| Depression is a psychiatric disease characterized by low mood,lack of interest,diminished will and slow thinking,especially major depressive disorder(MDD)has a long course and high recurrence rate,which seriously affects the quality of life of patients and even has suicidal tendencies.In the past 3 years,the pandemic of COVID-19,which affects people’s production and living,has greatly increased the number of people suffering from depression.First-line treatments of depression,including psychological and antidepressant medications,could only achieve a 50%remission rate.The onset of depression is mostly due to a combination of biological,social,psychological and somatic factors.The most common trigger is stress,and prolonged stress can lead to excessive activation of the hypothalamic-pituitary-adrenal axis(HPA axis)and secretion of excess glucocorticoids(GCs).Microglia expresses GCs receptors can be activated by GCs to the pro-inflammatory M1 phenotype,which secretes a variety of inflammatory cytokines.NLRP3 inflammasome was predominantly expressed in microglia and are associated with microglia-mediated neuroinflammation.NLRP3 inflammasome has been shown being correlation between stress and depression,and pyroptosis mediated by NLRP3 inflammasome can cause neuroinflammation,leading to the onset and progression of depression.NLRP3 inflammasome mediated classical pyroptosis may be a new target for depression treatment.Kai-Xin-San is a classical formula for the treatment of affective disorders,which has anti-inflammatory,antioxidant,autophagy-promoting,and monoamine neurotransmitters and HPA axis dysfunction regulating effects,and was widely used in the treatment of depression.As a formula of traditional Chinese medicine,Kai-Xin-San has many active ingredients and complex regulatory mechanisms,so the antidepressant mechanism of KaiXin-San needs further study.Whether it inhibited neuroinflammation by suppressing NLRP3-mediated pyroptosis and exerting antidepressant effect has not been reported so far.ObjectiveIn this study,we induced a depressive mouse model with neuroinflammation triggered by microglia activation as the pathological phenotype by repeated intraperitoneal injections of corticosterone(CORT,an exogenous glucocorticoid)and evaluated the ameliorative effect of Kai-Xin-San on depression-like behavior and the inhibitory effect on neuroinflammation in mice.We further validated the inhibitory effect of Kai-Xin-San on microglia-mediated neuroinflammation in vivo and in vitro by using LPS-induced classical neuroinflammatory animal model and LPS+IFNy induced BV2 microglia inflammation cell model,and explored the molecular mechanism of the antidepressant effect of Kai-Xin-San based on microglia pyroptosis by inhibiting neuroinflammation according to RNA-seq results.Methods1.The mouse model of depression was induced by CORT,and normal control group(Control group),model group(CORT group),fluoxetine group(FLX group),Kai-Xin-San low-dose group(KXS-L group),and Kai-Xin-San high-dose group(KXS-H group)were established.CORT was injected continuously for 3 weeks,of which the second and third weeks were treated by gavage with aqueous extract of Kai-Xin-San.Four behavioral tests,namely,open field test(OFT),elevated plus maze(EPM),forced swimming test(FST)and tail suspension test(TST),were performed to examine the depression-like behavior and the antidepressant effect of Kai-Xin-San.The hippocampal tissue samples from the model and normal groups,which were previously modeled in the same way by our team,were used to transcriptomic RNA sequencing(RNA-seq),in order to identify the possible molecular mechanisms of CORT causing neuroinflammation and leading to depression-like behavior in mice.To observe the pathological damage of neurons in depression-related brain regions of the mice by nissl staining.To observe monoamine neurotransmitter changes and HPA axis activation by detecting 5-hydroxytryptamine(5-HT)and adrenocorticotropin-releasing hormone(CRH)levels in brain tissue and serum by enzyme-linked immunosorbent assay(ELISA).To observe the activation of microglia in the brain by immunofluorescence.To evaluate the neuroinflammatory status in the brain through detecting the mRNA expression of M1-related pro-inflammatory cytokines and M2-related anti-inflammatory cytokines of microglia by RT-qPCR,and detecting the content of IL-1β and IL-18 in brain tissue by ELISA,intend to explore the possible molecular mechanism of anti-neuroinflammation of Kai-Xin-San.2.Lipopolysaccharide(LPS)was used to induce classical neuroinflammatory mouse model by intraperitoneal injection,and normal control group(Control group),model group(LPS group),dexamethasone group(DEX group),Kai-Xin-San low-dose group(KXS-L group)and Kai-Xin-San high-dose group(KXS-H group)were set up,and the experiment lasted 5 days,and the drug,intervention group was administered throughout the experiment,and the mice were injected with LPS besides the Control group between the third day and the fifth day.The study focused on the prefrontal cortex to explore the inhibitory effect ofKai-Xin-San on microglia-mediated neuroinflammation and to verify that NLRP3mediated pyroptosis was an important regulatory mechanism.To detect the neuroinflammatory status in the prefrontal cortex by assessing microglia activation through immunofluorescence,and detecting the mRNA expression of pro-inflammatory and antiinflammatory cytokines by RT-qPCR.To evaluate the role of pyroptosis in the inhibition of neuroinflammation of Kai-Xin-San through detecting the mRNA expression of the various molecules of NLRP3 inflammasome-mediated pyroptosis by RT-qPCR,and through detecting IL-1β and IL-18 levels in brain tissue homogenate by ELISA,and through observing NLRP3 and ASC expression in brain tissue by immunofluorescence,and through detecting the proteins expression of NLRP3,Cleaved-Caspase-1,Cleaved-GSDMD and IL1β in the tissue of prefrontal cortex by Western blot.RNA-seq was performed using brain tissues of prefrontal cortex to verify the molecular mechanism of-Kai-Xin-San in the inhibition of neuroinflammation by KEGG pathway enrichment and other analyses.The molecular mechanism of neuroinflammation inhibition by Kai-Xin-San was achieved by the regulating NLRP3 inflammasome mediated pyroptosis pathway.3.In the vitro level,BV2 microglia were treated with LPS/IFNy to produce the cellular inflammation model,and the protective effects of Kai-Xin-San on BV2 cells were evaluated by observing cell morphology,detecting cell viability and NO concentration;RTqPCR was performed to detect the expression of pro-inflammatory and anti-inflammatory cytokines mRNA,and immunofluorescence was performed to observe the expression of pro-inflammatory factor iNOS and anti-inflammatory factor IL-10.We also examined the changes of mRNA expression of NLPP3-mediated pyroptosis pathway to investigate the regulation of pyroptosis in BV2 cells,and to verify the molecular mechanism of Kai-XinSan’s anti-inflammatory effect on microglia through NLPP3-mediated pyroptosis.Results1.Study on the pharmacodynamics of Kai-Xin-San on CORT induced depression model mice and the mechanism of its antidepressant effect(1)Improvement effect of Kai-Xin-San on mice behavioral testsAnimal behavioral tests showed that the mice in the CORT group had a significant decrease in the distance of central area movement and activity time in the open field experiment.and a significant decrease in immobility time in the hanging tail experiment and forced swimming experiment,and in the elevated plus maze experiment,the times of entered the open arm and the time stayed in the open arm of the CORT group were significantly reduced,indicating that CORT successfully induced depression-like behavior in mice.In contrast.Kai-Xin-San improved many of these behavioral test indicators,suggesting that Kai-Xin-San improved depression-like behavior of the mice.(2)RNA-seq to determine the regulatory mechanisms of CORT-induced depressionlike behavior of the miceThe differentially expressed genes in the CORT and Control groups were enriched in pyroptosis and inflammation-related NOD-like receptor signaling pathway and TNF signaling pathway.The 10 KDAs of the differentially expressed genes between the two groups were mainly associated with immune and inflammatory responses.Based on the related theoretical basis and RNA-seq results,it is speculated that the mechanism of antidepressant effect of Kai-Xin-San may be related to the inhibition of neuroinflammation and pyroptosis.(3)Effect of Kai-Xin-San on biochemical indexes and brain histopathologyCompared with the control group,the monoamine neurotransmitter 5-HT in the brain tissue of CORT model mice decreased,while the HPA axis hormone CRH level increased,and Kai-Xin-San reversed these changes.The result suggested that CORT caused abnormal neurotransmitters related to depression and the over-activation of HPA axis,and Kai-XinSan improved the level of 5-HT and adjusted the overactivity of HPA axis,and Kai-XinSan may improve depression-like behavior in mice by modulating these two pathological mechanisms associated with the onset of depression.Nissl staining observed neuronal damage in the CORT group,and Kai-Xin-San ameliorated neuronal damage in brain tissue,especially in the hippocampal region.(4)Inhibitory effect of Kai-Xin-San on microglia activation and neuroinflammationIbal immunofluorescence showed that CORT increase the number of microglia in depression-related brain regions(prefrontal cortex and hippocampus)and altered the activation morphology,indicating that CORT induced microglia activation in the relevant brain regions,while Kai-Xin-San had an inhibitory effect on their activation.RT-qPCR results showed that the high expression of pro-inflammatory cytokines such as TNF-α and iNOS and low expression of anti-inflammatory cytokines such as IL-10 in the brain tissue of mice in the CORT group.The results of RT-qPCR showed that the mRNA expression of pro-inflammatory cytokines,such as TNF-α and iNOS,was high and the expression of antiinflammatory cytokines,such as IL-10,was low in the CORT mice,but the changes were reversed by Kai-Xin-San(no statistical difference in IL-10).The expression of proinflammatory and anti-inflammatory cytokines was partially down-regulated by Kai-XinSan,suggesting that CORT induced neuroinflammation in the model mice,and Kai-XinSan may inhibit neuroinflammation by regulating the different polarization states of microglia(M1 and M2 phenotype).(5)Effect of Kai-Xin-San on mRNA expression of NLRP3-related pyroptosis pathway moleculesThe RT-qPCR test showed that CORT caused high expression of NLRP3,Caspase-1.ASC,GSDMD and IL-1β mRNA in brain tissue,and Kai-Xin-San down-regulated the mRNA expressions of NLRP3,GSDMD and IL-1β,combined with the ELISA results of IL-1β and IL-18、these results indicated that CORT induced pyroptosis in the brain tissue of model mice,and Kai-Xin-San had an inhibitory effect on several molecules of NLRP3related pyroptosis pathway,which may be one of the molecular mechanisms of the antidepressant effect of Kai-Xin-San on neuroinflammation.2.Study on the inhibitory effect of Kai-Xin-San on neuroinflammation in the mice treated with LPS and its molecular mechanism(1)Inhibitory effect of Kai-Xin-San on neuroinflammationThe increased number of microglia and altered activation morphology in brain tissues(medial prefrontal cortex and hippocampus)of LPS group were observed by Iba1 immunofluorescence;LPS caused high expression of pro-inflammatory cytokines TNF-α,IL-6 and iNOS mRNA,as well as down-regulation of anti-inflammatory cytokine IL-10 and anti-inflammatory mediator Trem2 mRNA in prefrontal cortex tissues of the mice.The ELISA results showed elevated levels of IL-1β and IL-18 in prefrontal cortical tissues,KaiXin-San reversed these changes.It is suggested that LPS induced microglia activation and neuroinflammatory responses in model mice,while Kai-Xin-San suppressed these changes and responses.(2)Investigating the molecular mechanism of anti-neuroinflammation of Kai-Xin-San based on NLRP3 pyroptosis pathwayRT-qPCR showed that the expression of NLRP3,Caspase-1,ASC,GSDMD,IL-1βand IL-18 mRNAs were increased in the prefrontal cortex of the LPS group(no statistical difference in IL-18),while the expression of NLRP3,Caspase-1 and IL-1β mRNAs were significantly down-regulated by Kai-Xin-San.Immunofluorescence results showed that the number of NLRP3 and ASC-positive cells in the relevant brain regions of the LPS group were increased,and Kai-Xin-San down-regulated their positive expression;Western blot results showed that the protein expression of NLRP3,Cleaved Caspase-1,Cleaved GSDMD and IL-1β was increased(Cleaved Caspase-1 was not statistically different),and Kai-Xin-San significantly down-regulated the expression of NLRP3 and IL-1β among them,combined with the ELISA results of IL-1β and IL-18,these results indicated that Kai-XinSan inhibited pyroptosis in the prefrontal cortex,which confirmed the effect of Kai-XinSan on suppressing neuroinflammation by inhibiting pyroptosis.(3)RNA-seq verify the molecular mechanism of Kai-Xin-San on anti-inflammatory effectSimilar to the CORT experiment,the differentially expressed genes in the LPS model group and Control group were mainly enriched in the NOD-like receptor pathway,and TNF pathway and chemokine pathway related to pyroptosis and inflammation.The differentially expressed genes between Kai-Xin-San group and LPS model group were mainly enriched in the TNF signaling pathway.By KDA analysis,10 target genes regulated by Kai-Xin-San were identified,and after literature study,we hypothesized that Ctss may be a key target related with Kai-Xin-San’s anti-inflammatory effect.3.Kai-Xin-San exerted anti-inflammatory effect and influence on pyroptosis in BV2 cells in vitroThe observation of cell morphology,cell viability and NO concentration showed that Kai-Xin-San had a protective effect on BV2 cells treated with LPS/IFNy.LPS/IFN y caused an increase in the expression of pro-inflammatory cytokines TNF-α,IL-6 and iNOS mRNA in BV2 cells,as well as a decrease in the expression of anti-inflammatory cytokines IL-10,Argl and the anti-inflammatory mediator Trem2.Immunofluorescence experiments showed that LPS/IFNy induced high expression of iNOS and low expression of IL-10 in BV2 cells,and again Kai-Xin-San reversed these changes,indicating that Kai-Xin-San inhibited the LPS/IFNy induced inflammatory response in BV2 cells.LPS/IFNy induced an increase in NLRP3,ASC and IL-1β mRNA expression,while Kai-Xin-San down-regulated their expression.This suggests that the inhibitory effect of Kai-Xin-San on the inflammatory response of BV2 cells associated with its regulation of NLRP3-mediated pyroptosis.ConclusionKai-Xin-San exerts antidepressant effect by suppressing neuroinflammation through inhibiting microglia activation and regulating inflammatory cytokine expression.The mechanism of its anti-neuroinflammation is associated with the regulation of NLRP3-mediated microglia pyroptosis. |
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