| Depression as one of the most common psychiatric disorders is characterized by depressed mood,anhedonia and loss of interest,accompanied by anxiety,cognition deficit,sleep disorders,weight loss and other nonspecific somatic symptoms and brings heavy burden to the patients and society.Stress exposure can lead to serious illnesses such as depression and post-traumatic stress disorder.However,some individuals exposed to similar stressors maintain normal psychological and physical functioning and escape serious mental illness,who are considered to be resilient.The resilience is mediated not only by the absence of important molecular abnormalities that exist in susceptible individuals to impair their adaptability,but also by the presence of key molecular adaptations that occur specifically in resilient individuals to enhance their strain capacity.However,the neural substrates and molecular mechanisms that mediate susceptibility and resilience to stress remain unclear.It is necessary to study stress-susceptible and stress-resilient related proteins and biological processes by proteomic approach.The hippocampus is vulnerable to the psychological stressors,and aberrant structural and functional changes are found in the hippocampus of patients with depression and animal model of depression.As a part of the mesolimbic dopamine reward circuit,the nucleus accumbens and ventral tegmental area are demonstrated to be related to development of depression.Interestingly,the hippocampus has neuronal fibres directly projecting to this pathway and controls the activity and function of this pathway.Moreover,the hippocampus plays an important role in the formation of recognition and dampening hypothalamic-pituitary-adrenal(HPA)axis responses to stress.On the basis of the role of the hippocampus in depression,hippocampal proteomics study from stress-susceptible and stress-resilient rats is urgently needed.In this study,8-week-old male SD rats accepted 7 weeks’ chronic unpredicted mild stress(CUMS)exposure.Rats were exposed to a variable sequence of mild,unpredictable stressors consisting of 3 or 4 different stressors per day,including water or food deprivation(24 h),empty water bottles(2 h),cold room(4 ℃,2 h),hot room(45 ℃,15 min),cage tilt(16 h);continuous lighting(12 h),soiled cage(200 mL of water spilled onto the bedding,12 h),grouped housing in one cage(4-5 per cage,12 h),strobe lighting(200 flashes/min,4 h),intermittent white noise(85 dB,6 h).Before and after CUMS,all rats received SPT,FST and OFT to evaluate their state.The CUMS-exposed rats were divided into stressed-susceptible(CUMS-S)and stressed-resilient(CUMS-R)group.Then,the area of neuron and density of dendritic spine in the hippocampal CA1,CA3 and DG regions were detected by Nissl staining and Golgi staining respectively.The alterations of morphology of microglia were detected and analyzed by immunohistochemical staining.Levels of IL-1(3,TNF-a and MDA,and the activity of SOD in hippocampus were also detected.Further,the hippocampal proteins were analyzed and identified by Mass Spectrometry.The differential proteins were obtained and bioinformatics analysis for these proteins including gene ontology(GO)annotations and GO functional enrichment was carried out.The related proteins were verified by Western blotting,immunohistochemical staining and immunofluorescence staining.Here,we found that in CUMS-S rats the sucrose preferences was 45%,while the sucrose preferences of Ctrl and CUMS-R rats were more than 80%.In FST,the immobility time of CUMS-S rats was 220 s longer than those in Ctrl rats(78 s)and CUMS-R rats(82 s).After CUMS,numbers of zone crossing and rearing times of CUMS-S rats were significantly decreased,less than those in Ctrl and CUMS-R rats respectively.There was no difference in these tests between Ctrl and CUMS-R group.The area of neuron of the hippocampal CA1,CA3 and DG regions significantly decreased in CUMS-S group compared with Ctrl and CUMS-R group.By Golgi staining,we found that CUMS induced significant decreases in density of dendritic spine and density of mushroom-type spine of the hippocampal CA1 region in CUMS-S rats compared with Ctrl and CUMS-R rats.However,there was no difference in those between Ctrl and CUMS-R rats.The percentages of ramified,hypertrophied,amoeboid microglia were 16.4%,35.2%,48.4%in hippocampus of CUMS-S rats,those in Ctrl and CUMS-R rats were 68.7%,24.3%,7%and 45.2%,45.8%,9%.The percentage of amoeboid microglia in CUMS-S rats was more than those in Ctrl and CUMS-R rats,while hypertrophied microglia in CUMS-R rats was more than those in Ctrl and CUMS-S rats.Obvious increased levels of IL-1β and TNF-a were detected by ELISA in the hippocampus of CUMS-R rats compared with Ctrl and CUMS-R rats.Further,we observed that in hippocampus of CUMS-S rats the MDA(a lipid peroxidation maker)level was increased and the SOD(an antioxidant enzyme)activity was decreased compared with Ctrl rats,whereas in hippocampus of CUMS-R rats the SOD activity was increased compared with Ctrl and CUMS-S rats.From the proteomics study,4,690 proteins were identified and 3,645 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,164 differentially expressed proteins were obtained.In the comparison of CUMS-S with Ctrl,20 upregulated proteins and 12 downregulated proteins were obtained,and these proteins mainly participated in inflammation process,material and energy metabolism,response to stress and synapse plasticity.In the comparison of CUMS-R with Ctrl,21 upregulated proteins were upregulated and 43 proteins were downregulated,and these proteins mainly participated in inflammation process,signaling,material and energy metabolism,response to stress and synapse plasticity.In the comparison of CUMS-S with CUMS-R,12 upregulated proteins were upregulated and 46 proteins were downregulated,.and these proteins mainly participated in material transport,inflammation process,signaling,material and energy metabolism,response to stress,development process and synapse plasticity.By Western blotting,levels of mGluRl,mGluR2 and NR1 were significantly decreased in CUMS-S compared with Ctrl.CUMS-S rats had 37%increase of tau phosphorylation at Ser396,127%increase at Ser404 and 75%increase at Thr205 compared with Ctrl rats.Compared with Ctrl rats,the EAAT2 level was decreased 37%in CUMS-S rats and increased 33%in CUMS-R rats.Astroglial plasticity was disrupted in CUMS-S rats and enhanced in CUMS-R rats.The phosphorylation level of Nrf2 at Ser40 in CUMS-R rats was significantly higher than Ctrl rats,while that in CUMS-S rats was significantly lower than Ctrl rats.CUMS-S rats had 44%decrease of ERK at Thr202/204,71%decrease of AKT at Ser473 compared with Ctrl rats.CUMS-R rats had 390%decrease of ERK at Thr202/204,47%decrease of AKT at Ser473 compared with Ctrl rats.In conclusion,CUMS induced depressed rats show decreased area of neuron and density of dendritic spine in hippocampus,increased microglia-dependent proinflammatory response and oxidative stress response,disrupted astroglial plasticity,decreased EAAT2 level and disrupted antioxidant capability.CUMS-R rats show normal state,normal area of neuron and density of dendritic spine,enhanced astroglial plasticity,increased EAAT2 level and increased antioxidant capability.Proteomics study showed that there were many proteins involved in synapse plasticity,inflammation process,signaling,material and energy metabolism,response to stress,development process and material transport.Major depressive disorder(MDD)is a mood disorder with depressed mood,anhedonia and loss of interest as three core symptoms.While the underlying mechanisms are not completely understood,accumulating data have suggested that inflammation and oxidative stress play important roles in the pathophysiology of neurological and psychiatric diseases including major depression and bipolar disorder.Oxidative stress and inflammatory pathways are known to interact with each other at various levels to lead pathophysiological outcomes.This status quo emphasizes the need for antidepressant medications with inhibition of inflammation and oxidative stress.Emodin(1,3,8-trihydroxy-6-methylanthraquinone),a natural anthraquinone derivative found in the roots and rhizomes of various plants,has been reported to exhibit a number of biological activities such as anti-inflammation,antioxidantion,anti-cancer,immunosuppression,hepatoprotection,neuroprotection,and so on.Based on these previous findings,we hypothesize that emodin may have antidepressant-like effects.In this study,8-week-old male SD rats accepted 5 weeks9 chronic unpredicted mild stress(CIJMS)exposure.Rats were exposed to a variable sequence of mild,unpredictable stressors consisting of 3 or 4 different stressors per day.After CUMS for 5 weeks,all the rats accepted SPT to evaluate their status.We defined depressive tendency as a more than20% decrease in sucrose water intake.Under this evaluation criterion,the CUMS-exposed rats were divided into depressive tendency group(DeT,n = 30)and stxessed-resilient(R,n=34)group,and the DeT group would be used for the next experiment.The DeT and Ctrl rats daily received emodin(Emo)(80mg/kg)or the same volume of Tween-20(Veh)by intragastric administration for 2 weeks(Ctrl + Veh = 15,Ctrl + Emo=15,DeT + Veh = 15,DeT + Emo = 15).Next,all rats in DeT groups continued to be exposed to CUMS for 2weeks.Finally,all the 60 rats accepted SPT,FST,OFT to detect their status.Then,the area of neuron and density of dendritic spine in the hippocampal CA1,CA3 and DG regions were detected by Nissl staining and Golgi staining respectively.The alterations of morphology of microglia were detected and analyzed by immunohistochemical staining.Levels of IL-1β,TNF-α and MDA, and the activity of SOD in hippocampus were also detected.The levels of proteins in hippocampus were detected and analyzed by Western blotting,Immunohistochemical staining and immunofluorescence staining were used to observe the levels and distributions of proteins.Using quantitative real-time PCR,miRNAs were detected.Here,the vehicle-treated DeT(DeT + Veh)group were shown lower sucrose preference(<50%)compared with the Ctrl(Ctrl + Veh)group at 7 weeks.However,emodin treatment improved sucrose preference of emodin-treated DeT(DeT + Emo)group compared with DeT + Veh group.In FST,the immobility time of DeT + Veh group(210 s)was significantly longer than those in Ctrl + Veh group(79 s),but this increase was reversed in the DeT + Emo group(78 s).The number of zone crossing and rearing times were significantly decreased in DeT + Veh group compared with Ctrl + Veh group.However,emodin treatment prevented the decreases in DeT + Emo group.The area of neurons of the hippocampal CA1, CA3 and DG regions significantly decreased in DeT + Veh group compared with Ctrl + Yeh group,while emodin rescued the decrease in DeT + Emo group.By Golgi staining,we found that CUMS induced significant decreases in spine generation(density of dendritic spine)and maturation(density of mushroom-type spine)of the hippocampal CA1 region in DeT + Veh group compared with Ctrl + Veh group,whereas the effects were partly abolished by emodin.We found that more microglia with higher solidity(0.25-0.31,>0.31)value was in DeT+ Veh group than Ctrl + Veh group,whereas treatment of emodin abolished the effect.Obvious increased levels of IL-1(3 and TNF-a were detected by ELISA in the hippocampus of DeT + Veh group,while the increases were abolished by emodin.We also observed that in hippocampus of DeT + Veh group the MDA(a lipid peroxidation maker)level was increased,whereas the SOD(an antioxidant enzyme)activity was decreased,suggesting an imbalance between oxidation and antioxidation.The treatment of emodin attenuated the abnormity.The total level of Nrf2 was not changed,but its phosphorylation level at Ser40 was significantly decreased in the hippocampal extracts of DeT rats without emodin treatment.When emodin was administrated for the last 2 weeks,the phosphorylation level of Nrf2 at Ser40(p-Nrf2)was significantly higher than DeT + Veh and Ctrl groups.Then,decreased phosphorylation level of Nrf2 at Ser40 in the nuclear fraction of DeT + Veh group was detected,whereas emodin could increase its level in DeT + Emo group.Immunohistochemistry study also showed the decreased phosphorylation level of Nrf2 at Ser40 in hippocampus(CA1,CA3, and DG)of the DeT + Veh rats,and the more enhanced staining of phosphorylated Nrf2 at Ser40 in DeT + Emo rats than DeT + Veh and Ctrl rats.We further found that phosphorylated Nrf2 at Ser40 were mainly expressed on neurons and emodin could avoid reduced co-localization percentage of p-Nrf2 and neuron in the hippocampal CA1,CA3 and DG by double-label immunofluorescence staining.By Western blotting,we found the total levels of hippocampal GSK-3P were equal in all groups,while its phosphorylation level at Ser9 was significantly decreased in DeT + Veh group,indicating increased activity of GSK3 p.When emodin was administrated for the last2 weeks,the phosphorylation of GSK3 P at Ser9 was restored to a normal level.Then,we observed the decreased phosphorylation level of GSK3 P at Ser9 in both cytoplasmic and nuclear fractions of DeT + Veh group compared with Ctrl + Veh group.Emodin could increase its phosphorylation level compared with DeT + Veh group.Further,the decreased phosphorylation level of AKT at Ser473 and in DeT + Veh rats and increased PI3 K regulatory subunit p85 p level were observed in DeT + Veh group,while emodin could rescue their levels in in DeT + Emo group.The elevated level of 5-LO was found both in DeT + Veh and DeT + Emo groups compared their respective control groups,while a significant increase in leukotriene B4 level was only found in DeT + Veh.In addition,the 5-LO level was lower in DeT + Emo than in DeT + Veh.By immunohistochemistry,the 5-LO mainly stained in the cytoplasm of hippocampus in Ctrl group.In DeT + Veh group,the staining of 5-LO significantly increased,especially in the nucleus of hippocampal CA1, CA3 and DG regions,while emodin treatment completely removed the increase of 5-LO in the nucleus and did not have a significant effect on the cytoplasmic 5-LO level compared with DeT + Veh group.Then we detected the higher level of 5-LO in nuclear fractions of DeT + Veh hippocampus than that of Ctrl + Veh hippocampus,whereas emodin treatment decreased 5-LO level in nucleus to a normal level and increased 5-LO level in cytoplasm compared with and Ctrl +Emo and DeT + Veh group.We further found that in DeT + Veh group 5-LO were more distributed in nucleus of neurons and in DeT + Emo group 5-LO were mainly distributed in cytoplasm of neurons in the hippocampal CA1 by double-label immunofluorescence staining.DeT + Veh rats displayed significantly decreased levels of miR495,miR139-5p and miR126-3p,except miR135-5p compared with Ctrl + Veh rats.Emodin treatment increase the decreased levels of miR495,miR139-5p and miR126-3p in the hippocampus of DeT +Veh rats compared with those treated with the vehicle.However,emodin treatment had no significant effect on these miRNAs in Ctrl rats.In conclusion,emodin could inhibit up-regulated activity of GSK3(3 via PI3K/AKT signaling pathway in the hippocampus of depressive tendency rats,which promotes up-regulated activity of Nrf2 leading to antioxidation.Emodin treatment may also inhibit inflammation through regulating 5-LO activity via miR495 and miR139-5p.Thus,emodin rescues the decreasing the area of neuron and the number of dendritic spine in hippocampus,and eventually improves the depression-like behaviors induced by-CUMS through inhibiting oxidative stress and inflammation. |
PDF Full Text Request