The Role And Mechanism Of HMGB1-mediated Microglial NLRP3 Inflammasome Activation In Regulating Stress Sensitivity

Depression,as a mental disease with emotional disorders,is characterized by significant and lasting low mood,lack of interest,slow thinking and movement,and is caused by the interaction of psychological and physiological factors.Stress as an inducing and aggravating factor of depression.When individuals suffer the same intensity of stress events,they will produce different stress responses,which is because of differences in genetic background,social experience,personality traits and so on.Individuals who are sensitive to stressful events show social avoidance and anxiety behavior in the early stage of experiencing stressful events.When individuals show significant sensitivity to stress,they are at high risk for depression.Recent studies have found that,microglia as immune cell,actively participate in the regulation of stress sensitivity in CNS,which is the pathological basis of depression.However,the molecular mechanism by which microglia regulate stress sensitivity remains unclear.This dissertation explored the mechanism of the NLRP3 inflammasome of hippocampus microglia in regulating stress sensitivity.The main findings are as follows:1.Chronic mild stress(CMS)model was used to analyze and screen stress-sensitive mice(SS)and stress-resistant mice(SR).Mice were performed to CMS for 3 weeks,and about 40%of mice showed symptoms of anhedonia and behavioral despair at the same time,which was defined as stress-sensitive mice.RNA-seq analysis showed that genes related to immune response and inflammation regulation were significantly up-regulated in hippocampus of SS mice.While genes related to microglial homeostasis and neuroplasticity were significantly down-regulated.By analyzing the microglial morphology and phenotype in hippocampus,it was showed that the number of CD68~+microglia in SS mice was significantly increased compared with the control mice(Ctrl)and the SR mice,and the cell soma of microglia was enlarged and the branches were shortened,which is a typical activation state.Phenotypic analysis showed that compared with the Ctrl mice and SR mice,the pro-inflammatory cytokines IL-1β,IL-6,TNF-α,i NOS and IFN-γin the hippocampus of SS mice were significantly up-regulated,while the anti-inflammatory cytokines IL-4,IL-10,TGF-β,Arg1 and Ym1 were significantly down-regulated.It is suggested that microglia is a pro-inflammatory activated phenotype in the hippocampus of SS mice.Correlation analysis showed that the number of CD68~+microglia and the expression level of IL-1βin the hippocampus were significantly positively correlated with anhedonia and behavioral despair in mice.Minocycline inhibited of microglial pro-inflammatory activated phenotype,and significantly reduced stress sensitivity and improved depressive-like behavior in stressed mice.Therefore,the differences in the morphology and function of microglial activation in the hippocampus are the key factors that regulate the sensitivity and resistance of mice to stress.In conclusion,pro-inflammatory activation of hippocampal microglia is associated with stress sensitivity.2.To explore the microglial NLRP3 inflammasome activation in stress sensitivity.In this dissertation,RNA-Seq technology was used to analyze the differences in the enrichment of signal pathways related to immune response in stressed mice by gene enrichment method.It was found that inflammatory response pathway to stress and NLRP3 inflammasome activation pathway were significantly enriched in SS mice,which was different from SR mice.Several behavioral tests demonstrated that NLRP3inflammasome activation was involved in the regulation of SS and SR in mice,and it was found that treatment with the inhibitor MCC950 eliminated behavioral differences in mice subjected to stress.Inhibition of NLRP3 inflammasome activation improved the sensitivity of mice to stress.Then,the differences of NLRP3 inflammasome in the hippocampus of SS and SR mice were analyzed at m RNA and protein levels.Similarly,this dissertation found that NLRP3 inflammasome activation in hippocampal microglia of SS mice were significantly enhanced and positively correlated with stress sensitivity in mice.This suggests that the activation of NLRP3 inflammasome enhances microglial proinflammatory activation in the hippocampus,thereby regulating stress-sensitive and resistant behavior in mice.Subsequently,this dissertation used RNA-Seq technology to analyze the hippocampal transcriptional profile of stressed mice after inhibition of NLRP3 inflammasome activation by using MCC950.It was found that the treatment of the MCC950 regulated the expression of inflammatory transcription profile in stressed mice,and the molecular characteristics of the transcription profile were like that of Ctrl mice,restoring the homeostasis imbalance of microglia in stressed mice.Finally,this dissertation initially explored the effect of NLRP3 inflammasome activation on the function of microglia,and found that stress-induced NLRP3 inflammasome activation enhanced the expression of CD68 in microglia.Further in vivo and in vitro experiments demonstrated that NLRP3 inflammasome activation enhanced the microglial phagocytosis.In conclusion,this dissertation revealed that NLRP3 inflammasome activation is the core molecular pathway for microglia to participate in the regulation of stress sensitivity in mice.3.To explore the effect of microglial NLRP3 inflammasome activation on adult hippocampal neurogenesis(AHN).This dissertation reveals that the pattern recognition receptor NLRP3 regulates stress-related immune responses mediating AHN and is involved in stress response.Firstly,immunofluorescence staining proved that there were significant differences in neurogenesis between SS mice and SR mice in the DG of the hippocampus,in which the neurogenesis of SS mice was severely damaged.Minocycline can significantly enhance hippocampal neurogenesis by inhibiting microglial proinflammatory activation,and correlation analysis has also demonstrated that microglia-mediated neuroinflammation regulates neurogenesis and participates in stress sensitivity.Secondly,MCC950 was used to prevent the activation of the NLRP3inflammasome in microglia,thereby improving neurogenesis damage in stressed mice.Correlation analysis showed that up-regulated neurogenesis by MCC950 was negatively correlated with stress sensitivity in mice.Then,this dissertation further explored the relationship between neurogenesis and stress sensitivity by using neurogenic inhibitor TMZ,and the results showed that TMZ reduced the number of proliferating new neurons in stressed mice,and blocked the effect of MCC950 on enhancing neurogenesis in stressed mice.Finally,through various behavioral analysis experiments,TMZ can significantly hinder the effect of MCC950 on the relief of depression-like behavior in stressed mice.In conclusion,ANH is important regulatory pathway of NLRP3inflammasome activation leading to stress sensitivity in mice.4.To explore the role of the alarm HMGB1 in mediating the microglial NLRP3inflammasome activation.Here,this dissertation revealed that HMGB1 induces microglial NLRP3 inflammasome activation,which injury neurogenesis is involved in regulating the response to stress in mice.In stressed mice,the expression of HMGB1 in hippocampus was significantly reduced by MCC950 treatment,while the number of HMGB1~+-IBA1~+microglia was decreased,and this was negatively correlated with the degree of anhedria in mice,suggesting that NLRP3 inflammasome activation was closely related to the release of microglia HMGB1 in hippocampus.Then,recombinant HMGB1(ds HMGB1)was injected into the hippocampus of mice by stereotactic injection technique.The results showed that ds HMGB1 induced the pro-inflammatory activation phenotype of microglia in the hippocampus,such as the significant increase of IL-1β,IL-6,TNF-αand other pro-inflammatory factors.Cytokines related to NLRP3inflammasome activation were also significantly increased in stressed mice.ds HMGB1severely impaired adult neurogenesis in the dorsal and ventral hippocampus regions,causing the mice to exhibit depression-like behavior.These results suggest that AHN with increased HMGB1-induced NLRP3 inflammasome activation in the hippocampus is closely related to depression-like behavior in mice.Finally,in this dissertation,HMGB1-targeted inhibitor GZA was used to evaluate the mechanism of HMGB1’s regulation of stress response.The results showed that GZA could inhibit the activation of microglia NLRP3 inflammasome and improve the proinflammatory activation of microglia-mediated neurogenesis damage.In summary,GZA repairs damaged neurogenesis by inhibiting activation of the pro-inflammatory microglia NLRP3 inflammasome,alleviating depression-like behavior in mice.In conclusion,NLRP3 inflammasome induces microglial proinflammatory activation phenotype,which can affect stress susceptibility or stress resistance in mice by regulating hippocampal neurogenesis.The alarm hormone HMGB1 produced by stress is the core molecule that induces the microglial activation of the NLRP3 inflammasome.