Effects Of Mrp8/14 On Depressive-Like Behavior And Its Mechanism Study

Background:Depression is one of the most common mental disorders characterized by significantly and enduringly low mood.At present,more than 300 million patients worldwide suffer from depression.In general,the prevalence rate of depression in the United States and other developed countries is higher than that in the developing countries.The prevalence rate of depressive disorder is about 3%-5%according to the data provided by National Health and Family Planning Commission?3.59%?and World Health Organisation?4.2%?in 2017.Female patients with depression are significantly more than male patients.People with depression experience a high rate of disability.Depression not only damages the physical and mental health of the patients,but also results in a significant economic burden for the society.In the early 1950s,Selikoff and Robidzek found that an anti-tuberculosis medicine,called iproniazid could improve mood.Thereafter,generations of antidepressants have made great progress from monoamine oxidase inhibitors?MAOIs?and tricyclic antidepressants?TCAs?to selective serotonin reuptake inhibitors?SSRIs?and selective norepinephrine reuptake inhibitors?SNRIs?.However,even compared with the first generation of classic antidepressants,the widely used first-line antidepressants?SSRIs and SNRIs?are not perfect.About a third to a half of depression patients are still not responding to the classic antidepressants.Besides,most of the antidepressants work after continuous treatment of 2-4 weeks,which is quite slow and infaust to these depression patients who have suicidal feelings.The disease mechanism is very complex.There are various hypotheses about depression,such as the monoamine neurotransmitter,neuroinflammation,neural plasticity,stress/hypothalamic-pituitary-adrenal axis,the balance of glutamic acid and GABA,anhedonia/opioids,the balance of choline and adrenaline etc.Although the etiology and pathophysiology of depression remain unknown,recent evidence suggests that neuroinflammation affects the brain signal patterns involved in the psychopathology of depression.Microglia are the primary participants in neuroinflammation.Primed microglia have the similar surveillance function as resting microglia.When they are attracted by the secondary stimulation,the proinflammatory action will be extraordinarily enhanced.Besides,animals with primed microglia are more vulnerable to depressive-like behavior.Intense or durable immunity stimulation can also induce microglia activation,promote a large secretion of pro-inflammatory cytokines,evoke neuroinflammation response and then result in depressive-like behavior.Studies from our and other labs have found that some damage associated molecular patterns?DAMPs?play a vital role in the development of depression.Take HMGB1 for example,it induces the priming of hippocampal microglia and significantly amplifies the proinflammatory response to LPS.Ds-HMGB1 can evoke neuroinflammation mediated by TLR4 and RAGE,and then induces depressive-like behavior in mice.Mrp8 and Mrp14 are two important members of the Ca²⁺binding S100 protein family and have been identified as crucial endogenous DAMPs.Under physiological conditions,these two proteins exist and function mainly as a heterodimeric complex of Mrp8/14.Mrp8/14 can be actively or passively released into the extracellular matrix.After binding with TLR4,RAGE and other pattern recognition receptors?PRRs?,Mrp8/14 can induce proinflammatory effects.Mounting evidence shows that Mrp8/14 is involved in various inflammatory diseases,but no study about Mrp8/14 and depression has been conducted.Therefore,this study aims to explore the effects of Mrp8/14 in depression and further reveal the potential molecular mechanisms.Contents:1 The effects of Mrp8/14 in inflammatory primingWe explored the role of Mrp8/14 in microglial priming in rat hippocampus using acute tail shocks.The inhibitor of Mrp8/14?ABR-215757?was injected to test the changes of microglial priming.Besides,after central injection of recombinant Mrp8/14 protein?rMrp8/14?,hippocampal microglia in rats were isolated to reveal the effects of rMrp8/14on microglial priming in the hippocampus.2 The changes of Mrp8/14 and related inflammatory signaling pathways in the mouse model of depressionThe mouse model of depression was established using chronic unpredictable mild stress?CUMS?.Sucrose preference test and tail suspension test were used to evaluate the animal model.The change and source of Mrp8/14 after stress were detected and the changes of hippocampal TLR4 and RAGE signaling pathways were also determined.3 The effects of Mrp8/14 in CUMS-induced depressive-like behavior and inflammatory signaling pathwaysABR-215757 was used as the selective inhibitor of Mrp8/14.ABR-215757 was daily administrated via i.p injection to detect its effects on depressive-like behavior and the changes of TLR4 and RAGE signaling pathways.4 Effects of recombinant Mrp proteins on neuroinflammation and depressive-like behaviorRecombinant Mrp proteins were centrally administrated via i.c.v cannulation,and the change of depressive-like behavior was measured.After that,we further explored the microglia activation,TLR4 and RAGE signaling activation,and the release of central cytokines.5 Effects of TLR4 on rMrp8/14-induced neuroinflammation and depressive-like behaviorThe Mrp8/14 heterodimer was used to further study its mechanism on depressive-like behavior and neuroinflammation.A TLR4 inhibitor?TAK-242?was injected before the treatment with recombinant Mrp8/14 protein.The effects of the TLR4 inhibitor on Mrp8/14-induced neuroinflammation and depressive-like behavior were detected.6 Effects of TLR4 on rMrp8/14-induced BV2 microglia activationThe effects of Mrp8/14 on microglia activation were confirmed using BV2 microglia in vitro.Reactive oxygen species?ROS?and nitric oxide?NO?were used to determine microglia activation.Results:1 Mrp8/14 mediated neuroinflammatory priming evoked by acute tail shocks1.1 After 24 hours of acute tail shocks,hippocampal Mrp8 and Mrp14 mRNA was significantly increased in rats.Mrp14 protein was also markedly upregulated.1.2 The in vitro study showed that lipopolysaccharide?LPS?could rapidly increase the expressions of TNF-?,IL-1?and IL-6 in microglia.However,ABR-215757 at different concentration could not change the effects of LPS.1.3 After treatment with LPS,the expressions of TNF-?,IL-1?and IL-6 in microglia of stressed rats were elevated.ICM pretreatment with Mrp8/14 inhibitor prevented the increases of TNF-?,IL-1?,and IL-6 induced by stress and LPS.1.4 ICM administration with rMrp8/14 at a low dosage could significantly enhance the proinflammatory effects response to LPS.The expressions of TNF-?,IL-1?and IL-6 in microglia of rMrp8/14-treated rats were elevated.2 CUMS activated Mrp8/14 and related inflammatory signaling pathways2.1 After 4 weeks of CUMS,the sucrose preference was decreased,while the immobility duration in tail suspension test was increased.The stressed mice showed depressive-like behavior.2.2 Mrp8 and Mrp14 in serum and hippocampus were significantly upregulated in stressed mice,and the increased Mrp8 and Mrp14 might derive from hippocampal neurons.2.3 Hippocampal TLR4 was upregulated and the phosphorylation of NF-?B p65 was enhanced in mice subjected to CUMS.2.4 No significant effects of CUMS was found on the expression of RAGE.3 The Mrp8/14 inhibitor affected depressive-like behavior and inflammatory signaling pathways3.1 The Mrp8/14 inhibitor significantly improved the CUMS-induced depressive-like behavior?sucrose preference test and tail suspension test?.3.2 Mrp8/14 inhibitor could reduce the upregulation of TLR4 protein and the phosphorylation of NF-?B p65.RAGE did not change significantly after administration of Mrp8/14 inhibitor.4 Recombinant Mrp proteins could induce the neuroinflammation and depressive-like behavior4.1 Central administration of rMrp8,rMrp14 or rMrp8/14 could reduce the sucrose preference and extend the immobility duration in tail suspension test in mice.4.2 Central administration of rMrp8,rMrp14 or rMrp8/14 could induce the upregulation of TLR4 protein and the phosphorylation of NF-?B p65.RAGE did not change significantly after administration of recombinant Mrp proteins.4.3 The recombinant Mrp proteins promoted proinflammatory cytokines?TNF-?,IL-1?and IL-6?production,and promoted the expression of IBA-1.5 The inhibitor of TLR4 could improve rMrp8/14-induced neuroinflammation and depressive-like behavior5.1 The sucrose preference test and tail suspension test showed that pretreatment with the inhibitor of TLR4?TAK-242?could significantly improve the rMrp8/14-induced depressive-like behavior.5.2 Pretreatment with TAK-242 reduced rMrp8/14-induced TLR4/NF-?B signaling activation,decreased the generation of proinflammatory cytokines and the upregulation of IBA-1.6 The inhibitor of TLR4 could inhibit rMrp8/14-induced BV2 microglia activationrMrp8/14 increased the generation of nitric oxide and reactive oxygen species,and enhanced the expression of iNOS in BV2 microglia ex vivo.Pretreatment with TLR4inhibitor inhibited rMrp8/14-induced BV2 microglia activation.Conclusions:Mrp8/14 plays a key role in neuroinflammatory priming and depressive-like behavior.The Mrp8/14 inhibitor ABR-215757 improves acute stress-induced microglial priming and chronic stress-induced depressive-like behavior and TLR4/NF-?B signaling activation.The present study further enriches the neuroinflammation hypothesis of depression.Targeting the Mrp8/14 may be a novel promising antidepressant approach.