain aging injury in SAMP8 rapid aging mice ObjectiveTo study the protective effect and mechanism of ginsenoside Rg1 on brain aging injury in SAMP8 rapidly aging mice.This study may provide theoretical support for the prevention and treatment of Rg1 in the anti-aging process.MethodsMale 7-month-old SAMR1 mice were served as a control group,and SAMP8 rapidly aging mice were randomly divided into: model group,ginsenoside Rg1(5,10mg/kg)group,Tempol(50 mg/kg)and Apocynin(50 mg/kg)group.The mice were routinely raised and fed freely.The drugs were treated intragastrically with the corresponding drug(0.1 ml/10g)and continuously administered for 2 months.The control group and the model group were intragastrically administered with equal volume distilled water.The mice body weight was measured monthly.The moveing activity and exploratory behavior were examined in the open field experiment.New body recognition experiments were performed to observe the changes in learning and exploration ability of mice in each group.DHE fluorescence method was used to detect ROS production.Western Blot was used to detect the expressions of NOX2-relatedproteins,NOX2,p22 phox,p47phox and NLRP-1 inflammasome-associated proteins,NLRP1,ASC,caspase-1 and IL-1β.ResultsThe results showed that there was no significant difference in body weight before administration.The body weight of SAMR1 group increased slowly after administration,while the SAMP8 group showed a slow decrease in body weight.Compred with SAMP8 mice,Rg1(10 mg/kg)treatment significantly increased the body weight.The results of open field experiment and new body recognition experiment showed that compared with SAMR1 control mice,the activity was significantly reduced in SAMP8 group.There was an increasing trend in motor ability and exploration learning ability in Tempol,Apocynin and Rg1(5,10 mg/kg)treatment groups.DHE results showed that compared with the SAMP8 model group,the ROS production in the brain cortex and hippocampal CA1 and CA3 regions were significantly reduced in the Tempol,Apocynin and Rg1(5,10 mg/kg)groups.The results of HE staining showed that there was no obvious nuclear pyknosis and deep nuclear staining in SAMR1 mice.And in SAMP8 rapid aging model group there were markedly neuronal damage in cortex and hippocampal CA1 and CA3 areas.While treatment with Tempol,Apocynin,and Rg1(5,10 mg/kg)significantly attenuated the neuronal damage.Western Blot results showed that compared with the SAMR1 control group,the expressions of NOX2,p22 phox,p47phox,NLRP1,ASC,caspase-1 and IL-1β were significantly increased in SAMP8 group.Compared with SAMP8 group,tempol,apocynin and Rg1(5,10 mg/kg)treatment significantly reduced the expression of NOX2,p22 phox,p47phoxNLRP1,ASC,caspase-1 and IL-1β in SAMP8 mice.ConclusionThe experimental results show that with the age of mice,the learning and memoryfunction of mice begins to decline,and memory function decreases significantly at 20 and 24M,and the ROS production and β-galactoside enzymes increased significantly.Meanwhile,the expressions of NADPH oxidase and NLRP-1 inflammasome associated proteins were significantly increased.The study suggests that NADPH oxidase-mediated NLRP-1 inflammasome activation plays an important role in the aging process.The SAMP8 mice showed obvious brain aging at 9 months.Compared with the SAMP8 model group,treatment with Tempol,Apocynin and Rg1(5,10 mg/kg)significantly reduced the ROS production,attenuated neuronal damage in the cortex and hippocampus,and inhibited the expressions of NOX2 and NLRP1 inflammasome associated protein.The study suggsets that inhibition of NOX2 can inhibit oxidative stress and neuroinflammatory damage to neurons,and closely relate to brain protection and anti-neuronal aging.