| Background and objective:Depression is a serious harm to humans with high incidence and mortality.However,the pathological mechanism of depression remains mostly unclear,and clinical antidepressants with safety and effectiveness are relatively insufficient.Previous studies by us and others found that the occurrence and development of depression were closely related to neuroinflammation in the central nervous system(CNS).As the CNS resident immune cell,microglia can be activated quickly,to release a large number of inflammatory cytokines,and eliminate the danger signal to maintain internal environment homeostasis in response to endogenous and/or exogenous danger signals.Post-mortem studies detect the activated microglia with increased inflammatory cytokines in the dorsal anterior cingulate cortex of depression patients.Our research group previously observed similar phenomenon in prefrontal cortex in animal model of depression,suggesting that the microglia activation with inflammatory cytokine release might be the key pathological event in neuroinflammation during depression development.Stress is an important cause of depression.We previously confirmed the hypothalamic pituitary adrenal axis activation with elevated corticosterone levels in chronic mild unpredictable stress-induced depression in rats.But it is still unknown that whether there is any intrinsic link between elevated glucocorticoid levels and microglia activation or microglia-mediated neuroinflammation.As a classical immunosuppressive and anti-inflammatory agent,how could the glucocorticoid activate microglia and enhance microglia mediated immuno-inflammatory response?These questions need to be explored urgently.Previous studies have shown that,after chronic stress or exogenous glucocorticoid pretreatment,the separated microglia from rat hippocampus exerting stronger immuno-inflammatory response to immuno-stimulation,suggesting a "priming" effect besides classic anti-inflammatory effect by glucocorticoid to prime and enhance the immuno-inflammatory response.But the mechanism underlying is still unknown.On the other hand,clinical and basic studies have demonstrated that epimedium flavonoids could enhance immune function and have antidepressant activity,our group has reported that epimedium flavonoids exerted antidepressant activity by regulating hypothalamic pituitary adrenal axis.It still need to be explored and elucidated the possible reversal effect by these flavonoids constituents on pathological changes of microglia caused by stress level corticosterone and the molecular mechanism underlying.The present study intends to use the stress level glucocorticoid(corticosterone)to stimulate microglia in vitro,to confirm the possible mode and molecular mechanism of corticosterone priming microglia immuno-inflammatory response.Then this thesis intend to evaluate the regulatory effect and explore the mechanism of epimedium flavonoids with the aim to find potential antidepressant constituents based on new approaches against glucocorticoid priming microglia.Methods:BV2 cells were pretreated with corticosterone(25 nM)for 24 h,then treated with different concentrations of lipopolysaccharide(LPS)including 1,5,10,50,100 ng/mL for 4 h,real-time quantitative polymerase chain reaction(q-PCR)assay was used to detect interleukin 1 beta(IL-1β),tumor necrosis factor alpha(TNF-α)mRNA levels;BV2 cells were pretreated with corticosterone(25 nM)for 24 h,then treated with LPS(5 ng/mL)for 2 h,western blotting assay was used to determine nuclear transcription factor NF-κB p-p65 protein level in cell nucleus and whole cell;BV2 cells were pretreated with corticosterone(25 nM)for 24 h,then treated with LPS(5 ng/mL)for different time,western blotting assay was performed to detect total IL-1β,mature IL-1β,NOD-like receptor pyrin domain containing 3(NLRP3),Caspase-1 protein levels;BV2 cells were treated with corticosterone(25 nM)for 24 h,western blotting assay was performed to evaluate NLRP3 protein level,q-PCR assay was used to measure glucocorticoid receptor target genes including NLRP3,SGK,GILZ and MKP-1 mRNA levels.Primary microglia were extracted and purified from mixed glial cells of Sprague-Dawley newborn rat,immunofluorescence method labeling ionized calcium binding adaptor molecule-1(Iba-1)was performed to determine purity of primary microglia obtained;Primary microglia were pretreated with corticosterone(25 nM)for 24 h to detect NLRP3 mRNA level,or then treated with LPS(5 ng/mL)for 4 h to determine IL-1β,TNF-a mRNA levels;Primary microglia were pretreated with corticosterone(25 nM)for 24 h,then treated with LPS(5 ng/mL)for 2 h to measure p-p65 protein level or treated with LPS(5 ng/mL)for 8 h to measure total IL-1β,mature IL-1β,NLRP3 and Caspase-1 protein levels.In addition,C57BL/6 mice were subcutaneous injected by corticosterone(20 mg/kg)for three weeks,detecting NLRP3 and IL-1β mRNA levels by q-PCR assay,Iba-1 and NLRP3 expression by immunofluorescence of microglia,NLRP3,p-p65,total IL-1β mature IL-1β and Caspase-1 protein levels by western blotting in the hippocampus of mice.B V2 cells were treated with epimedium flavonoids(icariin,baohuoside I,epimedin A,epimedin B,epimedin C)for 24 h,testing cell viability by MTT assay,and determining intervention dose of these epimedium flavonoids;BV2 cells were pretreated with corticosterone(25 nM)and epimedium flavonoids for 24 h,then treated with LPS(5 ng/mL)for 4 h to investigate IL-1β mRNA level by q-PCR assay;BV2 cells were pretreated with corticosterone(25 nM)and epimedium flavonoids for 24 h,then treated with LPS(5 ng/mL)for 2 h to test p-p65 protein level or treated with LPS(5 ng/mL)for 8 h to test total IL-11β,mature IL-1β,NLRP3 and Caspase-1 protein levels by western blotting assay;BV2 cells were treated with corticosterone(25 nM)and epimedium flavonoids for 24 h to determine NLRP3 mRNA and protein levels by q-PCR and western blotting method.Results:Compared with normal control group,the IL-1β(p<0.001)and TNF-a(p<0.001)mRNA levels in LPS-vehicle group increased significantly while there was no obvious change in corticosterone treatment group(p>0.05);Compared with LPS-vehicle group,the IL-1β(p<0.05)and TNF-α(p<0.05)mRNA levels in corticosterone+ LPS group increased more sharply.In line with these results,compared with normal control group,the p-p65(p<0.01)protein level of cell nucleus and whole cell in LPS-vehicle group was upregulated significantly;Compared with LPS-vehicle group,the p-p65(p<0.05)protein level of cell nucleus and whole cell in corticosterone + LPS group increased obviously.BV2 cells were pretreated with corticosterone for 24 h,treated with LPS for 8 h,compared with normal control group,the total IL-1β(p<0.01),mature IL-1β(p<0.001),NLRP3(p<0.01)and Caspase-1(p<0.001)protein levels in LPS-vehicle group were up regulated significantly;Compared with LPS-vehicle group,the total IL-1β(p<0.01),mature IL-1β(p<0.001),NLRP3(p<0.05)and Caspase-1(p<0.001)protein levels in corticosterone + LPS group increased steadily.Of note,compared with normal control group,BV2 cells pretreated with corticosterone(25 nM)for 24 h induced the NLRP3(p<0.05),SGK(p<0.05),GILZ(p<0.05),MKP-1(p<0.05)mRNA and NLRP3(p<0.01)protein levels raised significantly.On the other hand,a similar phenomenon was observed in primary microglia:Primary microglia were pretreated with corticosterone(25 nM)for 24 h,then treated with LPS(5 ng/mL)for 4 h,compared with normal control group,the IL-1β(p<0.05)and TNF-α(p<0.001)mRNA levels in LPS-vehicle group were up regulated significantly,there was no significant change in corticosterone treatment group(p>0.05);Compared with LPS-vehicle group,the IL-1β(p<0.01)and TNF-α(p<0.01)mRNA levels in corticosterone + LPS group were augmented obviously.And compared with normal control group,the p-p65(p<0.05)protein level in LPS-vehicle group increased sharply,compared with LPS-vehicle group,the p-p65(p<0.05)protein level in corticosterone + LPS group increased remarkably.Primary microglia were pretreated with corticosterone(25 nM)for 24 h,then treated with LPS(5 ng/mL)for 8 h,compared with normal control group,the total IL-1β(p<0.01),mature IL-1β(p<0.05),NLRP3(p<0.01)and Caspase-l(p<0.05)protein levels in LPS-vehicle group were up regulated notably;Compared with LPS-vehicle group,the total IL-1β(p<0.05),mature IL-1β(p<0.01),NLRP3(p<0.01)and Caspase-1(p<0.01)protein levels in corticosterone + LPS group increased significantly.And compared with normal control group,primary microglia cells treated with corticosterone(25 nM)for 24 h led to NLRP3(p<0.01)mRNA and NLRP3(p<0.01)protein levels increased obviously.C57BL/6 mice were subcutaneous injected(5 mL/kg)by corticosterone(20 mg/kg)for three weeks,compared with normal control group,the NLRP3(p<0.01)mRNA and NLRP3(p<0.001)protein levels were augmented significantly in mice hippocampus,the NLRP3 expression level in microglia increased by immunofluorescence assay,and there were no obvious change of IL-1β mRNA level,p-p65,total IL-1β,mature IL-1β and Caspase-1 protein levels in hippocampus of C57BL/6 mice.The MTT method was performed to determine the non-cytotoxic doses of epimedium flavonoids after the treatment with BV2 cells for 24 h,and select the dose by the cell viability was more than 80%:Icariin and Baohuoside I was 2.5,5 and 10μM;Epimedin A,Epimedin B and Epimedin C was 0.25,0.5 and 1 μM.BV2 cells were pretreated with corticosterone(25 nM)and epimedium flavonoids for 24 h,then treated with LPS(5 ng/mL)for 4 h,icariin(p<0.01),baohuoside I(p<0.001),epimedin A(p<0.01),epimedin B(p<0.05)and epimedin C(p<0.01)downregulated IL-1β mRNA levels significantly.Simultaneously,BV2 cells were pretreated with corticosterone(25 nM)and epimedium flavonoids for 24 h,then treated with LPS(5 ng/mL)for 2 h,epimedium flavonoids of icariin,baohuoside I,epimedin A,epimedin B and epimedin C suppressed p-p65(p<0.01)protein levels significantly,or treated with LPS(5 ng/mL)for 8 h,epimedium flavonoids of icariin,baohuoside I,epimedin A,epimedin B and epimedin C downregulated NLRP3(p<0.01),Caspase-1(p<0.01),total IL-1β(p<0.01)and mature IL-1β(p<0.01)protein levels obviously.BV2 cells were treated with corticosterone(25 nM)and epimedium flavonoids for 24 h,epimedin A,epimedin B and epimedin C reduced NLRP3 mRNA(p<0.05)and protein(p<0.05)levels significantly,yet there was no significant change by icariin or baohuoside I.Summary:These results illustrated the effect of corticosterone priming microglial immuno-inflammatory response by inducing NLRP3 mRNA and protein expression,making microglia to exert stronger and faster immuo-inflammatory response to the subsequent immuno-stimulation such as LPS.These results also demonstrated the improvement of epimedium flavonoids including icariin,baohuoside I,epimedin A,epimedin B and epimedin C through downregulating p-p65 protein level and NLRP3 inflammasome activation against corticosterone priming microglial immuno-inflammatory response in microglia.And epimedin A,epimedin B and epimedin C inhibiting NLRP3 inflammasome activation by down-regulating the high levels of NLRP3 mRNA and protein induced by corticosterone.Our findings elucidated the possible mechanisms how corticosterone priming microglial immuno-inflammatory response and found the reversal effect of epimedium flavonoids,providing the experimental basis for using epimedium flavonoids to prevent and treat neuroinflammation and stress-related depression. |
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