| Depression is a common and potentially life-threatening mental illness characterized by anhedonia,energy loss,loss of appetite,cognitive impairment,delayed movement,and suicidal tendencies.The main pathological features of depression include significant reduction in the volume and weight of cortical hippocampal brain area,significant reduction in the number and density of astrocytes,and significant reduction in the mRNA level and protein expression of glial fibrillary acidic protein(GFAP)and calcium-binding protein S100?.At the same time,it was accompanied by extensive neuroinflammatory reaction,and the levels of inflammatory factors such as tumor necrosis factor(TNF-?)and interleukin-family members(IL-1,IL-6)and prostaglandin PGE2 were significantly increased.A large number of studies have shown that depression is caused by genetic factors,environmental factors and individual factors,but its exact pathogenesis is still unclear.There are some deficiencies in the clinical use of antidepressants,such as individual differences,long duration of medication and slow onset,etc Therefore,it is urgent to deeply explore the pathogenesis of depression and provide directions for the development of new treatments for depressionStudies have shown that there is an interaction between neuroinflammation and central nervous system function in psychiatric diseases.Data from clinical studies of patients with major depression and bipolar disorder showed significant increases in concentrations of cytokines activated by NLRP3(nod-like receptor 3)in cerebrospinal fluid and serum.The downstream secretion of IL-1? or IL-18 can stimulate the release of other inflammatory cytokines such as IL-6 and TNF-? through autocrine or paracrine mechanisms,and the trend of cytokine expression in the serum of MDD patients is consistent with this process.The transformation process from resting to activated morphology of microglia mediated by NLRP3 inflammasome and its correlation with depression-like behavior induced by stress model in mice.Clinical and basic experiments have shown that NLRP3 inflammasome may mediate the process of neuroinflammatory elevation in depression,suggesting that NLRP3 inflammasome may develop potential effective therapeutic targets for depression In Central nervous system(CNS),astrocyte underwent processes such hyperplasia and activation,and finally transformed into Reactive astrocytes(RAS).Recent studies have shown that RAS has great heterogeneity,which can be divided into different subtypes,which may have both promoting and inhibiting effects on the pathological process of CNS.In the model of neuroinflammation induced by systemic LPS injection and ischemic stroke induced by middle cerebral artery occlusion,significant differences in the gene expression of RAS were found,which were referred to as A1 phenotype(A1s)and A2 phenotype(A2s).Als highly up-regulated gene expression in the classical complement pathway may be detrimental to synaptic damage,while A2s up-regulated the expression of neurotrophic factor,which may have a neuroprotective effect.It has been found that A1s appear in a large number of human neurodegenerative diseases,including Alzheimer's disease(AD),Huntington's disease(HD),Parkinson's disease(PD),and play an important role in the course of the disease.However,it has not been reported in depression and other mental diseases,so the study on the role of A1/A2 phenotypes in depression and its specific signaling mechanism will provide a new target for the prevention and treatment of depression.Disorders of central nervous system(CNS)homeostasis,such as generalized inflammation of the brain during neurodevelopmental and psychiatric disorders in traumatic neurodegenerative diseases.It was found that under long-term and chronic stress,microglia were activated and transformed into a proinflammatory phenotype,releasing proinflammatory cytokines and inhibiting the activity of astrocytes.The interaction between glial cells and neurons in the brain region of the rodent model increased significantly,including the decreased expression of CX3CL1 and its receptor CX3CR1 on microglia,while the expression of high-mobility group protein B1(HMGB1)and ATP released by reactive astrocytes increased.In addition,the proinflammatory cytokines IL-1? and TNF-? secreted by NLRP3 inflammasome in microglia cause molecular changes in neurons that trigger depression-like behavior.In addition,C3 complement proteins can be identified by microglia cells by marking the targeted synapses,and then play a phagocytic function,thus triggering synaptic pruning.Therefore,to explore the correlation between glia-neuron network and the expression and function of Als/A2s in depression is helpful to deepen our understanding of astrocytes and neural networks of microglia in depression.Based on the above research,the first part of this paper establishes a model of Chronic mild stress(CMS)to explore the correlation between the expression of A1/A2 phenotypes and depression in vivo level.The results showed that the expression level of A1 phenotype marker was upregulated in the hippocampus of CMS mice after 6 weeks,and the expression level of A2 phenotype marker remained unchanged.Fluoxetine(FLX)could reduce the expression level of A1 phenotype marker without affecting the expression level of A2 phenotype marker.The results of the temporal CMS model showed that the A1 phenotype markers in the hippocampal area of mice showed a dynamic change level during the whole temporal CMS period(2,4 and 6 weeks),and the A1 phenotype markers showed the highest expression and the largest range at 4 weeks of stress,while the expression level of A2 phenotype markers remained unchanged.The morphology of microglia was continuously activated,the total number of neurons was basically unchanged,but the density of synaptic morphology damage decreased.The above results indicated that A1/A2 phenotype astrocytes existed in the hippocampus of CMS mice,but only A1 phenotype astrocytes showed dynamic changes in the expression level during the development of CMS.In addition,microglia activation and synaptic morphology were changed during the development of CMS as well.It was found that the expression level of NLRP3 inflammasome protein in the hippocampus of mice in a temporal CMS model was continuously increased.Based on this phenomenon and combined with the results of the first part,the second part of this paper constructed and selected NLRP3flox/flox mice,GFAPcre/+ mice,CDllbcre/+mice,NLRP3loxp/loxpGFAPcre+ mice,NLRP3loxp/loxpCD11bcre/+ mice,WT mice and NLRP3 KO mice,aiming to compare the regulatory effects of three different NLRP3 knockout mice on depression-like behavior and expression level of A1/A2 phenotype markers in CMS mice.To investigate the correlation between NLRP3 inflammasome and the expression and function of A1/A2 astrocytes in CMS model in vivo level.The results showed that the expression levels of representative Al marker C3 and representative A2 marker S100a10 in the hippocampus of NLRP3loxp/loxpGFAPcre/+ mice in CMS model group did not change,the activation morphology of microglia and the number of neurons remained unchanged,while the synaptic morphology improved the dendrite spine density.In NLRP3 KO mice and NLRP3loxp/loxpCD11bcre/+ mice,the expression level of representative A1 marker C3 decreased,but the expression level of representative A2 marker S100a 10 did not change,the activation level of microglia was decreased,while the synaptic morphology and the dendrite spine density were improved.The above results indicated that NLRP3 inflammasome in the hippocampus of CMS mice,especially the NLRP3 inflammasome from microglia,affected the expression level and neurotoxic function of A1 astrocytes,while the expression level of A2 astrocytes was not affected.Based on the above conclusions,the third part of this paper further elucidates the molecular mechanism by which NLRP3 inflammasome regulated the expression and function of A1/A2 phenotype astrocytes in CMS model.The results showed that NLRP3 inflammasome from astrocyte did not affect the expression level of A1/A2 phenotype markers under the stimulation of various inflammatory models(TNF-?+IL-1?+C1q,microglia conditioned medium,IL-6).The application of MCC950 to inhibit NLRP3 inflammasome or knocking out the NLRP3 gene showed that inhibition of NLRP3 inflammasome on microglia could reduce the secretion and release of TNF-?,IL-1? and Clq.The expression level of representative A1 marker C3 was significantly decreased,and its neurotoxic effect was reduced,while the expression level of A2 phenotype markers were not affected.Further studies of the mechanism revealed that microglia conditional knockout caspase-1 reduced the secretion and release of TNF-?,IL-1? and C1q.Inhibition of the NF-?B pathway upstream of NLRP3 inflammasome can significantly inhibit the activation of NLRP3 inflammasome in caspase-1 precursor cleavage and the release of downstream inflammatory cytokine IL-1?,reducing the secretion and release of TNF-?,IL-1?and C1q.The above results indicated that activated microglia stimulated the downstream NLRP3-Caspase-IL-1? pathway through NF-?B pathway,promoted the secretion and release of TNF-?,IL-1?,C1q and induced the expression of A1 phenotype astrocytes.Finally,primary neuronal toxicity experiments showed that NLRP3 inflammasome on microglia needed to induce the neurotoxic function of A1 phenotype astrocytes through astrocyte conditioned medium.In summary,this study found that A1 phenotype astrocytes are correlated with the development of depression.Elucidating the regulatory role of NLRP3 inflammasome on the expression and function of A1 phenotype astrocytes and related molecular mechanisms.The NLRP3 inflammasome on microglia is expected to be a therapeutic target for drug development targeting A1 astrocytes in depression.Part ? Study on the A1/A2 phenotype of astrocytes in depressionObject:To investigate the role of A1/A2 phenotype astrocytes in the development of depressionMethods:A time-dependent CMS model was developed in Wide type(WT)mice,and the depression-like behavior and the efficacy of FLX drug treatment were evaluated by behavioral evaluation.qRT-PCR,Immunofluorescence and Western blot were used to evaluate the mRNA level,fluorescence expression level and protein expression level of representative A1 marker C3 and representative A2 marker S100a10 in the hippocampus of CMS mice respectively.Western blot was used to detect the protein expression levels of microglia marker Iba-1 and neuronal marker neun in the hippocampus of CMS mice.Immunofluorescence was used to detect the double-labeling condition of microglia activation markers(Iba-1/CD68)and neuronal density markers(PSD-95/Synaptophysin)in hippocampal slices of CMS mice.The fluorescence expression levels of neuronal index neun and neuronal synaptic morphological index MAP2 were detected by immunofluorescence respectively.Results:1)After two weeks of CMS stress,the expression of A1 phenotype markers in the hippocampus were upregulated,and the expression level of A2 phenotype markers were unchanged.2)After four weeks of CMS stress,the expression level of representative A1 marker C3 was the highest in the hippocampus of the mice,and the expression level of representative A2 marker S100a10 was unchanged.3)At six weeks of CMS,the expression of A1 phenotype markers were upregulated,while the expression of A2 phenotype markers were not changed.4)Treatment with fluoxetine at six weeks of stress alleviates depression-like behavior.5)FLX can reduce the expression level of type A1 marker,but not affect the expression level of type A2 marker.6)The microglia in the hippocampus of time-dependent CMS mice were continuously activated.7)The number of neurons in the hippocampus of time course CMS mice was basically unchanged,but the synaptic morphology was injured and the density was decreased at six weeks of CMS.Conclusion:1)In the hippocampus of CMS mice,A1/A2 phenotype markers were expressed.2)The expression level of A1 phenotype markers was correlated with the occurrence and development of CMS.3)During the development of CMS,microglia was continuously activated and the number of neurons is not changed.4)At the end of CMS,the density of synapse was decreased and the morphology of synapse was damaged.Part ? The role of NLRP3 inflammasome in regulating the A1/A2 phenotype of astrocytes in depressionObject:To explore the regulatory effect of NLRP3 inflammasome on the phenotype and function of A1/A2 astrocyte.Methods:CMS model was prepared using WT mice,NLRP3 KO mice,GFAPcre/+ mice,CD11bcre/+ mice,NLRP3loxp/loxp mice,NLRP3loxp/loxpGFAPcre/+ mice and NLRP3loxp/loxpCD11bcre/+mice.The depressive-like behavior of mice was evaluated by behavior.The mRNA,fluorescence and protein expression levels of the representative A1 phenotype marker C3 and the representative A2 phenotype marker S100a 10 of astrocytes in the hippocampus of CMS mice of the above strains were evaluated by qRT-PCR,immunofluorescence and Western blotting,respectively.Immunofluorescence was used to detect the co-labeling of Iba-1 with CD68,PSD-95 with Synaptophysin,and the fluorescence expression levels of neun and MAP2 in the hippocampal slices of CMS mice.Golgi staining was used to evaluate dendritic spine density and length in hippocampal neurons of CMS mice.The protein levels of TNF-?,IL-1a and C1q in the hippocampus of CMS mice were detected by ELISA and Western blotting.The co-labeling of Caspase-1 p10/GFAP/PI in the hippocampus of GFAPcre/+mice,NLRP3loxp/loxp mice,and NLRP3loxp/loxpGFAPcre/+ CMS mice was detected by TSA multi-staining,and the protein expression levels of NLRP3,Caspase-1,Caspase-11,GSDMD and IL-1? in the hippocampus of GFAPcre/+mice,NLRP3loxp/loxp mice,and NLRP3loxp/loxpGFAPcre/+ CMS mice were detected by Western blotting.The mRNA and protein expression levels of inflammatory factors(IL-6,IL-1?,TNF-?)and anti-inflammatory factors(IL-10)in the hippocampus of GFAPcre/+mice,NLRP3loxp/loxp mice,and NLRP3loxp/loxpGFAP/+CMS mice were detected by qRT-PCR and ELISA,respectively.The mRNA and protein expression levels of trophic factors(BDNF,GDNF,NGF,VEGF,IGF-1)in the hippocampus of GFAPcre/+mice,NLRP3loxp/loxp mice,and NLRP3loxp/loxpGFAPcre/+ mice in CMS group were detected by qRT-PCR and Western blotting,respectively.Western blotting was used to detect the expression levels of autophagy index(p62,LC3?,LC3?)and endoplasmic reticulum stress index(GRP78,CHOP,Caspasel2)in the hippocampus of GFAPcre/xmice,NLRP3loxp/loxp mice,and NLRP3loxp/loxpGFAPcre/+ mice in CMS group,respectively.Results:1)Compared with the respective control groups of WT mice,CDllbcre/+mice,GFAPcre/+ mice and NLRP3loxp/loxp mice,the above strains of CMS mice all produced depressive behaviors,and the expression levels of the representative A1 phenotype marker C3 in the hippocampus were significantly increased,while the expression levels of the representative A2 phenotype marker S100a 10 were unchanged.The activation degree of microglia in hippocampus of CMS mice increased,the number of neurons remained unchanged,but the synaptic morphology was damaged and the density of dendritic spines decreased.The expression levels of TNF-?,IL-1? and C1q protein in the hippocampus of CMS mice were increased.2)Compared with the control group of NLRP3 KO mice,the depressive-like behavior of mice in CMS group was partially improved,the expression level of C3,a representative A1 phenotype marker in hippocampus,was decreased,and the expression level of S100a 10,a representative A2 phenotype marker,was unchanged.In CMS mice,the activation of microglia decreased,the number of neurons remained unchanged,but the morphology and function of synapses improved,and the density of dendritic spines increased.The expression levels of TNF-?,IL-1? and C1q proteins in the hippocampus of CMS mice were decreased.3)Compared with the control group of NLRP3loxp/loxppGFAPcre/+ mice,the depressive-like behavior of mice in the CMS group was partially improved,and the expression levels of A1/A2 phenotype markers in the hippocampus were unchanged.The activation of microglia,the number of neurons,the improvement of synaptic morphology and function,and the increase of dendritic spine density in the hippocampus of CMS mice remained unchanged.The expression levels of TNF-? and IL-1?protein in the hippocampus of CMS mice decreased,but did not affect the expression of C1q protein.4)Compared with the control group of NLRP3loxp/loxpCD11bcre/+ mice,the depressive-like behavior of mice in the CMS group was partially improved,and the expression level of C3,a representative A1 phenotype marker,was decreased in the hippocampus,while the expression level of S100a10,a representative A2 phenotype marker,was unchanged.The activation degree of microglia in hippocampus of CMS mice decreased,the number of neurons remained unchanged,the morphology and function of synapses improved significantly,and the density of dendritic spines increased significantly.The expression levels of TNF-?,IL-1a and C1q protein in the hippocampus of CMS mice were significantly decreased.5)Compared with the control group of NLRP3loxp/loxpGFAPcre/+ mice,the expression levels of hippocampal pyroptosis index and proinflammatory factors in CMS group mice were significantly decreased.The expression levels of anti-inflammatory factors and nutritional factors increased.The expression level of autophagy index protein increased and that of endoplasmic reticulum stress index protein decreased significantly.Conclusion:1)NLRP3 inflammasome,especially NLRP3 inflammasome on microglia,upregulates the expression level of A1 phenotype marker and its neurotoxic function in the hippocampus of CMS mice,and mediates the occurrence of depressive-like behavior in mice.2)NLRP3 inflammasome on astrocytes mediates depression-like behavior in mice by affecting environmental homeostasis in the hippocampus of CMS mice,including levels of pyrosis,inflammation,nutrition,autophagy and endoplasmic reticulum stress.Part ? The molecular mechanism of NLRP3 inflammasomeregulating A1 phenotype astrocyte Object:To elucidate the molecular mechanism of NLRP3 inflammasome regulating A1/A2 astrocyte expression and function.Methods:Primary astrocytes from WT mice and NLRP3 KO mice were cultured in vitro and stimulated with TNF-?(30 ng/mL),IL-1?(3 ng/mL),C1q(400 ng/mL)for 24 h,or IL-6(100 ng/mL)alone for 24 h,or primary microglia from WT mice were cultured in vitro and stimulated with LPS(100 ng/mL)for 6 h.Microglia conditional medium(MCM)was collected from each group.Primary astrocytes of WT mice and NLRP3 KO mice were incubated of the above stimulants for 24 h,and the levels of mRNA,fluorescence and protein expression of the representative A1 marker C3 and the representative A2 marker S100a 10 were evaluated by qRT-PCR,immunofluorescence and Western blotting,respectively.Primary microglia of WT mice and NLRP3 KO mice were cultured in vitro,stimulated with LPS(100 ng/mL)for 6 h,and stimulated with ATP(5 mM)for 30 min.MCM of each group was collected,and primary astrocytes of WT mice were incubated for 24 h.Representative A1 marker C3 and representative A2 marker S100a 10 were evaluated by qRT-PCR,immunofluorescence and Western blotting,respectively.ELISA and Western Blot were used to evaluate the protein expression levels of TNF-?,IL-1? and C1q.Primary microglia of WT mice were cultured in vitro,incubated with selective NLRP3 inhibitor MCC950(100 ?M)for 30 min,stimulated with LPS(100 ng/mL)for 6 h,and stimulated with ATP(5 mM)for 30 min.MCM from each group was collected to stimulate primary astrocytes(WT)for 24 h,The mRNA,fluorescence and protein expression levels of the representative A1 marker C3 and the representative A2 marker S100a10 in astrocytes were evaluated by qRT-PCR,immunofluorescence,Western blotting,respectively.The protein expression levels of TNF-?,IL-1?,C1q and IL-1? were evaluated by ELISA and Western Blot,respectively.The primary microglia of WT mice and Caspase-1-/-mice were cultured in vitro and stimulated with LPS(100 ng/mL)for 6 h.The conditioned medium MCM of each group was collected,and the primary astrocytes of WT mice were incubated for 24 h.The protein expression levels of TNF-?,IL-1?,Clq and IL-1? were evaluated by ELISA and Western Blot.Primary microglia of WT mice were cultured in vitro,pre-incubated with JSH-23(10 M),an inhibitor of NF-?B,for 30 min,then stimulated with LPS(100 ng/mL)for 6 h,and then stimulated with ATP(5 mM for 30 min.Cell supernatants and total proteins were collected.Western Blot and immunofluorescence were used to evaluate the expression levels of NF-kappa B pathway indicators(p-p65,p-IKK?,p65,IKK?)and p65 nuclear fluorescence,Western Blot was used to evaluate the expression levels of downstream indicators of NF-kappa B pathway(NLRP3,Caspase-1,IL-1?),ELISA and Western Blot were used to evaluate the expression levels of TNF-?,IL-1?,C1q proteins.Primary microglia of WT mice and NLRP3 KO mice were cultured in vitro,stimulated with LPS(100 ng/mL)for 6 h,stimulated with ATP(5 mM)for 30 min,and MCM(NLRP3 gene knockout group)of each group were collected.Primary microglia of WT mice were cultured in vitro,pre-incubated with MCC950(100 ?M)for 30 min,stimulated with LPS(100 ng/mL)for 6 h,stimulated with ATP(5 mM)for 30 min,and collected from each group of MCM(pharmacologically inhibiting NLRP3 inflammasome).The primary astrocytes of WT mice were incubated for 24 h,and the conditional media(ACM)of each group were collected,which were ACM(NLRP3 gene knockout-MCM group)and ACM(pharmacologically inhibited NLRP3 inflammasome-MCM group).Primary neurons of WT mice were cultured in vitro and incubated with MCM and ACM collected from the above groups for 24 h.CCK8 and LDH kits were used to detect the effects of MCM and ACM on neuronal injury in the above groups.Immunofluorescence was used to detect the level of Hoechst staining in neuronal nuclei and the level of MAP2 fluorescence expression in neuronal synaptic morphology.Results:1)Three different stimuli(TNF-?+IL-1?+C1q,IL-6,MCM)were given to primary astrocytes in vitro,and it was found that the NLRP3 inflammasome on astrocytes did not affect their own expression levels of A1/A2 markers.2)In vitro administration of LPS+ATP stimulation to primary microglia,gene knockout of NLRP3 and pharmacological inhibition of NLRP3 inflammasome together demonstrated that NLRP3 inflammasome on microglia up-regulated the expression level of representative A1 marker C3 by upregulating the content of TNF-?,IL-1? and C1q in its supernatant,but did not affect the expression level of A2 marker.3)In vitro,primary microglia were stimulated with LPS+ATP,and the results of Caspase-1 gene knockout indicated that NLRP3 on microglia increased the contents of TNF-?,IL-1? and C1q in their supernatant by upregulating its downstream Caspase-1/IL-1? pathway.4)LPS+ATP stimulation of primary microglia in vitro inhibited NF-?B entry,which indicated that activated microglia activated the downstream NLRP3/Caspase-1/IL-1? axis through the NF-?B pathway,promoted TNF-?,IL-1?,Clq cytokine secretion,and upregulated the expression level of C3,a representative A1 marker.5)MCM and ACM were given to primary neurons in vitro.The results of neurotoxicity test showed that the conditioned medium of primary microglia before and after NLRP3 gene knockout both had no neurotoxic effect.The NLRP3 inflammasome on microglia induced neurotoxic effect of A1 phenotype astrocytes by primary astrocyte conditioned medium.Conclusion:1)NLRP3 inflammasome in Primary astrocyte is not involved in regulating its own A1/A2 phenotype expression level.2)NLRP3 inflammasome on primary microglia up-regulates the expression level of A1 phenotype astrocytes without affecting the expression level of A2 phenotype astrocytes.3)Activated microglia upregulated the NF-?B pathway,then activated the downstream NLRP3/Caspase-1/IL-1? pathway,and promoted the secretion of TNF-?,IL-1? and C1q.4)NLRP3 inflammasome in primary microglia upregulates neurotoxic effects of A1 phenotype astrocytes via astrocyte conditioned medium.The major contribution of the present study lie in:1.We reveal the expression level of A1 phenotype astrocytes and the important role of their neurotoxic function in the pathological process of depression,and to expand new understanding of the glial-neuron microenvironment in depression.2.We found that NLRP3 inflammasome,especially NLRP3 inflammasome in microglia,regulated the expression level and neurotoxic function of A1 phenotype astrocytes,and further elucidated its molecular mechanism,providing a new perspective for further understanding the neuroinflammation hypothesis in NLRP3 inflammasome-mediated depression.3.The antidepressant effect of NLRP3 inflammasome in microglia was elucidated by a variety of experimental techniques to provide new evidence for accelerating the development of antidepressant drugs targeting NLRP3 inflammasome in microglia. |
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