Roles Of NOX2 And Dextromethorphan In LPS-induced Sickness Behavior

Object:As a safe and widely use antitussive in clinic,Dextromethorphan is regarded as morphine drug with little addiction,because of its’dextrorotation structure and lower affinity with opioid receptor.Previous studies verified that Dextromethorphan reduce the neuro-inflammation and prevent the neuro-degeneration via NOX2inhibition,decreaseing the production of ROS and other pro-inflammatory,resulted in microglial activation suppression.Even,Dextromethorphan reduced the mortality of liver failure in sepsis model.However,this advantage mentioned above is long-term effect,the role of NADPH oxidase 2（NOX2）in sickness behavior and brain IL-1βand IL-6 production after infection remains elusive.Materials and Methods:1.Animals and treatment The C57BL/6J mice and NOX₂^-/-mice were obtained from the Jackson Laboratory（Bar Harbor,ME）.LPS（2.5 mg/kg）or saline（2.5 mg/kg）were intraperitoneally administrated to 8-week male C57BL/6J mice and NOX₂^-/-mice initially,and sickness behavior was observed every 3hs after LPS injection until24h.Dextromethorphan（12.5mg/kg）or saline（12.5 mg/kg）were also intraperitoneally injected to C57BL/6J mice three times（3h,6h,9h after LPS injection）and sickness behavior was accessed every 3hs after LPS injection until 24h.2.Sickness behavior Assessment We applied behavior test to evaluate the mice’s sickness behavior after treatment,including the response,the moving distance,ptosis,piloerection and excretion in eyes.3.Immunohistochemistry Mice were euthanized using fetal plus at the desired time points after injection.We used the following primary antibodies for immunohistochemistry:antibodies against CD11b（1:400）.Immuno-staining was visualized by using 3,3’-diaminobenzidine（DAB）and urea-hydrogen peroxide tablets.4.Quantification of the immuno-histochemistry staining density The quantification CD11b immunohistological staining in SN was performed by Image J software based on a protocol for quantifying western blots.5.Cytokine and analysis The brain tissues from WT and NOX2^-/-mice were measured for mouse IL-1β,IL-6,and TNF-αaccording to the manufacture’s instructions.Results1.Effect of NOX2 on sickness behavior in 24hs after LPS administration In WT mice,the sickness behavior score increased over time until 15h after LPS injection,then it show a significant decreased trend until the end of observation.The sickness score of NOX2^-/-mice was displayed similar tendency after LPS injection.However,comparing to WT mice,a significant reduction in sickness behavior score in NOX₂^-/-mice was observed at 9h,12h,15h（p<0.05）by analysis of ANOVA.2.Deficiency of NOX2 suppresses inflammation in brain The level of IL-1βand IL-6 in brain tissue of WT mice at 9h was higher after LPS injection than that in vehicle-induced mice.Notably,the level of IL-1βand IL-6 in brain tissue from NOX2deficiency mice at 9h after LPS administration was significant lower than in LPS treated WT mice.3.Deficiency of NOX2 alleviated the activation of microglia in SN At the end of 24h-observation,treatment with LPS in WT mice induced morphological change in microglia,regarded as activation.Activated microglia exhibited significant enlargement of their cell bodies,irregular shapes.Dramatically,this morphological activation was significant alleviated in NOX2 deficiency mice,characterized by a small and round shape comparing with WT mice which was analysed by gray quantification.4.Effect of Dextromethorphan on sickness behavior induced by LPS In LPS treatment mice,the sickness behavior score increased gradually until 15h after LPS injection,then it decreased significantly until the end of observation.Interestingly,DM attenuated the sickness behavior significantly at 9h,12h,15h（p<0.05）by analysis of ANOVA,comparing to LPS treatment mice.5.Effect of Dextromethorphan on inflammation and the microglial activation The level of IL-1β,IL-6 and TNF-αin brain tissue from mice of post-treatment DM group at 9h after LPS administration were significant lower than in LPS treated WT mice respectively.The mice were sacrificed at the end of 24hs,the brain tissue were immunostained with an antibody against the CD11b antigen.Activated microglia showed significant enlargement of their cell bodies in LPS group,whereas,microglia was less motivated in post-treatment DM group,characterized by smaller shape.There was significant difference in activation of microglia between two groups analysis by gray quantification（p<0.05）.Conclusion Sickness behavior measured by animal response scores after LPS peritoneal injection was significantly relieved in NOX2^-/-mice compared to wild type mice.LPS-induced brain mature IL-1βand IL-6 levels and prolonged microglia activation were also decreased in NOX2^-/-mice.Dextromethorphan,an antitussive drug,was recently found NOX2^-/-inhibition effect.Post-administration of dextromethorphan decreased LPS-induced C57BL/6J mice sickness behavior scores and brain mature IL-1β,IL-6 and TNF-αconcentrations significantly.Furthermore,dextromethorphan alleviated microglia activation at 24h after LPS dosing indicated by CD11b staining.These findings demonstrated that NOX2 is a novel therapeutic target for sickness behavior and the application of dextromethorphan is promising in this setting.