| At present,depression has become the most common mental illness in the world.Its clinical symptoms include poor emotional state,slow thinking response,loss of interest and pleasure,loss of volitional activity,self-mutilation,suicide and so on [1].Due to the high prevalence rate,recurrence rate and suicide mortality rate,depression has become a heavy burden in our society and family[2].The main pathological features of depression are atrophy of cortex and hippocampus,shortening of neuronal axons,disorder of nerve regeneration in hippocampus,and reduction of glial cell number [3][4].At present,the theory of the pathogenesis of depression mainly includes four aspects: 1.Neuroplasticity hypothesis;2.Excitatory toxicity hypothesis;3.Monoamine hypothesis;4.Immunoinflammation hypothesis[5].Although there has been a lot of research on depression,much of it remains phenomenologically relevant.Therefore,this study intends to further explore the mechanism of the occurrence and development of depression and explore new targets of it.GPCR plays an important role in various physiological and pathological processes,and many clinical drugs use it as a target to play a therapeutic role [6].Β-arrestins are a class of GPCR negative regulators that can bind to G protein coupled receptor kinase(GRK),desensitize the receptor,and participate in the cascade of cell signal transduction[7].At present,it has been found that there are four major members of the Arrestin protein family,which can be divided into two categories: one is mainly distributed in the visual system,the other type is β-arrestins,which are widely distributed in various tissues [8].The present task was to study β-arrestin1,which shared 80% homology with β-arrestin2 and affected pathological process of lung cancer,diabetes and so on.It has been reported that the levels of β-arrestin1 protein and m RNA in monocytes of patients with depression are significantly reduced compared with those of healthy people,which can be improved after antidepressant treatment [10].β-arrestin1 may be an important target of antidepressant action.Antidepressants increase the release of glial cell-derived neurotrophic factors by glial cells in a way dependent on β-arrestin1,thereby promoting neurogenesis and neuronal plasticity [11].At the same time,a study in 2015 measured the levels of β-arrestin1 protein in peripheral blood monocytes of non-pregnant women during the luteal phase of menstruation and compared it with the Hamilton Depression Rating Scale score.The higher Hamilton’s depression score,the less expression of the protein β-arrestin1 in peripheral blood monocytes,and the more severe the depression [12].These studies suggest that the level of β-arrestin1 can be assessed to some extent to reflect the severity of depression.However,the specific role and mechanism of β-arrestin1 on the effect of depression are not clear,so we intend to explore the role of β-arrestin1 in the development of depression and its mechanism in basic research.OBJECTIVE: To explore the correlation between β-arrestin1 and depression and its mechanism on cells;To explore the mechanism of β-arrestin1 regulating the activation of NLRP3 inflammasome in microglia cells.METHODS:CMS depression model was established in WT mice,and the protein level of β-arrestin1 in the hippocampus of CMS mice was detected by Western blotting.The expression of β-arrestin1 in hippocampal glial cells and neurons of CMS mice was observed by immunofluorescence assay.CMS depression model was established in WT and β-arrestin1-/-mice,and then the following experiments were performed: 1.Behavioral tests were performed in each group: to detect the effect of β-arrestin1 on depression-like behavior of CMS model mice by sucrose preference experiment,novelty feeding test,forced swimming test,tail suspension test and open field;2.To explore the effects of β-arrestin1 on monoamine neurotransmitters and amino acid neurotransmitters in the hippocampus of CMS model mice by high performance liquid chromatography;3.Immunofluorescence assay was used to detect the effects of β-arrestin1 on the number of Neu N-positive cells and the fluorescence intensity of synaptic morphological index MAP2 and synaptic density index SYP in the hippocampal region of CMS mice;4.Golgi experiment was used to detect the effects of β-arrestin1 on the number of dendrites and the density of dendritic spines of neurons of CMS mice;5.To explore how β-arrestin1 affect the release of inflammatory cytokines IL-6,IL-1β and TNF-α in the hippocampus and peripheral serum of CMS model mice by RT-PCR and ELISA;6.To explore how β-arrestin1 affect the loss of astrocyte in CMS model mice by Immunofluorescence;7.To explore how β-arrestin1 affect the activation microglia activation in CMS model mice by immunohistochemistry;8.To detect the expression levels of NLRP3,pro-caspase1/caspase1 and pro-IL-1β /IL-1β in hippocampal tissue of CMS mice by western blotting,which explored the effect on activated NLRP3 in depression by β-arrestin1.Primary microglia of WT and β-arrestin1-/-mice were cultured and stimulated with LPS+ATP.1.The protein levels of NLRP3,pro-caspase1,pro-IL-1β,caspase1 and IL-1β in cell supernatant were detected;2.The expression level of NLRP3 m RNA in primary mouse microglia was detected by RT-PCR assay;3.Co-IP experiment to further explore the ubiquitination of β-arrestin1 knockout on NLRP3 inflammasome of primary microglia.After the primary WT microglia were stimulated with LPS+ATP,the protein interaction between NLRP3 and β-arrestin1 was detected by CO-IP and immunofluorescence.RESULTS:1.The effects of β-arrestin1 on behavioral and pathological characteristics of CMS miceCompared with the control group,the protein level of β-arrestin1 in the hippocampus was significantly increased in the CMS model mice.The results of brain slices fluorescence were consistent with the results of Western blotting.The expression of β-arrestin1 was up-regulated in the DG region of mouse hippocampus,and the expression was mainly on microglia cells,suggesting that β-arrestin1 may be correlated with depression.Then,WT and β-arrestin1 knockout mice was modeled by CMS to detect their depression-like behavior.It illustrated that β-arrestin1 knockout mice improved the depression-like behavior of CMS mice,which included increased immobility in tail suspension and forced swimming,decreased sucrose preference,and decreased body weight gain.The results of high-performance liquid phase experiment also showed that β-arrestin1 knockout alleviated the abnormal levels of monoamine neurotransmitters in the hippocampus after CMS but had little effect on the levels of amino acid neurotransmitters.The number of Neu N-positive cells in the nucleus of neurons,the synaptic morphological index MAP2 and the synaptic density index SYP were detected by immunofluorescence.It was found that β-arrestin1 could relieve the morphological damage of neurons but did not affect the number of nuclei of neurons.Golgi staining was further used to confirm that β-arrestin1 knockout could alleviate the downregulation of the number of dendrites and the density of dendritic spines after CMS.In addition,β-arrestin1 knockout also alleviated the loss of astrocytes after CMS.These results indicated that β-arrestin1 could participate in the pathological process of CMS mice.2.Effect and preliminary mechanism of β-arrestin1 on inflammatory response and NLRP3 activation in CMS miceIn CMS mice,immunohistochemical experiments showed that the β-arrestin1 knockout inhibited the increase in the number and volume of microglia after CMS.At the same time,the results of RT-PCR and ELISA showed that β-arrestin1 knockout alleviated the elevated levels of inflammatory cytokines after CMS.Furthermore,β-arrestin1 knockout also inhibited the up-regulation of NLRP3 inflammasome,IL-1β and caspase1 expression in the hippocampus of CMS mice.At the cellular level,β-arrestin1 knockout also inhibited the expression of NLRP3 in primary microglia cells,and the secretion of mature caspase1 and IL-1β in supernatant after stimulation.These results indicated that β-arrestin1 could inhibit the activation of NLRP3 inflammasome.And then we conduct mechanism research.By RT-PCR,we found that β-arrestin1 knockout did not affect the LPS+ATP-induced upregulation of NLRP3 m RNA levels in microglia.It suggested that β-arrestin1 may not affect NLRP3 synthesis.It is speculated that β-arrestin1 may affect the degradation of NLRP3 inflammasomes.Therefore,we detect the binding of UB to NLRP3 by CO-IP and it was found that LPS+ATP treatment promoted the ubiquitination of NLRP3 inflammasome in microglia,and β-arrestin1 knockout could further enhance the ubiquitination,which promoted the degradation of NLRP3.What’s more,the binding of β-arrestin1 and NLRP3 increased after the stimulation of LPS+ATP by CO-IP.At the same time,immunofluorescence also showed that β-arrestin1 and NLRP3 colocalization increased in microglia under the stimulation of LPS+ATP.These results indicate that β-arrestin1 can inhibit NLRP3 ubiquitination and degradation by binding to NLRP3.Conclusion:1.β-arrestin1 is associated with depression,and β-arrestin1 knockout can improve the behavioral and pathological characteristics of CMS mice.2.β-arrestin1 is involved in the inflammatory response of NLRP3 inflammasome and the activation of NLRP3 in microglia in depression.The mechanism may be that β-arrestin1 can interact with NLRP3 protein to inhibit NLRP3 ubiquitination and thus affect its degradation.To sum up,the innovation of this paper is as follows:1.It was found that β-arrestin1 was up-regulated in depression and mediated depression-like behavior and pathological changes.β-arrestin1 was involved in the regulation of neuroinflammation in CMS mice,which deepened the understanding of the pathophysiological mechanism of depression.2.It is revealed that β-arrestin1 can regulate the activation of NLRP3 inflammasome in depression and preliminarily elucidates that β-arrestin1 can inhibit the degradation of NLRP3 inflammasome to regulate its activation.This study provides a new idea for β-arrestin1 as a target for the treatment of depression. |
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