Taurine Protects Spatial Learning And Memory Ability In Paraquat And Maneb Co-exposure Mouse Through Inhibition Of NOX2

Modern agricultural production has exposed human to pesticides and upgraded the degree of environmental pollution.Chronic exposure to pesticides is associated with a series of mechanisms of neurodevelopmental and neurodegenerative diseases,including attention deficit/hyperactivity disorder,Alzheimer’s disease（AD）,Parkinson’s disease（PD）and autism spectrum disorders.There is the clear evidence that these mechanisms affecting the learning and memory function,placing a heavy burden on numerous families and the entire society.The effects of chronic exposure to pesticides on central nervous system（CNS）are still understood in the limited vision.There has not yet effective method to prevent and cure the impairment of learning and memory function caused by long-term and low-dose pesticides exposure.Therefore,it is of great significance to study the mechanism of nerve system injury caused by pesticides exposure.Combined exposure to pesticides is closer to the human’s actual exposure to a variety of environmental pollutants.Paraquat（PQ）and Maneb（MB）are the regular herbicide and fungicide respectively.Epidemiological investigations and laboratory studies have found that exposure to PQ+MB can cause pathological changes in the brain,and the long-term and low dose exposure to various types of pesticides including PQ and MB significantly increases the prevalence rate of PD.Many studies and autopsies of patients show these are associated with the significant chronic neuroinflammation of CNS caused by PQ and MB.The neuroinflammation in the brain is correlated with the abnormal microglia aggregation and activation.Moderate microglia activation has a protective effect on neurons,but overactivation can lead to a sustained release of cytotoxic factors,including IL-6,IL-1β,NO and TNF-α,which results in the neuronal damage and loss.If this change occurs in areas of the brain associated with learning and memory ability,such as the cortex and hippocampus,it can lead to the impairment of learning and memory function.PET research confirmed hippocampal microglial activation could induce hippocampal atrophy and neuronal damage in PD dementia patients.NADPH oxidase（NOX）plays an important role in microglia activation.NOX is the main enzyme that produces reactive oxygen species（ROS）in vivo,and NOX2 is a subtype of NOX family expressed on CNS microglia with high expression.Previous studies have found that combined exposure to PQ and MB（P+M）can activate microglia through activating NOX2.Inhibition of NOX2 now gradually becomes a promising therapeutic strategy for neurodegenerative disorders,but it currently cannot be used to clinical application since current inhibitors of NOX2 lack nonspecificity and display toxicity at regular used dose.Hence,elucidating the mechanism behind NOX2 activation is particularly attracting.We expect to find the upstream signal that activates NOX2 and to explore whether inhibition of NOX2activation by regulating the upstream signal can alleviate the learning and memory impairment in mice.Pattern recognition receptors（PRRs）play an important role in the innate immune response by recognizing and binding pathogen-associated molecular patterns（PAMPs）or injury-associated molecular patterns（DAMPs）.Chronic activation of PRRs can lead to inflammatory diseases.Studies have shown that scavenger receptors（SRs）and complement receptor 3（CR3,also known asα_Mβ₂or CD11b/CD18）are two members of the PRR family and are involved in regulating NOX2 activation.However,whether SRs or CR3 is involved in NOX2 activation in PD is unclear.Taurine is the most abundant sulfur-containing free amino acid in excitable tissues such as brain,retina and cardiac muscle in mammals,which has anti-oxidation and other neuroprotective effects.Levels of taurine in hippocampal environments significantly declined in 3x Tg-AD mouse,and taurine concentrations in plasma decreased in PD afflicted vs.unafflicted patients.These findings influenced our desires to study if taurine could improve deficits in cognitive function in PD mice.Part IA Study on the Mechanism of NOX2 Activation in Microglia induced by Paraquat and ManebObjective:1.To observe whether BV2 exposure to P+M leads to an increase in ROS level.2.To confirm whether the increase of ROS in BV2 cells induced by P+M co-exposure is due to NOX2 activation.3.To investigate whether the upstream signal of NOX2 activation in microglia induced by co-exposure to P+M is CR3 or SRsMethod:1.To explore the exposure dose of P+M causing excessive activation of BV2microglia:BV2 cells were exposed to P+M at doses of 5+0.3,10+0.6 and 50+3 umol/L,respectively.Intracellular ROSlevel was detected using DHE method to respond to activationdegree of NOX2.MTT method was used to tested cell viability to determine P+Mco-exposure dose that could cause NOX2 abnormal activation without significantly impairing cell viability.2.BV2 cells were administrated NOX2 inhibitor apocynin（Apo）or DPI for an hour,then BV2 cells were exposed to P+M for24hours,intracellular ROS content was detected by DHE method,extracellular superoxide content by WST-1 method.After Apo or DPI administration,BV2 cells were exposed to P+M for an hour,Western blot was used to detect changes in p47phox level in membrane and cytoplasm respectively to determine whether NOX2 activation was the main source of P+M causing increased reactive oxygen levels in BV2 cells.3.One hour after administration of SRs inhibitor fucoidan,BV2 cells were exposed to P+M for 24 hours,intracellular ROS content was detected by DHE method,and extracellular superoxide content by was detected by WST-1 method.After an hour of P+M exposure,changes of p47^phoxcontent in cell membrane and cytoplasm were detected by Western blot to confirm whether the activation of NOX2 induced by P+M was regulated by SRs.4.Mixed-glia culture was prepared from whole brains of 1-3 days-old wild type（WT,C57BL/6J）and CR3^-/-fetal mice.Hence,the primary cells were used in subsequent experiments.5.One hour after administration of RGD peptide or anti-CD11b antibody,BV2cells were exposed to P+M for 24 hours,or CR3^-/-mice primary mixed-glial cells were exposed to P+M for 24 hours.Cellular ROS content was detected by DHE method,extracellular superoxide content by WST-1 method.After exposed to P+M for an hour,p47^phoxcontent in membrane and cytoplasm was detected by Western blot,respectively,and whether the activation of NOX2 was regulated by CR3 was observed.6.BV2 cells were exposed to P+M for 24 hours after administration of Saracatinib（Sara）and Erk inhibitor U0126.Intracellular ROS content（intracellular superoxide）was measured by DHE method,extracellular superoxide contentby WST-1 method.After an hour of P+M exposure,changes of p47^phoxcontent in membrane and cytoplasm were detected by Western blot to confirm whether the activation of NOX2 was via the Src-Erk pathway was observed.Result:1.Exposure to BV2 cells at a dose of 10+0.6μmol/L resulted in a significant increase in intracellular superoxide and extracellular superoxide induced by P+M.The cytoplasmic p47^phoxcould be transferred to membrane.Theresults indicated that NOX2was activated by P+M exposure.Moreover,at this exposure dose,the cell viability was not significantly affected,so the dose of 10+0.6μmol/L was applied in subsequent experiments.2.After administration of NOX2 inhibitors Apo and DPI,exposure to P+M did not cause significant changes in p47phox levels in BV2 cytoplasm and cell membrane,and significant changes in ROS and extracellular superoxide levels in BV2 cells,proving that P+M exposure caused oxidative stress comes from the activation of NOX2.3.After SRs inhibitor fucoidan was administered,P+M still significantly increased the intracellular ROS content and extracellular superoxide content in NV2cells,and the cytoplasmic p47phox shifted to the cell membrane also significantly increased,indicating that SRs are not involved in the process of P+M activating NOX2.4.After administration of CR3 inhibitor RGD,anti-CD11b antibody or CR3knockout,exposure to P+M did not cause significant changes in ROS and extracellular superoxide levels in BV2 cells,and p47phox levels in cytoplasm and cell membrane did not change significantly.It was proved that CR3 is an upstream signal receptor that P+M leads to activation of NOX2.5.After administration of Src inhibitor Sara and Erk inhibitor U0126,exposure to P+M did not cause significant changes in ROS and extracellular superoxide content in BV2 cells,and the cytoplasm and cell membrane p47^phoxlevels did not change significantly.It is proved that Src-Erk is involved in the activation of NOX2 by P+M.Conclusion:1.P+M co-exposure resulted in over-activation of NOX2 in mouse CNS microglia;2.The excessive activation of NOX2 in microglia caused by P+M co-exposure is mediated by CR3;3.Microglia CR3 regulates NOX2 activation by P+M combined exposure via Src-Erk pathway.Part II Taurine decreased NOX2levelto improve the spatial learning and memory function of mice induced by Paraquat and ManebObjective:1.To clarify the protective effect of taurine on spatial learning and memory impairment in mice exposed to P+M;2.To explore the protective effect of taurine on mouse cortical and hippocampal neurons and its mechanism3.To elucidate the mechanism of taurine inhibiting P+M-induced abnormal microglial activation;Method:1.Animal dosing:Male C57BL/6J mice（12 weeks old）were randomly divided into three groups:control（Con）,exposure group（P+M）and intervention group（Tau+P+M）,12～15 mice in each group.In P+M group,the mixture of 5ul/g PQ（10mg/kg）and MB（30mg/kg）was injected intraperitoneally.In Tau+P+M group,150mg/kg of taurine was injectedintraperitoneally 30 minutes before P+M injection.Thecontrol mice were injected intraperitoneally with 5ul/g of 0.9%saline.Continuous injection for 6 weeks,twice a week.Two days after the last injection,Morris water maze（MWM）test was performed in 10～12 mice in each group.After MWM test was finished,mice were euthanized and hippocampus were dissected preserved at-80 C.For Immunohistochemistry,6mice whole brain was collected by perfusion of 4%paraformaldehyde after anesthesia.2.MWM test:including the navigation test and the space exploration test for a total of 5 days.On the 1st to the 4th day,the navigation test was conducted to evaluate the learning ability.On the fifth day,the space exploration test was performed to evaluate the spatial memory ability of mice.3.The levels of cortical and hippocampal tissue Iba-1,Neu-N and PSD-95expression were detected by immunohistochemicalin three groups of mice to reflectmicroglia activation,number of hippocampal neurons and synaptic plasticity levels,respectively.The mechanism of taurine remission P+M combined exposure of mice with learning and memory ability was clarified.4.Western blot was used to detect the expression levels of Neu-N,active caspase-3,p47^phox,gp91phox,CR3,p Src/Srcandp Erk/Erk in the hippocampus of three groups of miceand to investigatethe mechanism of taurine inhibiting NOX2inalleviating the learning and memory ability of mice exposed to P+M.Result:1.In MWM test,the escape latency and swimming distance of mice in P+M group were significantly higher than those in control group and Tau+P+M group,and the cumulative time of target quadrant swimming was significantly shorter than those in the other two groups.These results suggest that taurine can effectively alleviate the impairment of spatiallearning and memory caused by P+M.2.The results of immunohistochemical experiments showed that the levels of Iba-1in the cortex and hippocampus of the P+M group were significantly higher than those of the control group and the Tau+P+M group,and the levels of Neu-N and PSD-95were significantly lower than those of the other two groups.This shows that P+M leads to excessive activation of mouse cortex and hippocampal microglia,decreased number of neurons and impaired synaptic plasticity.Taurine has a significant relief effect.3.Western blot results showed that,compared with the P+M group,taurine significantly increased the Neu-N expression level in the hippocampus of the Tau+P+M group and significantly reduced the active caspase-3 level,indicating that the hippocampus caused by P+M The decrease in the number of neurons is due to an increase in the neuronal apoptosis level,and taurine can reduce the hippocampal neuron apoptosis level caused by P+M.Western blot results showed that taurine significantly increased the expression of Neu-N in the hippocampus of Tau+P+M group and decreased the level of active caspase-3,suggesting that the decrease in the number of hippocampal neurons caused by P+M was due to the increase of the apoptotic level of neurons,and taurine could reduce the apoptotic level of hippocampal neurons induced by P+M.4.Taurine significantly reduced the levels of p47phox,gp91phox,CR3,p Src/Src,and p Erk/Erk in the hippocampus,indicating that the protective ability of taurine on the learning and memory of P+M-exposed mice is due to the inhibition of CR3expression,and thus via Src-Erk Signaling pathways reduce NOX2 expression,relieve neuroinflammation and protect hippocampal neurons and synapses.Conclusion:1.Impaired spatial learning and memory in mice caused by P+M combined exposure is related to a decrease in the number of hippocampal and cortical neurons and impaired synaptic plasticity.2.The damage of hippocampal neurons and synapses in mice combined with PM exposure is related to the increase of microglia NOX2 level.3.Taurine can improve spatial learning and memory function in mice by reducing hippocampal CR3 content and regulating NOX2 expression in P+M exposed mice.4.Taurine reduced hippocampal CR3 in mice exposed to P+M and regulated NOX2 through the src-erk pathway.