| Background:Depression is caused by the combined effects of social,physical and psychological factors,where stress is an important cause.According to recent data released by the World Health Organization,there are currently about 300 million people suffering from depression and 50 million dementia patients worldwide[1].Due to the lack of effective treatment,depression is expected to become the world's most burdensome disease by 2030.Therefore,exploring the pathogenesis of depression and effective interventions for depression have important medical and social implications.Magnetic resonance imaging(MRI)revealed a reduction in the volume of some brain regions,such as frontal lobe,hippocampus,temporal lobe,thalamus,striatumand amygdala,of depressed patients.Autopsy results of depressed patients also showed significant atrophy in the hippocampus,amygdala,prefrontal cortex and striatum.The hippocampus is the limbic system structure of the medial temporal lobe of the brain,which plays an important role in spatial learning and memory,navigation,and also participates in emotional regulation.The role of hippocampus in the pathogenesis of depression is receiving increasing attention.Continuous spatial positioning training increases hippocampal volume,the number of hippocampal neurogenesis,synapses and dendritic spines.After giving cognitive tasks related to cognitive function in depressed patients,the clinical symptoms of the patients are partially improved.Therefore,research on improving depressive behavior by improving hippocampal function is of great significance.It is also important to identify key molecules to elucidate the pathogenesis of depression and to develop effective treatment strategies.MicroRNAs(miRNAs)are a subset of non-coding RNAs of about 22-24 nucleotides(nt)in length that function primarily by affecting the expression of target messenger RNAs(mRNAs).Studies have shown that miR-191 is involved in neuronal development,neuronal activity,learning or memory processes,neuronal apoptosis,and activation of protein kinase A activity.However,the role of miR-191 in the development of depression is unclear.Aims:To explore whether spatial learning can improve depressive behaviors in depressed rats;to analyze the hippocampal proteomic basis of spatial learning to improve depression in rats;to elucidate the role and mechanism of miR-191 in spatial learning to improve depression in rats.Methods:Chronic unpredictable stress(CUS)was used to replicate rat depression models;open field test(OFT),forced swim test(FST),sucrose preference test(SPT)to assess rat behavior.Depression assessment criteria:Rats with a 30%reduction in sucrose water intake in SPT and a 50%increase in immobility time in FST were depressed rats.1)120 2-month-old male Sprague-Dawley rats(200 gą20 g)were randomly divided into control group(n=25,CON)and CUS group(n=95).After 21 days,38 rats in the CUS group exhibited depressive behavior.Fifteen depressed rats were randomly selected as post-depression learning group(D-L)and given a 7-day Morris water maze training.At the same time,15 depressed rats were selected as the post-depression swimming group(D-S)for swimming training(the daily swimming time was the same as the average delay time of the D-L group).Behavioral testing and other analyses were performed after 7 days.2)miR-191 study:84 SD rats were randomly divided into CUS group(n=60)and unstressed control group(n=24).After one week of stress,26 rats with depression tendency were screened by SPT(Rats with<65%sucrose water intake for 2consecutive days are defined as pre-depression).Pre-depression rats were randomly divided into two groups:one group of rats was injected with Agomir-miR191(n=15,CUS+Agomir-miR191)in bilateral hippocampal CA3,and one group of rats was injected with PBS in bilateral hippocampal CA3(n=11,CUS+PBS).Stress continued for 2 weeks after injection.Unstressed rats were randomly divided into two groups:one group of rats was injected with Antagomir-miR191(n=12,Antagomir-miR191)in the bilateral hippocampal CA3,and one group of rats was injected with PBS into the bilateral hippocampal CA3(n=12,PBS).After two weeks,the above four groups of rats were subjected to behavioral testing and other analysis.Other tests:Proteomics based on isobaric tags for relative and absolute quantification(iTRAQ)were used to detect differentially expressed proteins in hippocampus of each group.Other tests such as Western blotting(WB),immunofluorescence,immunohistochemistry,and quantitative PCR(Q-PCR)have also been employed.Results:After 21 days of CUS,rats'spontaneous activity in OFT was significantly reduced.In FST,the immobility time was prolonged from 65 s to 114 s.The sucrose water intake in the SPT was reduced from 84%to 52%,indicating that CUS can induce depressive behaviors in rats.After 7 days learning,the spontaneous activity of the D-L rats in the OFT was significantly enhanced,the immobility time was reduced to 43 s in the FST,and the sucrose water intake was 72%in the SPT;while in the group of D-S rats,the spontaneous activity was still weak in the OFT,the immobility time was 92 s in the FST and sucrose water intake was 45%in the SPT.These results show that spatial learning can significantly improve CUS-induced depressive behaviors in rats.Using proteomic analysis,4,576 proteins were identified from the rat hippocampus,and 3482 proteins were quantified.When setting quantification ratio of>1.2 coupled with p<0.05 as upregulated threshold and<0.83 coupled with p<0.05 as downregulated threshold.In the comparison of D-S with CON,11 upregulated proteins and 11 downregulated proteins were obtained.In the comparison of D-L with CON,42 upregulated proteins and 11 downregulated proteins were obtained.In the comparison of D-L with D-S,26 upregulated proteins and 8 downregulated proteins were obtained.WB results showed that SNAP25 and PSD95 in the D-L were significantly higher than those in the D-S(32%and 53%)and reached the level of the normal control;the levels of NR1,GluR1,and GluR2 in the D-L were higher than those in the D-S(33%,55%and 57%).The level of NR2B in the D-L increased by 45%compared with the control,increased by 45%compared with the D-S PLP1 in D-L increased by 48%compared to the control;MAG increased by 22%and PLP1 increased by 85%compared to D-S.The level of PKA?in the D-L was significantly decreased,44%lower than that in the control,and 41%lower than in the D-S.The level of GSK-3?in the hippocampus of the D-S and the D-L was higher than that of the control,respectively.The level of PP2Ac in the D-S was 42%higher than those in the control;PP2A-Y307 in the D-L was decreased by 34%and 33%compared with the control or the D-S,respectively;the PPP1A level in the D-L was decreased by 48%and 42%,respectively,compared with the control and the D-S.Nissl staining showed that the number of neurons in the hippocampus CA1,CA3 and DG areas of the CUS rats decreased by 20%,29%and 22%,respectively,compared with the CON rats.WB results showed that compared with CON,death-associated protein kinase 1(DAPK1)increased by 48%,Caspase 3 increased by 32%,Cleaved caspase 3 increased by 50%,Beclin1 increased by 33%,and LC3 II/I ratio increased by 53%in the hippocampus of depressed rats,suggesting increased apoptosis and autophagy.After GC(10~-44 M)treatment,compared with control neurons,DAPK1increased by 62%,Caspase 3 increased by 83%,Cleaved caspase 3 increased by 53%,Beclin1 increased by 36%,and LC3 II/I ratio increased by 35%.Q-PCR showed that miR-191 levels in the D-S were lower than CON rats(45%),while the level of miR-191 in the hippocampus of the D-L was increased by 131%compared with the D-S,which was not different from the CON rats.After transfection miR-191 mimic in N2a,DAPK1 increased by 52%,Caspase 3 increased by 44%,Cleaved caspase 3 increased by 31%,Beclin1 increasedby 45%,and LC3 II/I ratio increased by 78%.Dual luciferase reporter assays verified that DAPK1 is a miR-191target gene.Compared with PBS rats,Antagomir-miR191 rats had 44%,55%,and 46%reductions in the number of zone crossing,the number of rearing and the total distances travelled in the OFT;the immobility time in FST was 113 s;sucrose water intake in SPT decreased by 29%.Compared with CUS+PBS rats,CUS+Agomir-miR191 rats increased the number of zone crossing and the total distances travelled by 94%and96%,respectively,in the OFT;the immobility time decreased from 125 s to 70 s;the sucrose water intake in SPT increased by 55%.Compared with PBS rats,the number of neurons in hippocampal CA1,CA3 and DG of Antagomir-miR191 rats decreased by 21%,25%and 34%,DAPK1 increased by40%,Caspase 3 increased by 54%,and Cleaved caspase 3 increased by 68%.Beclin1increased by 47%and the LC3 II/I ratio increased by 27%.Compared with CUS+PBS rats,the number of neurons in hippocampal CA1,CA3 and DG of CUS+Agomir-miR191 rats increased by 35%,35%and 38%,respectively;DAPK1decreased by 24%and Caspase 3 decreased by 32%.Cleaved caspase 3 decreased by31%,Beclin1 decreased by 26%,LC3 II/I ratio decreased by 17%.Conclusion:Spatial learning improves CUS-induced depressive behavious by up-regulating miR-191 levels in the hippocampus of depressed rats,targeting down-regulation of DAPK1 levels,and reducing hippocampal neuron loss. |
PDF Full Text Request