Study On The Function And Mechanism Of Inflammasome Mediated Astrocytic Pyroptosis In Major Depressive Disorder

Depression is the most common mental illness.According to the statistics of the Chinese Mental Health Association,the incidence of depression in China is about 3%to 5%.At present,more than 26 million people suffer from depression,resulting in an economic burden of about 62.191 billion yuan,which seriously affects the quality of life of patients.Studies have shown that patients with depression have no significant loss of neurons in their brains,which is in stark contrast to the phenomena observed in other neuropsychiatric and neurodegenerative diseases[5].Autopsy results of depressive patients showed reduced volume and weight of frontal cortex and hippocampus,decresaed number and density of astrocytes with remarkable downregulation of mRNA and protein expressions of markers,such as GFAP and S100β.At the same time,neuroinflammation is widespread in brain regions of depressive patients,manifested as markedly upregulating levels of inflammatory factors such as TNF-α,IL-1,IL-6 and PGE2[5].Consistently,in the animal depression models,the expression of GFAP expression was down-regulated in related brain regions[12],with neuroinflammation widespread.Therefore,depression is a neuropsychiatric disorder characterized as pathological changes and dysfunction in astrocytes,accompanied by neuroinflammation.Finding interventions targeting the inhibition of astrocyte loss and restoration of astrocytic normal function,and at the same time avoiding excessive inflammation activation are expected to be applied to depression treatment and drug development.Both basic and clinical evidence indicates a significant role of neuroinflammation in the development of depression1[26].When the body is stimulated by the DAMP/PAMP,inflammatory factors of peripheral monocytes are over-released,entering the brain through the increased permeabled blood-brain barrier,which results in excessive neuroinflammation.In addition,peripheral in:flammatory factors,chemokines and tryptophan metabolites can also activate glial cells and trigger neuroinflammation.Inflammatory factors affect the metabolism of monoamine neurotransmitters,glutamate metabolism and toxicity,synaptic plasticity and excitability of HPA axis[5](Figure 1).These results suggest that inhibition of neuroinflammation may exert antidepressant effects.Pyroptosis is a new type of programmed cell death discovered and confirmed in recent years,mediated by Caspase 1/11/4/5.It is characterized by GSDMD cleavage and release of a large number of pro-inflammatory factors[10-13].The morphological characteristics,regulation mechanism of pyroptosis are different from other kinds of cell death such as apoptosis and necroptosis[14,15].The occurrence of pyroptosis is closely related to the NOD-like receptor protein as the core.Activated inflammasome transfer pro-Caspasel to mature Caspase 1 with enzymatic activity,leading to the cleavage of GSDMD and pyroptosis subsequentlTly[16].With development of research,non-classical mechanisms of pyroptosis have been gradually reported:Professor Shao Feng’s team found that Caspase3 activated by damaged mitochondria-released cytochrome acted as scissor to cleavage GSDME and triggered pyroptosis[17].In addition,TNF-a activated Caspase8-medieated GSDMD splicing,resulting in pyroptosis[18].In 2018,the NCCD revised focal death as a regulated cell death that relies on gasdermin family proteins to form plasma membrane pore,often but not always due to the activation of inflammatory Caspase[19].KYN is an intermediate product of tryptophan metabolism.About 5%of tryptophan is used to synthesize 5-HT in intestinal chromaffin cells,and most of other tryptophan is metabolized to canine uridine by IDO enzyme in peripheral tissues(such as liver and kidney)and central nervous system(such as astrocytes and microglia).At present,the increase of canine uridine production and the decrease of 5-HT production are considered to be important causes of depression[20-22].Long-term stress causes the activation of IDO enzyme,promoting the conversion of tryptophan into kynurenine.As a result,the total amount of tryptophan is reduced and the production of 5-HT is insufficient,which results in decreased inhibition of amygdala and increased production of CVRT,leading to depression[23].The release of pro-inflammatory factors,interferon and oxygen free radicals under stress can also induce metabolism of tryptophan to kynurenine,thereby affecting the synthesis of 5-HT[23].Studies have shown that healthy mice administrated by kynurenine exhibited depressive behavior,while in well-trained mice in which kynurenine can be rapidly converted into canine uric acid,triggering a protective mechanism,so these animals are not susceptible to depression[24].In conclusion,kynurenine exerts an important role in depression.However,the regulation and mechanism of kynurenine on astrocyte inflammation and the occurrence and development of depression are still unclear.With the development of research,FLX can also play an antidepressant role through 5HTR.Astrocytes widely express various subtypes of 5HTR.Studies have shown that fluoxetine can act on 5-HT2BR in astrocytes,affecting its synapse formation,glial transmitter release,and participate in the treatment of depression.FLX,the classic drug of SSRIs,was initially used to increase the concentration of 5-HT in intercellular space to treat depression.With the development of researches,FLX can also play an antidepressant role through 5HTR,and further studies show that FLX plays an antidepressant role through astrocyte 5-HT2BR.5-HT2BR is a class of GPCRs,which can regulate downstream molecular signals through classical G-protein signaling pathways.Recent studies have shown that non-classical pathways mediated by β-arrestin also play an important role in GPCR-mediated signaling pathways.On the basis of previous studies,we used CMS model mice to verify the correlation between astrocyte pyroptosis and depression in the first part.The death patterns of astrocytes in hippocampus of CMS mice were studied by immunofluorescence,western blotting and multiple staining.The results showed that the astrocyte in hippocampus was lost mainly caused by pyroptosis.The pyroptosis of astrocyte in four-weeks CMS model mice accounted for 56.7%of the dead cells.Subsequently,CMS models were made by Caspasel-/-mice and GSDMD-/-mice.Behavioral results showed that Caspasel and GSDMD knockout alleviated depression-like behavior in mice,suggesting that inhibiting pyroptosis could alleviate depression-like behavior in CMS mice.In addition,GSDMD-N adeno-associated virus containing GFAP promoter was overexpressed in the hippocampus of GSDMD-/-mice.The CMS model was established four weeks later to further confirmed that astrocytic GSDMD-mediated pyroptosis was involved in the occurrence and development of depression.These results suggested that astrocyte pyroptosis existed in hippocampus of CMS in a high percentage.Preventing astrocyte pyroptosis alleviated depression-like behavior in depression model mice and inducing astrocyte pyroptosis leads to depression-like behavior in mice.The second part is based on the results in the first part that the level of L-KYN in hippocampus and peripheral blood of CMS model mice is increased significantly,we studied the relationship between L-KYN and depression in depth.Firstly,primary astrocytes were cultured in vitro and stimulated by L-KYN.Firstly,primary astrocytes were cultured in vitro and stimulated by L-KYN.It was found that L-KYN combined with ATP could induce pyroptosis of astrocytes.The mechanism was that L-KYN promoted nuclear p65 to enter the nucleus and up-regulated the expression of NLRP2.In vivo studies showed that L-KYN could induce depression-like behav:ior in mice,and significantly up-regulated the expression of NLRP2 inflammasome,activated Caspasel and IL-1β cleavage in the hippocampal region of mice.This indicated that L-KYN could induce pyroptosis of cells in the hippocampal region of mice by activating inflammasome.Subsequently,adeno-associated virus was used to knock down the expression of NLRP2 in astrocyte of hippocampus of mice.It was found that specific knockdown of NLRP2 in astrocyte could significantly improve the depression-like behavior of mice induced by L-KYN intraperitoneal injection,alleviating the activation of Caspasel and decreasing the expression of IL-1β.These results suggest that L-KYN+ATP activates NLRP2 to induce astrocyte pyroptosis and participates in the development of depression.Knocking down NLRP2 can improve L-KYN-induced depression-like behavior in mice.As is shown in the first part,FLX can inhibit astrocyte pyroptosis.In the third part,we studied the mechanism of SSRIs on regulating astrocyte pyroptosis in depression,which was found that β-arrestin2-/-cancelled the alleviation effects of FLX in depression-like behavior,inhibitory role of Caspasel activation and GSDMD-N,IL-1β expression in hippocampus of CMS model mice.We concluded that β-arrestin2 was the main target of FLX-included SSRIs.Primary astrocytes cultures of p-arrestin2+/+and β-arrestin2-/-mice were carried out,and we found that 5-HT2BR inhibitor SR127445 abolished the inhibitory effect of FLX and Cit on L-KYN+ATP-induced pyroptosis of astrocytes.Further research results showed that SSRIs promoted the combination of P-arrestin2 and NLRP2 to play an anti-pyroptosis effect.These results suggested that SSRIs such as FLX can promote the binding of β-arrestin2 to NLRP2 by acting on 5-HT2BR to inhibit the pyroptosis of astrocytes mediated by NLRP2 inflammasome,and as a result,playing an antidepressant role.It provides a new molecular target for the development of drugs targeting pyroptosis to treat depression.Part I Correlation between pyroptosis of astrocyte and depressionOBJECTIVE:To investigate the role of astrocyte pyroptosis in the development of depression.METHODS:CMS models were established in WT mice,Caspasel-/-mice,GSDMD-/-mice and GSDMD-/-mice with astrocytic overexpressed GSDMD-N.The effects and mechanisms of pyroptosis in depression were studied by behavioral tests,immunohistochemical,western blotting and multiple staining methods.RESULTS:1)Astrocytes were activated in hippocampus of CMS mice at two weeks,but no pyroptosis occurred;2)Astrocyte pyroptosis existed in hippocampus of CMS mice at the fourth week;3)Astrocyte pyroptosis in hippocampus of CMS mice at six weeks was more obvious;4)No microglia and neuron pyroptosis in hippocampus of CMS mice at six weeks;5)The expression of Caspasel and IL-1β increased significantly with the prolongation of modeling time.6)FLX could improve depression-like behavior in CMS mice;7)Astrocyte pyroptosis in hippocampus of CMS mice was inhibited by FLX;8)FLX decreased the expression of pyroptosis-related proteins in hippocampus of CMS mice;9)Caspasel knockout could significantly improve the sugar-water preference,forced swimming immobility time and tail immobility time and general open-field exercise route of CMS mice;6)Caspasel knockout alleviates astrocyte pyroptosis in hippocampus of CMS mice;7)GSDMD knockout significantly improves sugar preference,forced swimming immobility time,tail hanging immobility time and general open-field route of CMS mice;8)GSDMD knockout alleviates astrocyte pyroptosis in hippocampus of CMS mice;9)Astrocyte-specific overexpression of GSDMD-N in GSDMD knockout mice hippocampus aggravated the depressive behavior of GSDMD knockout CMS mice.CONCLUSIONS:1)Astrocyte pyroptosis exists in hippocampus of CMS mice;2)Astrocyte pyroptosis leads to depressive behavior in mice.Part II The role and molecular mechanism of astrocyte pyroptosis in depressionOBJECTIVE:To elucidate the correlation between L-KYN and depression-like behavior and explore the molecular mechanisms.METHODS:The levels of serum inflammatory factors and hippocampal homogenate of CMS mice were detected by ELISA and HPLC.WT primary astrocytes were cultured in vitro and stimulated with different concentrations of L-KYN(0,62.5 μM,125 μM,500 μM,1 mM and 2 mM)to detect the expression levels of NLRP2,NLRP3,Caspasel,GSDMD and IL-1β.Immunofluorescence was used to observe the expression and distribution of NLRP2 inflammasome in hippocampus of mice.Western blotting and YO-pro-1 iodid/EthD2 double staining were used to study the effect of L-KYN+ATP on astrocyte pyroptosis.The expression of NLRP2 or NLRP3 in astrocytes was interfered by siRNA,then stimulated by L-KYN+ATP,the expression of Caspasel,GSDMD and IL-1β were detected by Western blotting.YO-pro-1 iodid/EthD2 double staining was used to observe the number of positive cells.SiRNA interfered with AhR expression in astrocytes,then L-KYN+ATP was given to stimulate the expression of NLRP2.Western blotting was used to detect the expression level of NLRP2,and Chip was used to detect the binding of NLRP2 to AhR.Western blotting was used to detect the expression levels of p65,p-p65,IKKβ and p-IKKβ.The distribution of p65 was detected by cytoplasmic nucleus separation kit.Western blotting and immunofluorescence were used to detect the effects of JSH-23,an inhibitor of NF-kB,on the expression of p65 and p-p65.WT mice were i.p.L-KYN to observe depressive behavior.Western blotting was used to detect the expression of NLRP2 inflammasome,Caspasel and IL-1β in hippocampus of mice.Adeno-associated virus was interfered by NLRP2 containing GFAP promoter in hippocampus of mice.L-KYN was injected intraperitoneally to detect depression-like behavior.Western blotting was used to detect the expression levels of p65,p-p65,Caspasel,GSDMD and IL-1β.RESULTS:1)The contents of KYN and KYNA in peripheral serum and hippocampus of CMS mice were significantly increased,and FLX inhibited the contents of KYN and KYNA in peripheral serum and hippocampus of CMS mice;2)L-KYN+ATP stimulated pyroptosis in primary astrocytes in vitro;3)L-KYN alone increased the expression of NLRP2 in astrocytes.(4)Knocking down NLRP2 inhibits L-KYN+ATP-induced astrocyte pyroptosis,while knocking down NLRP3 does not inhibit L-KYN+ATP-induced astrocyte pyroptosis;5)L-KYN activates p65 and promotes the expression of NF-κB in the nucleus;6)L-KYN can induce depressive behavior in mice;7)L-KYN significantly increased the content of IL-10 in peripheral serum of mice;8)L-KYN up-regulated the expression of NLRP2 inflammasome,Caspasel and IL-1β in the hippocampus of mice,;9)The sugar preference of AAV-eGFP L-KYN mice was significantly lower than that of control group,and the immobility time of forced swimming and tail suspension were significantly higher than that of control group.While the specific knockdown of NLRP2 in astrocyte improved the sugar preference,the immobility time of forced swimming and tail suspension in mice,and alleviated the expression of GSDMD,Caspasel and IL-1β.CONCLUSIONS:1)L-KYN+ATP activates NLRP2 inflammasome and induces astrocyte pyroptosis,which are involved in the pathological process of depression;2)L-KYN+ATP activates p65 to up-regulate the expression of NLRP2 and activates inflammasome-induced Caspasel maturation and astrocyte pyroptosis.Part III The role and mechanism of SSRIs mediating astrocyte pyroptosis in depressionOBJECTIVE:To elucidate the new mechanism of 5-HT reuptake inhibitors(SSRIs)for depression treatment.METHODS:CMS models were established in WT and β-arrestin2 KO mice.Western blotting was used to detect the expression levels of Caspasel,GSDMD and IL-1β in hippocampus of β-arrestin2 KO mice.In vitro cultured primary astrocytes were treated with SR127445,FLX or Cit,LPS+ATP or L-KYN+ATP,CCK8 and YO-pro-1 iodide/EthD2 double staining were used to detect cytotoxicity and pyroptosis.Western blotting was used to detect the expression of Caspasel,GSDMD and IL-1β in cell supernatant and cytoplasm.Co-IP was used to detect the binding of β-arrestin2 and NLRP2,and immunofluorescence was used to confirm the interaction of β-arrestin2 and NLRP2.RESULTS:1)Compared with the control group,the activation of Caspasel/11,GSDMD cleavage and IL-1β secretion increased significantly in CMS group,these effects were suppressed by FLX.β-arrestin2 knockout cancelled the anti-pyroptosis effect of FLX.2)Compared with the control primary astrocytes in vitro,the activation of Caspasel,GSDMD cleavage and the secretion of Caspasel and IL-1β in the supernatant increased significantly in the L-KYN+ATP group.While the activation of Caspasel.GSDMD cleavage and the secretion of Caspasel and IL-1β in the supernatant were inhibited significantly in the FLX.3)β-arrestin2 knockout cancelled the effects of FLX on Caspasel activation,GSDMD cleavage,supernatant Caspasel and IL-1β secretion.4)SSRIs promoted the binding of β-arrestin2 with NLRP2 and inhibited the activation of NLRP2 in astrocytes.CONCLUSIONS:SSRIs can promote the binding of β-arrestin2 and NLRP2 through 5-HT2BR receptor,thus inhibit the pyroptosis mediated by NLRP2 inflammasome in astrocytes.and play an antidepressant role.The major contributions of the present study lie in:1.We revealed the important role of astrocyte pyroptosis in the pathological process of depression and broaden the pathological mechanism of depression.2.We found that endogenous metabolite L-KYN mediated astrocyte pyroptosis through NLRP2 inflammosome,which further confirmed the neuroinflammatory hypothesis of depression and provided theoretical basis for the development of anti-depressant drugs targeting inflammosome.3.We elucidated the role of P-arrestin2 in the anti-pyroptosis effect of SSRIs on astrocytes,expanding the pharmacological mechanism of SSRIs.