Epigenetic Factors BRD4 And EZH2 Regulating Neuroinflammation Involved In Post-traumatic Stress Disorder And Depression

BackgroundPost-traumatic stress disorder(PTSD)and depression have a high incidence worldwide,which brings heavy mental and economic burdens to society and individuals.Traumatic and chronic stresses are critical involved in the pathogenesis of these disorders.Till now the mechanism is still unclear,which make treatment difficult and chronic course.It's meaningful to explore the underlying mechanism and search for some new pharmacological targets in stress-induced PTSD and depression.Accumulated studies have proved that stress could activate the inflammatory immune response,causing the inflammatory microenvironment disorder and the imbalance of the expression of inflammatory cytokines in the brain,which affects the synaptic plasticity and neurogenesis.These processes are close related to the pathogenies of PTSD and depression.Microglia are the main innate immune cells in the central nervous system,which involved in the regulation of brain environmental homeostasis by releasing inflammatory mediators and neurotrophic factors.Under physiological conditions,it participates in synapse formation and pruning,nutritional support,chemotaxis and neurogenesis.When the homeostasis of the internal environment is disturbed by psychological stress,infection,injury,neurodegeneration,the structure and function of microglia will change and status of microglia is defined as "activated".Both traumatic and chronic stress could cause over-activation of microglia and neuroinflammation,which involved in stress-induced PTSD and depression.Plenty of molecules involved in the regulation of the activation of microglia,and epigenetics plays an important role in this process.Bromodomain-containing protein 4(BRD4)may involve in the regulation of microglial activation and various inflammatory genes such as interleukin(IL)-1?,IL-6 and tumor mecrosis factor(TNF)-a.Methylation of lysine on histone is involved in the regulation of activation and silencing of genes.Enhancer of zeste homolog 2(EZH2),targeting the trimethylation level of lysine 27 on histone H3,has been reported to regulate microglial activation and polarization and neuroinflammation.However,whether BRD4 and EZH2 regulating neuroinflammation are involved in PTSD and depression?This is not yet clear.Based on animal models of PTSD and depression,this study focuses on traumatic stress and chronic stress on depression/anxiety-like behaviors,conditional fear memory,spatial learning and memory,phenotype of microglial activation,levels of pro-inflammatory cytokines,BRD4,EZH2 and H3K27me3 in stress-associated areas of the brain.The aim of the study is to examine whether BRD4 and EZH2 regulating neuroinflammation are involved in PTSD and depression.Objective(1)To observe the effects of traumatic stress on PTSD-like behaviors,the levels of BRD4 and pro-inflammatory cytokines,microglial activation and NF-?B,the transcription level of immediate early genes and the dendritic spine density of pyramidal neurons in the prefrontal cortex(PFC),hippocampus and amygdala of animals.To observe the effects of BRD4 inhibitor on the changes of the above mentioned indicators,and to explore the mechanism of BRD4 regulating neuroinflammation involved in traumatic stress-induced PTSD.(2)To observe the effects of chronic mild stress on depression-like behaviors and learning and memory,pro-inflammatory cytokines,microglial activation,and the levels of NF-?B,EZH2,H3K27me3 and SOCS3 in the PFC and hippocampus of animals.To observe the effects of EZH2 inhibitor on the above behavioral and molecular changes,and to clarify the role of EZH2 regulating of neuroinflammation involved in chronic stress-induced depression.Methods and results1 Part ?1.1 Experimental animal and grouping82 male Wistar rats(2-month-old)were divided into 4 groups:Control group,PTSD group,JQ1 group,and PTSD with JQ1 group.After one week of acclimatization,PTSD model was built and the rats formed fear conditioning.Rats in the medication group were administrated with BRD4 inhibitor JQ1 intraperitoneally.Then behavior tests were conducted subsequently according to the requirement,and tissues were sampled and trimmed for molecular biology experiments.1.2 Experimental methods(1)The PTSD model and conditioned fear memory were conducted with inescapable foot shocks(IFS)twice a day for six days,and foot shocks were composed of 15 shocks in 30 minutes accompanied by sound cues.(2)The conditioned fear memory test,sucrose preference test,open field test and elevated plus maze test were used to detect conditioned fear memory,depression-/anxiety-like behaviors in animals,and Y maze test was use to evaluate the spatial memory.(3)Enzyme-linked immunosorbent assay was used to detect the levels of pro-inflammatory cytokines in the PFC,hippocampus and amygdala of animals.(4)Immunohistochemistry was used to detect the level of microglia marker ionized calcium-bingding adaptor protein-1(Iba-1)in the PFC,hippocampus and amygdala of animals.(4)Real-time quantitative PCR was used to detect the transcriptional levels of immediate early genes in the PFC,hippocampus and amygdala of animals.(5)The activation of NF-?B in the PFC,hippocampus and amygdala of animals were detected by western blotting.(5)Golgi-cox staining was used to detect the dendritic spine density of pyramidal neurons in the PFC,hippocampus and amygdala of animals.1.3 Results(1)The IFS could induce conditioned fear memory,anhedonia,anxiety-like behavior and memory impairment in animals,while BRD4 inhibitor JQ1 could reduce the freezing time and improve behavioral impairment.(2)The IFS could significantly increase the levels of BRD4 mRNA and protein in the PFC,hippocampus and amygdala of animals.(3)The IFS could increase the levels of pro-inflammatory cytokines,the activation of microglia and NF-?B in the PFC and hippocampus of animals.However,the effect of stress on the levels of pro-inflammatory cytokines,the activation of microglia and NF-?B in the amygdala was not significant.BRD4 inhibitor JQ1 could significantly reduce the level of pro-inflammatory cytokines,the activation of microglia and NF-?B in the PFC and hippocampus.(4)The IFS could reduce the activation of immediate early genes in the PFC and hippocampus of the animals,and increase the level of immediate early genes in the amygdala.BRD4 inhibitor JQ1 could restore the levels of immediate early genes.(5)The IFS could decrease the dendritic spine density of pyramidal neurons in the PFC and hippocampus,while increase the dendritic spine density of pyramidal neurons in the amygdala.BRD4 inhibitor JQ1 could restore the dendritic spine density of pyramidal neurons.2 Part ?2.1 Experimental animal and groupingThirty male C57BL/6 mice(2 months old)were randomly divided into three groups of ten mice per group:control group,chronic stress group,chronic stress with EPZ-6438 group.After adaptation for one week,mice in the stressed groups received the CUMS procedure and the mice in the medication group were intraperitoneally administrated with EPZ-6438 once a day(1 mg/kg).Mice in the control and the chronic stress-only groups were administrated daily with the vehicle to balance the error.Then behavior tests were conducted subsequently according to the requirement,and tissues were sampled and trimmed for molecular biology experiments.2.2 Experimental methods(1)The chronic unpredictable mild stress(CUMS)procedure in 21 days was used to build animal model of depression.(2)Sucrose preference test,open field test,and elevated plus maze test were used to detect depression and anxiety-like behaviors of animals.Morris water maze test was used to evaluate the spatial learning and memory of animals.(3)Enzyme-linked immunosorbent assay was used to detect the levels of pro-inflammatory cytokines in the PFC and hippocampus of animals.(4)Immunohistochemistry was used to detect the number of Iba-1 positive cells and inducible nitric oxide synthase(iNOS)positive cells in the PFC and hippocampus of animals.(5)Western blotting was used to detect the levels of NF-?B,EZH2,H3K27me3 and suppressor of cytokine signaling 3(SOCS3)in the PFC and hippocampus of animals.2.3 Results(1)CUMS could induce depression/anxiety-like behaviors and spatial learning and memory damage in animals,while administration with EZH2 inhibitor EPZ-6438 could significantly reduce these behavioral impairments.(2)CUMS could increase the activation of microglia to M1,levels of pro-inflammatory cytokines and NF-?B in the PFC and hippocampus of animals,while administration with EZH2 inhibitor EPZ-6438 could significantly restore above changes.(3)CUMS could upregulate the expression of EZH2,H3K27me3 and SOCS3 in the PFC and hippocampus of animals,while administration of EZH2 inhibitor EPZ-6438 could decrease the level of EZH2 and H3K27me3,and increase the level of SOCS3.Conclusions(1)BRD4 could regulate the neuroinflammation in the PFC and hippocampus and dendritic spine density in the PFC,hippocampus and amygdala involved in the conditioned fear memory and PTSD-like behaviors.(2)EZH2 targeted H3K27me3 could regulate of microglial activation and pro-inflammatory cytokines involved in the chronic stress-induced depression-like behaviors in animals,which was partly mediated by affecting the level of SOCS3.