| Stress is a systemic,non-specific response of the organism in the face of multiple adverse factors in the survival environment.The body’s response to the stressor depends on the nature,intensity,and duration of the stressor,forming a continuous dose-effect curve of "inverted U" shape.In the process of long-term stress,the short-term stress in the early stage will make the body produce compensatory reactions,such as increased alertness and sensitivity;as the duration of stress continues to increase,various body systems continue to react to maintain homeostasis in the body,so as to actively adapt to the stressors.Under further stress loads,the cumulative effects lead to disturbances in body homeostasis,finally resulting in pathophysiological damage.The brain is the primary organ of stress and is adaptive to social and physical stressors because it identifies threat factors,stores memories,and regulates physiological and behavioral responses that may be harmful or protective.In a broad sense,stress has a wide range of effects on the entire brain,and the effects of stress on cognitive function do so primarily through three brain regions(hippocampus,amygdala,and prefrontal cortex).Research suggests that chronic stress may have deleterious effects on the hippocampus in rodents.As one of the key brain regions mediating the stress response,the hippocampus is primarily responsible for learning,memory,cognition,and emotion.Under acute stress,acute compensatory responses are generated in the hippocampus to facilitate the body’s active response to stress,counteracting the adverse effects of stress to restore the body to a new homeostatic state.These compensatory responses focus on the rapid activation of the sympathetic nervous system and the HPA axis,resulting in the release of norepinephrine and epinephrine from widely distributed synapses in the brain,growth of dendritic branches in CA1 and CA3 regions,and increased synaptic density.However,long-term stress leads to decreased plasticity of hippocampal neurons and an imbalance between apoptosis and regeneration of hippocampal cells,resulting in atrophy and loss of neuronal cells,causing structural and functional damage to the hippocampus.In the process of chronic stress,along with the fluctuation and interaction of various stress media,the hippocampus produces an acute compensatory response in the early stages of stress to actively cope with the adverse effects of stress,then gradually forms a new homeostatic balance to adapt to the stressful state,and gradually loses its compensatory response to damage in the later stages,resulting in stress impairment,which includes cognitive dysfunction.The biological mechanisms underlying this transition from compensation to adaptation to damage are not yet clear.The development and application of Next Generation Sequencing(NGS)technology has brought great convenience and breakthroughs to biological research.The mRNA-seq technique can detect the whole transcriptome of tissues or cells in a single experiment,leading to an exhaustive analysis of gene expression networks from which to screen research targets and advance research progress.The use of this method to detect and analyze dynamic changes in hippocampal gene expression during chronic stress has the potential to be suggestive and helpful in revealing the biological mechanisms underlying the transition from adaptation to damage from stress.Therefore,this study intends to clarify the dynamic changes of hippocampal transcriptome during chronic stress by transcriptome sequencing technology,to perform functional analysis of hippocampal transcriptome according to the neurophysiological pattern of stress,to summarize the pattern of biological function transformation during chronic stress,and to screen the related gene Bmp7 that may play a neuroprotective role during chronic stress,to verify whether BMP7 plays a neuroprotective role in the process of stress-induced cognitive dysfunction,and to further clarify the regulatory mechanism of BMP7 expression changes during chronic stress.Ⅰ.Dynamic effects of chronic unpredictable mild stress on the transcriptome of the rat hippocampusTo investigate the extensive changes in hippocampal transcriptome under chronic stress and to reveal the dynamic patterns of hippocampal transcriptome at different time points under chronic stress,chronic unpredictable mild stress model(3 days,2 weeks,6 weeks)was established using 6-8 weeks old SD rats.Relevant behavioral methods(Open-field experiment,Morris water maze experiment,New object recognition experiment)were used to detect the cognitive changes in CUMS rats;mRNA-seq technique was used to analyze the hippocampal transcriptome of chronic unpredictable mild stress(CUMS)rats at different time points.Using GO analysis,we explored the potential mechanisms of the harmful effects of stress on hippocampus;using cluster analysis and trend analysis,we clarified the dynamic patterns of hippocampal transcriptome changes at different time points of CUMS.Compared with the control,501,442 and 235 differentially expressed genes were detected in the hippocampus of rats subjected to CUMS for 3 days and 2 and 6 weeks,respectively.Gene Ontology(GO)analysis was used to determine the potential mechanism underlying the dynamic harmful effects of stress on the hippocampus;Certain GO terms of the down-regulated DEGs in CUMS(3 days)rats were also found in the up-regulated DEGs in CUMS(6 weeks)rats.These results showed opposing regulation patterns of DEGs between CUMS at 3 days and 6 weeks,which suggested a functional change from adaptation to damage in during the early and late stages of chronic stress.GO analysis for upregulated genes in rats subjected to CUMS for 3 days and 2 weeks suggested significant changes in ’ extracellular matrix’and ’ wound healing’.Upregulated genes in rats subjected to CUMS for 2 weeks were involved in changes associated with visual function.GO analysis of DEGs in rats subjected to CUMS for 6 weeks revealed increased expression of genes associated with ’ apoptotic process’and ’ aging’and decreased expression of those associated with inhibition of cell proliferation and cell structure.Based on the results of DEGs trend analysis,combined with stress physiology patterns and literature findings,Bmp7,as a gene for early compensatory changes,may promote positive hippocampal responses to the adverse effects of stress and protect the organism from cognitive impairment.These results suggest that early and middle phases of chronic stress mainly promote adaptive regulation and damage repair in the organism,while late phases of chronic stress lead to damage in the hippocampus.Bmp7,as a gene for early adaptive changes,may play a protective role against stress-induced cognitive dysfunction.Ⅱ.Protective effects of BMP7 in stress-induced cognitive dysfunctionBy modulating the changes of BMP7 during chronic stress,we observed whether it exerts a neuroprotective effect to antagonize the onset of stress-induced cognitive dysfunction.At the animal level,a chronic unpredictable mild stress model was established using 6-8 weeks SD rats,the cellular localization of BMP7 in the rat hippocampus was clarified using immunofluorescence method,the pattern of BMP7 expression changes in the rat hippocampus was clarified using WB and RT-qPCR assays,and the changes of NE/GC levels in the rat plasma and brain were detected using ELISA kits(competition method).Expression changes of PSD95,GAP43,Syp protein and cleaved-Caspase3 in the hippocampus of chronically stressed rats using WB method;the changes of oxidative stress in the hippocampus of chronically stressed rats were detected using enzyme cycling assay kit.In addition,the rats were injected with BMP7 overexpressing/interfering AAV virus using brain stereotaxic technique,and the changes in cognitive function of rats were detected by behavioral experiments,and the levels of apoptosis,oxidative stress and synaptic damage in the hippocampus after virus injection were further detected.At the cellular level,PC12 cells were intervened with GC to induce the onset of cellular damage effects and detect changes in biological functions of PC12 cells.Furthermore,BMP7 overexpression/interfering lentivirus was transfected in PC12 cell,and then cells were intervened with GC or DMSO to detect changes in cell viability using CCK-8 kit.The changes of apoptosis and cell cycle changes were detected using flowcytometry.RT-qPCR was used to clarify the changes of Bax gene,WB experiment was used to clarify the changes of apoptosis-related protein cleaved-Caspase3,immunofluorescence was used to observe the changes of reactive oxygen species and cellular neuronal synapses in cells,and enzyme cycling kit was used to detect the changes of cellular oxidative stress level.The results showed that BMP7 was mainly localized in neuronal cells in the rat hippocampus,and its expression level increased significantly after 3 days of stress,then gradually decreased and returned to normal at 6 weeks;the trend of NE hormone in plasma was similar to that of BMP7,while GC hormone slowly increased with the increase of stress time.Chronic stress induced a decrease of synapse-related protein expression,an increase of apoptosis and oxidative stress in the hippocampus of rats.Overexpression of BMP7 improved the cognition function of CUMS rats,reduced apoptosis and oxidative stress level,and enhanced synaptic plasticity in CUMS rats.Interference with BMP7 expression impaired cognition function of rats,induced apoptosis,and increased apoptosis and oxidative stress level in CUMS rats,and reduced synaptic plasticity.High levels of GC induced a decrease in rat neuron-like PC 12 cell activity was reduced and apoptosis was increased.Overexpression of BMP7 antagonizes GC-induced oxidative stress and apoptosis levels in PC12 cells and antagonizes GC-induced neurosynaptic damage in PC12 cells;interference with BMP7 exacerbates GC-induced damage effects and oxidative stress levels in PC12 cells,induces neurosynaptic damage in PC12 cells,and exacerbates GC-induced neurosynaptic damage levels.The above results indicate that during chronic stress,the elevation of BMP7 in the early stage helps the body adapt to stress and protects the hippocampus from stress damage,while with the extension of stress time,the expression of BMP7 gradually decreases,the protective effect on stress response gradually decreases,and the stress changes from adaptive to damaging response.Ⅲ.Transcriptional regulatory mechanisms of BMP7 during chronic stressThe second part of the study demonstrated that BMP7 plays a protective role against stress-induced cognitive dysfunction,but its transcriptional regulatory mechanism is currently unclear.In order to further clarify the regulatory mechanism of BMP7 expression during chronic stress,we first used bioinformatic methods to clarify the promoter sequence of BMP7 and search for its possible binding transcription factors,and initially clarified the candidate transcription factor NFATc4 by searching the JASPAR database,mRNA-seq sequencing results and the change pattern of BMP7 during chronic stress.Then RT-qPCR and WB experiments were used to clarify the change pattern of NFATc4 during chronic stress,and it was found that its expression change trend was similar to that of BMP7.To further clarify the regulatory mechanism of chronic stress on NFATc4 and BMP7,the stress hormone GC/NE was used to intervene in PC12 cells,and the results showed that appropriate concentrations of NE hormone promoted the expression of BMP7 and NFATc4,while GC hormone had no significant effect on the expression of both.The specific adrenergic receptors involved in the regulation of NFATc4 were clarified by inhibiting the activity of individual NE receptors through inhibitors,and RT-qPCR and WB experiments were used to clarify the specific adrenergic receptors involved in the regulation of NFATc4.We initially screened three NE downstream signaling pathways:cAMP/PKA,Ca2+/CaN and PI3k/Akt pathways,and then further validated and screened the signaling pathways in PC12 cells and CUMS rats using WB experiments,and found that NE hormone promoted the expression of BMP7 and NFATc4 by activating the downstream cAMP/PKA and Ca2+/CaN pathways.To further clarify the relationship between the transcription factor NFATc4 and BMP7,we overexpressed or repressed NFATc4 expression in PC 12 cells and found that the expression of BMP7 was subsequently increased or decreased.The changes of the binding level of transcription factor NFATc4 to the promoter of Bmp7 gene were further examined by dual fluorescein reporter gene and ChIP assay,and it was found that transcription factor NFATc4 directly regulates the expression of BMP7 by binding to the promoter of Bmp7 gene,and NE hormone promotes the binding of NFATc4 to the promoter of Bmp7 gene and thus the transcription of Bmp7 gene.The above results show that in the process of chronic stress,NE hormone increases at the early stage of stress,and the downstream cAMP/PKA and Ca2+/CaN pathways are activated successively by binding with ADRB1 receptor to promote the nuclear transfer of NFATc4,and then NFATc4 combines with Bmp7 gene promoter to promote the transcription of Bmp7.In summary,hippocampal transcriptome sequencing results suggest that genes compensated for upregulation early in chronic stress play an important role in promoting adaptive regulation and damage repair in the organism,and BMP7,as a representative gene for early adaptive changes in stress,can significantly improve stress-induced hippocampal damage,enhance hippocampal synaptic plasticity,and ameliorate chronic stress-induced cognitive dysfunction.During chronic stress,BMP7 is regulated by NE hormone,which activates its downstream cAMP/PKA and Ca2+/CaN signaling pathways by binding to the β1-type adrenergic receptor and regulates the expression of NFATc4,which directly regulates BMP7 expression by binding to the Bmp7 promoter.The results of this study suggest that BMP7 plays a protective role in the occurrence of stress-induced cognitive dysfunction,and by increasing the expression of BMP7 in the brain,it can effectively protect cognitive function,providing a new target and idea for stress injury protection. |
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