| Depression is a common neurodegenerative disorder characterized by depressed mood,anhedonia,disrupted sleep,loss of appetite,lack of energy,low self-worth,susceptibility to develop suicidal thoughts and cognitive dysfunction.Meta-analysis showed that the prevalence of depression among college students in our country is 31.38% in the last decade,and the prevalence of depression among the elderly over 60 years is 25.55%.Depression still has a potential increasing trend in different age groups in our country,which needs to be paid attention to.The World Health Organization reported that currently,there are more than 350 million people suffered from depression worldwide,indicating that depression is an important global public health problem.Although the existing treatments for depression have some effect on patients,the efficacy is unsatisfactory.Approximately 33% of patients’ symptoms of depression are significantly eased after the use of antidepressant drugs,while about 30% of patients gain little benefit from conventional pharmacological and psychological treatments.Although a large number of studies have elucidated the pathophysiology of depression,the etiology of depression is complex,a high degree of heterogeneity exists in clinical manifestations,and its underlying pathogenesis at a molecular level remains unclear.The pathogenesis of depression is influenced by factors such as family genetics,social environment,individual personality,and psychological activity,all of which not only lead to changes in the metabolism of the brain but also affect changes in the structure and function of several parts of the center,as well as the immune regulation of the entire organism.However,the conventional diagnostic classification of depression omits the association between immune dysregulation and depression.Thus,examining the link between the two paves the path for studying depression’s causes.Numerous studies have shown that acute and chronic stress can disrupt the immune system,resulting in an inflammatory response that promotes the onset and progression of depression.Recent research has demonstrated that certain inflammatory factors affect protein modifications during neuroinflammation,including a regulatory effect on the microtubule-associated protein tau(Tau).Combined with the previous findings of our project "The hyperphosphorylation of Tau leads to cognitive impairment in depression",it is clear that neuroinflammation plays an important role in the pathogenesis of neurodegenerative diseases and further research is worth carrying out.The literature suggests that in depression,increased inflammatory factors,decreased anti-infective cytokines,and decreased secretion of brain derived neurotrophic factors will decline their protection on neuronal cells and cause neuronal loss in the cerebral cortex and hippocampus,resulting in reduced hippocampal function and cognitive dysfunction.In previous studies,we found that the significantly elevated expression of DAPK1 in the prefrontal cortex and hippocampus of mice evokes the hyperphosphorylation of tau in these regions,causing cognitive dysfunction in depression.While the regulatory factors of how the expression of DAPK1 is elevated in depression are poorly studied and need to be further explored.Bioinformatics analysis using the Animal Transcription Factor Data Base(Animal TFDB)revealed that nuclear factor kappa-B(NF-κB)may be a potential upstream transcription factor of death-associated protein kinase 1(DAPK1).Meanwhile,a search in the JASPAR database(http://jaspar.genereg.net)revealed that there are nine potential binding sites for NF-κB within 2000 bp upstream of 5’ of the DAPK1 gene,with two scoring above 10,further demonstrating that NF-κB may be the upstream transcription factor that regulates the DAPK1 expression.The NF-κB family plays a key role in innate and specific immunity,and its activation is frequently associated with inflammation.NF-κB proteins are present in the cytoplasm together with IκB(inhibitor of NF-κB)which are activated in response to several inducing factors.Elevated activity of the IKK(IκB kinase)phosphorylates the IκB protein,which is subsequently ubiquitinated and then degraded by the proteasome.Degradation of IκB allows NF-κB to translocate to the nucleus to bind to homologous DNA sites,thereby regulating the transcription of related genes.Combined with the above bioinformatics analysis results,it was hypothesized that there were multiple NF-κB binding sites in the transcribed region of the DAPK1 gene,and NF-κB entry could induce the gene expression of DAPK1 and increase its protein synthesis.In this study,we investigated the relation between inflammatory factors,proteins related to NF-κB and the elevated expression of DAPK1 when depressed mice showed cognitive dysfunction through the Depression like mouse model(C57BL / 6J mice were single fed in a single cage,and chronic unpredictable mild stimulation was not regularly performed during the depression model mice preparation).Thus,it will provide new ways for the study of the mechanism of cognitive dysfunction in depression and provide a theoretical basis for promoting the discovery of molecular targets for depression treatment and improving the current treatment situation of depression.Methods1.Make the Depression like mouse model.One month old male C56BL/6J mice weighing in the range of 16-18 g were selected and divided into two groups: a normal control group and a depression model group.The former were 4 per cage and fed normally,while the latter were single fed in a single cage,treated by chronic unpredictable mild stimulation for 4 months.Finally,whether the mice developed the symptoms of depression was preliminarily tested by the Sucrose Preference Test.2.Inhibitors of DAPK1 and NF-κB were injected in the CA3 region of the hippocampus,respectively,followed by various behavioral tests.The model group mice were divided into 4 groups,and saline or inhibitor was injected into the CA3 subregion of the hippocampus of depressed mice by stereotactic injection in the brain.(1)Injection of the saline model group(Depression);(2)Injection of DAPK1 inhibitor TC-DAPK 6 group(DAPK1 inhibitor,DI);(3)Injection of NF-κB inhibitor SN50 group(SN50);(4)Injection of two inhibitors mixed solution TC-DAPK 6 + SN50 group(DI + SN50).Two weeks after the injection of the inhibitor,the animals in each group were subjected to behavioral assays: Detection of mice for pleasure deficit by sucrose prefer test;Detection of depressive state in mice by tail suspension test and forced swimming test;social interaction test to detect the social interaction ability of mice;detecting the spatial working memory ability and spatial learning memory ability of mice by Y-maze and the Morris water maze;The anxiety status of mice was detected by the elevated plus maze testand the open field test.3.The mice were anesthetized and killed after behavioral testing.Then the expression of DAPK1’s m RNA in each group was detected by qPCR.After the behavioral testing,fresh hippocampal tissues were obtained from three mice in each group and total RNA was extracted from tissues.RNA was reverse transcribed into c DNA by Reverse Transcription System.The qPCR kit was followed to detect the expression of DAPK1’s m RNA in hippocampus of mice.4.Detection of protein expression changes in each group by immunohistochemis--try,immunofluorescence,HE staining,and Western blot.Mice were anesthetized at the end of behavioral assays in each group and subjected to tissue perfusion for brain extraction or direct extraction of fresh hippocampal tissue for molecular biology experiments.The expression changes of hippocampal inflammatory factors,NF-κB-related proteins,DAPK1 proteins,and Taurelated proteins were detected by Western blot;the distribution and expression changes of inflammatory factors and NF-κB-related proteins in cells were detected by immunohistochemical staining;the expression localization and interrelationship of NF-κB and DAPK1 in hippocampus were observed by immunofluorescence.HE staining was used to detect the changes of neurons in each group.Results1.The mice in depression model showed depression in the Sucrose Preference Experiment.In the Sucrose Preference Experiment,compared with the normal control group,the sucrose preference index of the depression model group was significantly reduced(P < 0.001),indicating anhedonia and depression of the depression model group,which meant the successful modeling of Depression like mouse.2.The mice in each group were subjected to behavioral assays,whose results showed that the mice in the model group were significantly depressed,which showed cognitive and social impairment compared with the normal control group,and the symptoms in each group were alleviated after the injection of inhibitors.(1)Compared with the normal control group,the sucrose preference rate was reduced in the model group mice(P < 0.001),indicating that the model group mice were pleasure deficient and showed a depressive state.Mice in the DI group,SN50 group,and DI+SN50 inhibitor group had higher sucrose consumption rates compared to the Depression group(P < 0.001),indicating that the inhibitors could alleviate the depressive state of the mice.(2)The mice in the Depression group showed longer immobility in the tail suspension test and the forced swimming test compared with the normal control group(P < 0.001)and showed behavioral despair,indicating that the mice in the model group showed significant depression-like behavior.Mice in the inhibitor intervention group spent significantly less time immobile compared to mice in the Depression group(P < 0.001),indicating that the inhibitor reduced behavioral despair and improved depression-like behavior in depressed mice.(3)Mice in the Depression group had a reduced alternate correct rate in the Ymaze test compared with normal controls(P < 0.001),indicating that mice in this group had reduced spatial working memory capacity and had cognitive dysfunction.Depression-like mice treated with inhibitors had significantly higher alternate correct rates(P < 0.001)and spatial working memory capacity was improved,suggesting that inhibitor treatment attenuates cognitive dysfunction in depression-like mice.(4)When tested on day 5 of the MWM,it was found that mice in the Depression group had a significantly longer escape latency(P < 0.01),a significantly lower number of platform crossings(P < 0.001),and a significantly shorter stay in the target quadrant(P < 0.001)on day 6 compared to the normal control group,indicating that the mice in the model depressed group could not accurately remember where the hidden platform was located and had impaired cognitive learning.Spatial learning memory ability was impaired and cognitive impairment occurred.After inhibitor treatment,the Depression group’s mice showed the same trend and no significant difference in escape latency as the control group in the first 5 days,and inhibitor-treated mice traversed the platform significantly more often(P < 0.001)and stayed in the target quadrant longer(P < 0.001)on day 6 compared with the Depression group.It showed that the mice in the inhibitor group were able to accurately remember where the hidden platform was located,the reduced spatial learning memory ability was improved,and cognitive dysfunction was ameliorated.(5)Compared with the normal control group,mice in the Depression group showed significantly lower social interaction rates in the social interaction test(P < 0.001)and developed social impairment.Depression-like mice with inhibitor intervention had significantly higher social interaction rates(P < 0.001)and normalized social competence.The experimental findings suggest that the inhibitor can improve social impairment in depression-like mice.(6)There was no difference in the percentage of time spent in the open arm exploration activity and the percentage of time spent in the central area in both the EPM and OFT(P > 0.05),indicating that the mice in each group were not anxious.3.Detection of DAPK1 m RNA expression in the hippocampal region of each group of mice after inhibitor treatment showed that the expression was increased in the depression group and decreased in each inhibitor group.DAPK1 m RNA expression was increased in the depressed group compared with the normal control group(P < 0.001),and significantly lower in the DI,SN50,and DI + SN50 groups compared with the Depression group(P < 0.01;P < 0.001),indicating that NF-κB regulates DAPK1 expression at the gene level.4.After inhibitor treatment,the results of the meristematic experiment revealed that the expression of proteins related to DAPK1 and NF-κB was declined in the hippocampi of depression group;The expression of proteins related to DAPK1 was decreased but the expression of proteins related to NF-κB had no change in DI group;In SN50 group,the expression of proteins related to DAPK1 and NF-κB was decreased.(1)In contrast to normal control group,the expression of inflammatory factors,TNF-α and IL-1β,were increased in depression group;Compared to depression group,the expression of TNF-α and IL-1β in DI group had no significant change,while the expression of TNF-α and IL-1β in SN50 and DI + SN50 groups was decreased(P < 0.01,P < 0.001).All these demonstrated that DAPK1 doesn’t regulate the expression of TNF-α and IL-1β,whereas NF-κB can influence the expression of TNF-α and IL-1β.(2)In depression,the expression of TNF-α and IL-1β will be increased.And TNF-α and IL-1β are widely regarded as activators NF-κB pathway.Therefore,they can be used to detect the proteins of NF-κB.In contrast to normal control group,the expression of p-IKK,p-IκBα and p-p65 was increased(P < 0.001,P < 0.01,P < 0.05),which demonstrated that the activation of IKK,IκBα and p65 was elevated.Because the overexpression of inflammatory factors elevates the activity of IKK,IκBα and NF-κB p65 in the NF-κB pathway.The expression of proteins was still significantly increased in DI group when compared with the normal control group(P < 0.001,P < 0.01,P < 0.01),and the expression of p-IKK,p-IκBα and p-p65 in SN50 and DI + SN50 groups was decreased when compared with depression group(P < 0.001,P < 0.01,P < 0.05).All these proved that DAPK1 have no influence on NF-κB pathway,whereas the inhibitor of NF-κB can effectively inhibit the activation of the NF-κB pathway.(3)According to the literature,there are several binding sites of NF-κB in the transcription area of DAPK1,which can be used for the detecting the expression of DAPK1.Compared with the normal control group,the expression of DAPK1 was increased(P < 0.001)and the expression of p-DAPK1 was decreased(P < 0.001)in depression group,demonstrating the high activity of DAPK1 in depression.After interference of the inhibitor,the expression of DAPK1 was reduced(P < 0.001)and the expression of p-DAPK1 was increased(P < 0.001)in depression group,proving that both TC-DAPK 6 and SN50 can effectively inhibit the activity of DAPK1.Thus,the activity of DAPK1 is regulated by NF-κB.(4)The previous studies of our work found that the increased expression of DAPK1 in Depression like mouse would increase the level of tau phosphorylation.The detection of tau phosphorylation in this study found that the expression of phosphorylated Tau increased in Depression like mouse,and the expression could be reduced by inhibitors.This result confirmed that the degree of Tau hyperphosphorylation was controlled by DAPK1.(5)Immunohistochemical detection showed that compared with the normal control group,the expression of TNF-α,IL-1β and p-p65 increased and nuclear translocation of p-p65 appeared in depression and DI groups;the expression of the above molecules decreased in SN50 and DI + SN50 groups;the expression of DAPK1 increased in depression group.The expression of DAPK1 decreased in DI,SN50 and DI+SN50 groups compared with depression group.The results of immunohistochemical detection were consistent with the results of Western blot.(6)Immunofluorescence experiments indicated that compared with the normal control group,the expression of p-p65 and DAPK1 were increased in the CA3 area of hippocampus in depression group and the expression of DAPK1 was decreased in DI Group.The expression of p-p65 was not significantly changed and the expression of DAPK1 and p-p65 was both less in SN50 and DI + SN50 groups compared with depression group.The result showed that the decreased expression of DAPK1 have no effect on the expression of proteins of NF-κB,whereas the decreased activity of NF-κB p65 is followed by a decrease in the expression of DAPK1,indicating that DAPK1 is regulated by NF-κB pathway.(7)The results of HE staining showed that compared with the control group,the number of neurons in prefrontal cortex,CA3,CA1 and DG regions of depression-like mice decreased.The number of neurons in DI,SN50 and DI + SN50 groups was more than that in depression group.Conclusions1.Laboratory rearing of solitary mice plus chronic unpredictable mild stimulation is an effective way to prepare depression-like mouse models in which depressive symptoms are associated with elevated inflammatory factors in the brain.2.In depression,there is increased expression of inflammatory factors that activate the NF-κB pathway.NF-κB p65 is activated to p-p65 and transferred to the nucleus to initiate DAPK1 transcription,which increases DAPK1 protein expression and causes Tau hyperphosphorylation.This eventually leads to cognitive dysfunction in depression. |
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