The Role Of Microglia In Sex Differences Of Chronic Stress-Induced Depressive-Like Behavior In Mice

Background and Objective:Depression,as one of the main sources of global disease burden,ranks first among mental disorders.The Lancet published a major report on depression by the World Psychiatric Association this year,arguing that the global crisis of depression has not been resolved,and calling on all sectors to take active measures to ease the burden of depression.However,there are sex differences in the incidence,clinical symptoms,and drug treatment of depression.For these sex differences in depression,although some studies have explained the psychological and biological reasons,the sex difference of depression is still unclear.The research on sex differences and their mechanisms of depression will not only help to deeply understand the pathophysiological mechanism of depression,but also help to promote the classification and diagnosis of depression,precise medical treatment,and improve the effect of antidepressant treatment.Chronic stress is the most important risk factor for depression,and dysregulation of the hypothalamic-pituitary-adrenal(HPA)axis plays an important role.Clinical and animal studies have shown that there are sex differences in the activation of the HPA axis induced by stress,but there is no uniform and sufficient explanation for the sex differences in chronic stress-induced depressive-like behavior and HPA axis activation in mice.As the main immune effector cells of the central nervous system,microglia play an important role in neuroinflammation.Activation of microglia can differentiate in two different directions and thus have different functions.The pro-inflammatory phenotype is a classical activation state,and microglia mainly produce a large amount of pro-inflammatory cytokines,namely M1 type polarization,while the anti-inflammatory phenotype is selectively activated,and microglia produce some anti-inflammatory cytokines,namely M2 type polarization.The pro-inflammatory and anti-inflammatory cytokines produced by microglia activation have been proved to be closely related to depression,and the expression of these cytokines helps us to observe the activation state of microglia.So,are there sex differences in microglia that cause neuroinflammation?As a key neurotrophic factor,brain-derived neurotrophic factor(BDNF)has been shown to play an important role in neural growth,survival,maturation,differentiation and synaptic plasticity in the adult hippocampus.Serum BDNF levels have been reported to be lower in patients with depression,while chronic stress reduces BDNF synthesis,and antidepressant treatment stimulates BDNF production.In addition,pro-inflammatory and anti-inflammatory cytokines also affect the expression of BDNF and its receptors in the hippocampus,which suggests that neuroinflammation may affect the development of depression by affecting the level of BDNF.Further,our previous research group showed that the NLRP3 inflammasome is closely related to the occurrence and development of depression.Inhibition of the NLRP3 inflammasome can effectively improve the activation of microglia and neuroinflammation in male mice,up-regulate the level of BDNF,and alleviate the depressive behavior of mice.It is speculated that "microglia-neuroinflammation-BDNF" may form a pathway and play a key role in the pathogenesis of depression.The polarization of microglia in the hippocampus can affect neuroinflammation and BDNF level.This may be the underlying mechanism of the sex differences in stress-induced depression-like behavior in mice.And the NLRP3 inflammasome may play an important role in this pathway.Therefore,this subject intends to study the role of microglial polarization in the sex difference of depression-like behavior in mice induced by chronic stress in animal and cell experiments.This study aims to find molecular mechanisms for sex differences in depression and explore new strategies for sex differences in depression treatment.Methods:1.Chronic stress-induced depression-like behavior in mice(1)Using chronic unpredictable mild stress(CUMS)as the main stress paradigm.The experimental period of stress is 4 weeks.(2)Through behavioral observation,including sucrose preference,tail suspension,and open field,the performance of depressive-like behavior in mice were evaluated.(3)Detection of serum corticosterone level in chronic stress mice by enzyme-linked immunosorbent assay(ELISA).2.Effect of chronic stress on "microglia-neuroinflammation-BDNF" in mice hippocampus(1)The microglial polarization in hippocampus of chronic stress mice was detected by reverse transcription-polymerase chain reaction(RT-PCR)and western blot(WB).(2)The levels of inflammatory cytokines in hippocampus of chronic stress mice were detected by RT-PCR.(3)The levels of BDNF and its receptors in the hippocampus of chronic stress mice were detected by RT-PCR and WB.3.Effects of estrogen on inflammatory stimulated microglia in vitro(1)BV2 microglia were selected as tool cells,and estradiol(E2)and Lipopolysaccharides(LPS)were used to establish a cell stimulation model.(2)The effects of E2 on microglial activation,inflammatory cytokines,BDNF were detected by WB,and verified by the estrogen inhibitor.(3)WB was used to detect the effect of E2 on NLRP3 inflammasome activation,and then the estrogen inhibitor was used to verify.(4)WB was used to detect the effects of NLRP3 inflammasome inhibitor on E2-induced microglia activation,inflammatory cytokines,BDNF changes.Results:1.Sex differences in chronic stress-induced depression-like behavior in mice(1)The speed and extent of weight gain in mice caused by chronic stress showed sex differences.The weight growth rate of female mice decreased earlier than that of male mice.The relative weight of female stress mice is lower than that of male stress mice.(2)The depressive-like behavior of chronic stress-induced mice showed sex differences,mainly in that female mice showed more obvious depressive-like behaviors in sucrose preference test and open field test.(3)Chronic stress-induced HPA axis activation in mice presents sex differences.The HPA axis of female mice is more sensitive to chronic stress.2.Sex differences in the effect of chronic stress on "microglia-neuroinflammationBDNF" in mice hippocampus(1)The polarization of microglia in hippocampus of mice induced by chronic stress showed sex difference.M1 type polarization increased significantly in both female and male mice.However,M2 polarization of male mice increased significantly,and that of female mice decreased significantly.(2)Chronic stress-induced pro-inflammatory and anti-inflammatory cytokines in the hippocampus of mice showed sex differences.The expression of pro-inflammatory cytokines in female mice and male mice increased.Anti-inflammatory cytokines in female mice decreased significantly,while no significant changes found in male mice.These changes are consistent with the results of microglia polarization.(3)The changes of BDNF and its receptor Trk B in hippocampus of mice induced by chronic stress showed sex differences.The expression level of BDNF and its receptor Trk B in female mice decreased more than that in male mice.In addition,BDNF in the hippocampus of mice is correlated with depressive-like behavior and inflammatory cytokines,and there are also sex differences.3.Effects of estrogen on inflammatory stimulated microglia in vitro(1)Both LPS and LPS+E2 stimulation can activate microglia,but there is no significant difference.(2)E2 can affect the expression of inflammatory cytokines,BDNF induced by LPS,and the effects can be reversed by fulvestrant.(3)E2 can further affect LPS-induced NLRP3 inflammasome activation,and its effect can be inhibited by fulvestrant.(4)The effect of E2 on LPS-induced inflammatory cytokine and BDNF can be blocked by MCC950.Conclusion:1.The depressive-like behavior induced by CUMS in mice showed sex differences,and female mice showed more obvious depressive-like behaviors induced by chronic stress.And the activation of HPA axis in female mice is higher.2.Chronic stress-induced microglial polarization,inflammatory cytokines level,BDNF level in mice hippocampus showed sex differences.The formation of “microglianeuroinflammation-BDNF” may be an important mechanism for the sex difference of depressive-like behavior in mice induced by chronic stress.3.Low concentration of E2 can enhance the pro-inflammatory response of microglia induced by LPS and reduce the expression of BDNF.This indicates that estrogen has a regulatory effect on inflammatory cytokines and BDNF in microglia.And NLRP3 inflammasome plays an important role in the influence of estrogen.