| BackgroundDepression is a common psychiatric disorder,which is mainly manifested as low mood,retardation of thinking,decreased volitional activities and cognitive impairment.Besides,depression is often accompanied by some somatic symptoms such as sleep disorder,appetite loss and fatigue.With high incidence,recurrence and disability rate,depression brings heavy disease and economic burden to patients and society.However,the etiology and pathogenesis of depression are complex and have not been fully elucidated.Therefore,it is of great significance to gain insight into the pathogenesis of depression for its treatment and prevention.A substantial number of studies manifest that stress is a prominent risk factor for depression and early life stress augments the risk for depression in adulthood.The reason is that during the early stages of life,the brain is highly malleable and exposure to stress during this period causes persistent alterations in the structure and function of the brain.Previous studies suggested that early life stress resulted in the hypoevolutism and dysfunction of brain regions associated with emotion and cognition,for instance,the prefrontal cortex,hippocampus and amygdala and resulted in the dysfunction of hypothalamic-pituitary-adrenal(HPA)axis.The aforementioned changes are considered as the important pathogenesis of increased susceptibility to depression induced by early life stress.The HPA axis is an important functional system that regulates the body’s responses to stress.The negative feedback regulation of glucocorticoid(GC)on the HPA axis needs to be mediated by glucocorticoid receptor(GR).The reduced GR expression or impaired GR function is considered as a pivot factor leading to the abnormality of HPA axis in depressed patients.GR acts as a ligand-dependent transcription factor that translocates from the cytosol to the nucleus to regulate gene transcription after ligand binding.This process requires the heat shock protein 90(Hsp90)as the chaperone protein to participate in and FK506 binding protein 51(FKBP5)as the co-chaperone protein to bind to the GR-Hsp90 complex.The receptor complex has lower affinity for its ligand.Upon ligand binding,the receptor complex disintegrates and translocates into the nucleus regulating gene transcription.Thus,FKBP5 regulates HPA axis responses to stress by regulating the sensitivity and nuclear translocation efficiency of GR.MicroRNAs(miRNAs)are a class of small noncoding RNAs,which are able to bind with the 3’-untranslated regions(3’-UTRs)of the target messenger RNAs(mRNAs).They can regulate gene expression by either repressing translation or inducing degradation of mRNA,thus participating in various biological processes.Studies show that some microRNAs are closely related to the stress response and can directly regulate the expression of related genes.For example,miR-124a in the corpus callosum and miR-18 in the hypothalamus of mice were associated with acute stress response by regulating the expression of GR protein.Studies reported that miR-124a and miR-18a reduced GR expression via binding to the 3’-UTRs of GR gene.In addition,a recent study found that the expression of miR-511 was upregulated under the induction of glucocorticoids and miR-511 could negatively regulate the levels of FKBP5 protein by binding to the 3’-UTRs of FKBP5 gene.Based on the above studies,we speculated that miR-124a.miR-18a and miR-511 might play key roles in the susceptibility to depression resulted from stress.Therefore,the current study focused on the short-term and long-term effects of adolescent chronic stress on depressive-like behaviours,GR and FKBP5 expressions,as well as miR-124a,miR-18a and miR-511 expressions in the basolateral amygdala,hippocampus and prefrontal cortex which are closely related to the emotion and cognition regulation,in order to reveal the roles of GR,FKBP5,as well as miR-124a,miR-18a and miR-511 in the susceptibility to depressive disorder induced by adolescent chronic stress.Objectives(1)To investigate the short-term and long-term effects of adolescent chronic stress and dexamethasone administration on depressive-like behaviours,cognitive function and the expressions of GR,FKBP5,as well as miR-124a and miR-18a in the basolateral amygdala of rats.(2)To investigate the short-term and long-term effects of adolescent chronic stress and dexamethasone administration on depressive-like behaviours,cognitive function and the expressions of GR,FKBP5,as well as miR-124a,miR-18a and miR-511 in the prefrontal cortex and hippocampus of rats.(3)To probe the roles of GR,FKBP5,as well as miR-124a,miR-18a and miR-511 in the susceptibility to depressive disorder induced by adolescent chronic stress.Materials and methods(1)Experimental animals and groupingA total of 100 male Wistar rats(21 days old)after 7 days of acclimatization were randomly divided into 5 groups(n = 20 in each group):control group(C),chronic unpredictable mild stress(CUMS)group(S),CUMS and RU486 group(S+R),dexamethasone group(D),dexamethasone and RU486 group(D+R).Both CUMS and subcutaneous injection of dexamethasone for 21 consecutive days were used to establish the animal models of depression.Meanwhile,the chronic subcutaneous injection of RU486,the antagonist of GR,was used as a means of intervention.After modeling,10 rats randomly selected from each group were sacrificed for molecular biology experiments after behavioural tests,while the others were raised into adulthood and sacrificed after behavioural tests.(2)Chronic unpredictable mild stressThe stressors were included as follows:food deprivation for 24 h;water deprivation for 24 h;noise(92 dB,1500 Hz)for 2 h;pinching tail for 1 min;hot stress in oven at 45℃ for 5 min;day and night reversal for 24 h;and unpredictable foot shocks(1 mA,one shock/5 s,10-s duration for a total of 10 min).The stress process lasted for 21 days.The time and form of stressor daily were at random and each type of stressor was guaranteed to be performed three times during the CUMS period.(3)Drug administrationRats in the S + R group were treated with a single daily administration of RU486 at a dose of 10 mg/kg 30 min before the CUMS procedure.Rats in the D group received a single daily injection of dexamethasone at a dose of 1.5 mg/kg.Rats in the D + R group were treated with RU486 at the dose of 10 mg/kg 30 min before the application of dexamethasone at the dose of 1.5 mg/kg.All drugs were administrated subcutaneously for 21 consecutive days.Rats in the nondrug groups and one-time drug administration groups received an equivalent volume of normal saline or propylene glycol to balance the systematic error.(4)Behavioural testsThe sucrose preference test was conducted to evaluate an anhedonia state,one of the core symptoms of depression.The open field test was used to measure spontaneous activity and exploratory behaviours.The elevated plus maze test was performed to assess anxiety-like behaviours.The Morris water maze test was used for evaluating spatial learning and memory.(5)ImmunohistochemistryAfter the behavioural tests,four rats in each group were randomly selected to be anesthetized and transcardially perfused.The brain was taken out and embedded in paraffin block.The paraffin-embedded basolateral amygdala,hippocampus and prefrontal cortex were sliced into 4-μm coronal sections to detect the distribution and expression of GR and FKBP5 by immunohistochemical staining.(6)Western blottingAfter the behavioural tests,the rats were anesthetized and sacrificed.The whole brain was removed and both sides of basolateral amygdala,hippocampus and prefrontal cortex were isolated(one side used for western blotting,the other side used for real-time PCR).The basolateral amygdala,hippocampus and prefrontal cortex tissues were homogenized and centrifuged at 12000 rpm for 25 min at 4℃.The supernatants were collected and the protein concentration was measured with a BCA Protein Assay Kit.After denaturation,20 μg protein samples were loaded into each electrophoresis channel and separated by electrophoresis,then transferred to polyvinylidene difluoride(PVDF)membranes.After blocked in 5%skim milk in Tris buffered saline with Tween-20(TBST),the PVDF membranes were incubated with appropriate primary antibodies overnight at 4℃.On the following day,the PVDF membranes were washed and incubated with secondary antibodies for 1 h at room temperature.The PVDF membranes were washed again and developed by using a chemiluminescent method.The grey value of stripes was analyzed with the use of Image J 14.0 software.(7)Real-time PCR assayThe total RNA was extracted from basolateral amygdala,hippocampus and prefrontal cortex tissues using Trizol reagent.RNA concentration and purity were measured.RNA was reversely transcribed to cDNA using the corresponding reverse transcription primers.Then real-time PCR was performed according to the standard procedures to detect expressions of miR-124a,miR-18a and miR-511.U6 was used as an internal reference and relative expression levels of miR-124a,miR-18a,and miR-511 were calculated using 2-ΔΔCT method.Results(1)Both adolescent and adult rats that exposed to adolescent CUMS and dexamethasone administration manifested obvious depressive-like behaviours,anxiety-like behaviours and memory impairment.(2)Both adolescent CUMS and dexamethasone administration resulted in decreased GR expression and increased FKBP5 expression in the basolateral amygdala of adolescent and adult rats.(3)Both adolescent CUMS and dexamethasone administration induced an increase in miR-124a expression in the basolateral amygdala of adolescent and adult rats,and an increase in miR-18a expression in the basolateral amygdala of adolescent rats.(4)Both adolescent CUMS and dexamethasone administration decreased GR expression and increased FKBP5 expression in the the prefrontal cortex and hippocampus of adolescent and adult rats.(5)Both adolescent CUMS and dexamethasone administration induced upregulated expressions of miR-124a and miR-18a in the prefrontal cortex and hippocampus of adolescent and adult rats,and downregulated miR-511 expression in the prefrontal cortex of adult rats.(6)RU486,the GR antagonist,could effectively ameliorate the depressive-like behaviours,anxiety-like behaviours and memory abnormalities,and normalize the expressions of GR,FKBP5,miR-124a and miR-18a.Conclusions(1)Adolescent chronic stress and dexamethasone administration resulted in depressive-like behaviours,anxiety-like behaviours and memory impairment from adolescence to adulthood.(2)The abnormal expression of GR and FKBP5 in the prefrontal cortex,hippocampus and basolateral amygdala plays an important role in the susceptibility to depressive disorder induced by adolescent stress.(3)MiR-124a is associated with the susceptibility to depressive disorder via regulating GR expression in the prefrontal cortex,hippocampus and basolateral amygdala,and miR-18a is related to the susceptibility to depressive disorder by regulating GR expression in the prefrontal cortex and hippocampus.(4)The decreased expression of miR-511 in the prefrontal cortex is involved with an increased susceptibility to depressive disorder induced by adolescent stress. |
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