| Background: Estrogen is an important steroid hormone in the body,mainly produced by the ovary,and is closely linked with brain function.A large number of studies have shown that estrogen has a variety of neuroprotective effects,such as reducing nerve cell apoptosis by by removing of oxygen free radicals,and by antagonizing oxidative stress and protecting mitochondria from damage and reducing excitatory amino acid toxicity,and and promoting nerve cell axons,dendritic growth,synaptic formation,and estrogen deficiency can lead to brain damage.Menopause is a process with low estrogen in women,who are more prone to cognitive impairment and suffer from neurodegenerative diseases such as vascular dementia(VD)and Alzheimer’s disease(AD),which are the main clinical manifestations of cognitive impairment.The risk of cognitive impairment or dementia and Parkinson’s disease(PD)was significantly increased in young women with bilateral ovariectomy.Rodent ovariectomy(OVX)is a widely used for a modeling method to study human menopause and ovarian function loss.OVX animals were characterized by estrogen deficiency and showed significant cognitive decline.Whether or not the effects of neurons in different brain regions during menopause are consistent and what characteristics they have have not been systematically studied.Meanwhile,how to improve the cognitive impairment caused by menopausal brain injury is a medical problem.Due to the target organ of estrogen is is extensive and estrogen mechanism is still not very clear,so that estrogen and estrogen replacement therapy in clinical application is limited.Chinese traditional medicine provides a research idea to solve this problem.Rhubarb,is the traditional Chinese medicine by the“Dachengqi Tang”,“Xiaochengqi Tang”,“Tiaowei Chengqi Tang” and other monarchs,those by “promoting bowel evacuating” and significantly improves the clinical cognitive and mental disorders.Emodin is the main monomer of rhubarb,to study whether it has the role of improving the cognitive impairment of OVX animals,and the corresponding role of the link,which is of great value to promote the application of related agents.Obejective: 1.To analyze depression and dementia related proteomic basis of different brain regions in young OVX rats.2.To study the effects of emodin on brain function(depression,learning and memory,feeding and other behaviors)of young OVX rats,and to probe the mechanism from the perspective of “brain-intestinal axis”.Methods: 1.Model establishment and treatment 12-week-old SD female young rats(weight 230-280 g)were divided into 2 groups: sham operation group(SHAM,n = 18)and OVX group(OVX,n = 18).In order to observe the effect of emodin in the treatment of young ovariectomized rats,12-week-old female SD rats were divided into 4 groups: SHAM group(n = 16),OVX group(n = 16),OVX +emodin group(Emodin group,n = 16)and OVX + 17β-estradiol group(Estradiol group,n = 16).2.Behavior detection Spontaneous activity was detected by open field test(OFT)weekly from the 2nd week after operation.At the 7th week after operation,sucrose preference test(SPT),forced swimming test(FST)and OFT was detected to evaluate the mental state of rats.The cognitive function of rats was detected by Mirror water maze test(MWMT)in the 8th week after operation.3.Proteomics analysis after MWMT,the tissue of prefrontal cortex(PFC),hippocampus(HIP),hypothalamus(HYP)and amygdala(AMY)of rats was collected from SHAM and OVX groups by means of an i TRAQ labeling,and the resulting peptides were analyzed by Q ExactiveTM Plus hybrid quadrupole-Orbitrap mass spectrometer(Thermo Fisher Scientific).The peptides were subjected to NSI source followed by tandem mass spectrometry(MS/MS)in Q ExactiveTM Plus(Thermo)coupled online to the UPLC.The resulting MS/MS data were processed using Max Quant with integrated Andromeda search engine(v.1.4.1.2).Tandem mass spectra were searched against Uniprot_rat database(32,983 sequences).Gene Ontology(GO)annotation proteome was derived from the Uni Prot-GOA database(http://www.ebi.ac.uk/GOA/).4.Intestinal tract contraction and diastolic function test The intestinal contractility of duodenum,ileum and colonic isolated from SD rats and OVX rats was measured by Powerlab physiological data acquisition and analysis system,and the effect of 17β-estradiol or emodin on intestinal contraction strength was also detected.5.ELISA and electrochemical ELISA The plasma level of 17β-estradiol was measured by ELISA.The plasma levels of actitve Glucagon-like peptide 1 [GLP-1(7-36)amide and GLP-1(7-37)] were determined by ultrasensitive electrochemiluminescence analysis.6.Micro-CT scan The abdominal fat of rats was detected by small animal in vivo micro-CT imaging technique.7.Nissl staining and Golgi staining 8 weeks after operation,the numbers of neurons in PFC,HIP,HYP and AMY of rats were detected by Nissl staining,and neuronal dendritic spine densities and mushroom-shaped dendritic spine ratio were detected by Golgi staining.8.Western blotting and immunohistochemistry(IHC)Western blotting was used to detect and analyse the levels of estrogen receptor α(ERα),ERβ,autophagy and apoptosisrelated proteins,Endoplasmic reticulum(ER)stress-related proteins,synapse-associated proteins,cytoskeletal proteins,GLP-1 receptor(GLP-1R),Stromolecule Interaction Molecule(STIM2),Transient receptor potential canonical 1(TRPC1),calcium/calmodulin dependent protein kinase Ⅱα(Ca MKⅡα)and its autophosphorylation and so on.IHC was used to detect and analyse the levels of microglia,astrocytes,ERα,GLP-1R,and Proopiomelanocortin(POMC)in rat brain.Results: 1.OVX rats showed depressive behavior and cognitive impairments In this research,OFT,SPT,and FST were used to detect the behavioral alterations of rats after the operation.Compared with SHAM rats,OVX rats showed decreased rearing number,moving duration,total distance and the number of zone crossing from the 4th week after operation in OFT.In SPT performed at the 7th week after the operation,the percentage of sugar consumption of OVX rats was significantly lower than that in SHAM rats.In FST,the immobility time of OVX rats within 5 mins was significantly longer than that of SHAM rats.These data supported that OVX rats showed depressive behaviors.OVX rats were tested their cognition in MWMT in the 8th week after the operation.In the learning phase,OVX rats took longer latencies to find the platform than SHAM rats.In the memory test,the latency of the first platform acrossing of OVX rats was significantly longer than SHAM rats,and mean annulus crossings within 60 s of OVX rats was significantly reduced.Those data suggested that OVX rats showed cognitive impairment.2.Neuron loss induced by ovariectomy in PFC,HIP,HYP and AMY When setting i TRAQ ratio of >1.3 coupled with p < 0.05 as up-regulated threshold and < 0.77 coupled with p < 0.05 as down-regulated threshold,146 differentially expressed proteins in OVX rats were obtained.We identified significant differences in 20 proteins of PFC(5 increased and 15 decreased),41 proteins of HIP(30 increased and 11 decreased),17 proteins of HYP(11 increased and 6 decreased)and 79 proteins of AMY(43 increased and 36 decreased)in OVX rats compared with sham-operated rats.In PFC of OVX rats,the down-regulated proteins were enriched in the processes related to signaling transduction and synaptic plasticity,but the up-regulated proteins were mainly enriched in axon ensheathment.In the HIP,the down-regulated proteins were enriched in calcium-mediated signaling,synapse transmission,and nervous development.The upregulated proteins were mainly enriched in response to oxidative stress,ER-associated ubiquitin-dependent protein catabolic process,autophagy,cell death and apoptosis.In HYP,the down-regulated proteins were markedly enriched in calcium-mediated signaling,nervous development,and Ras/ERK1/ERK2 and MAPK cascade.In AMY,the down-regulated proteins were markedly enriched in synapse transmission and neurotransmitter transport,nervous development and signal transduction,while the up-regulated proteins were mainly in axon ensheathment,response to oxidative stress,autophagy,cell death and apoptosis.The differentially expressed proteins were enriched in specific cells,namely neuron,oligodendrocyte,astrocyte and microglia.Most of the differentially expressed proteins in this study were from neuron and microglia.Compared with SHAM rats,the microglia(recognized by Iba1)in HIP and AMY of OVX rats were significantly increased by 29.49% and 23.15%,respectively,and the astrocytes(recognized by GFAP)in PFC,HYP and AMY were significantly increased by 12.35%,19.79% and 21.82%,respectively.Neuron loss was presented in all these regions of OVX rats,which might be a result of apoptosis(probed by increased AK2,Bax,cleaved caspase-3 and caspase-3,phosphorylated p53,and decreased HYPK,HK and phosphorylated Bcl-2).ER stress activation(probed by increased GRP78,cleaved caspase-12,and phosphorylated PERK,IRE-1 and ATF6)and mitochondria dysfunction(probed by increased cytochrome c and decreased SFXN1 and NDFA11)were observed in the HIP and AMY.Dysregulation of autophagy in brains of OVX rats was indicated by the alterations of some autophagy-related markers /regulators,including increased ATG7 in PFC and HIP,GABARAPL2 in PFC and AMY,ORP1 in the HIP and decreased levels of SCAMP5 in the HIP,HSPA1 A in HYP and AMY,and HYPK in PFC and AMY.In HIP and AMY,levels of LC3II/I,ATG5,and Beclin1 were increased obviously.3.Synapse impairments in OVX rats Synaptic impairments,especially glutamic synaptic dysfunction,were detected in the brain of OVX rats.Compared with SHAM rats,in HIP of OVX rats,Arf-GAP with SH3 domain,ANK repeat and PH domain-containing protein 1(ASAP1)and Septin-8(SEPT8)were up-regulated,brain acid-soluble protein 1(BASP1)was down-regulated.Calcium-dependent secretion activator 1(CAPS1)was down-regulated in PFC,HIP and AMYof OVX rats.The levels of PSD95(a postsynaptic marker),synapsin1(a presynaptic marker)were decreased in HIP and AMYof OVX rats compared to SHAM rats,and synaptotagmin(a presynaptic calcium sensor in neurotransmitter release)was reduced in AMY.Dysregulation of Ca2+ related signals,e.g.Ras/Raf1/Mitogen-activated protein kinase kinase(MEK)/ Extracellular signal-regulated protein kinase(ERK)(Ras/Raf1/MEK/ERK)and phosphoinositide-3-kinase/AKT(PI3K/AKT)pathway were potentially involved in.4.Impairments of cytoskeleton in OVX rats In the brain of OVX rats cytoskeletal abnormalities were observed.Microtubule-associated protein(MAP)was decreased in the HIP and increased in HYP and Tubulin α-1A chain(Tuba1a)increased in the HIP and decreased in HYP and AMY.Growth factor receptor-bound protein 2(Grb2),an important link between cellular signaling and the neuronal cytoskeleton,was down-regulated in HIP of OVX rats compared to SHAM rats,and HSPA1 A was decreased in HYP and AMY of OVX rats.Especially tau hyperphosphorylation with glycogen synthase kinase 3β(GSK3β)activation,were observed in OVX rats.5.Specific differential proteins in amygdala of OVX rats In AMY,which is important in emotional behaviors,an unbalance between excitatory and inhibitory neurotransmission of OVX rats was indicated by decreased the levels of GABAR-B2,Vglu T1(vesicular glutamate transporter 1),the phosphorylation of NR2 B at Y1472,Fyn(an important kinase enhancing the activity of N2B-containing NMDARs),and SHANK3(SH3,and multiple ankyrin repeat domains 3,a postsynaptic scaffolding protein enriched in the density of excitatory synapses),and increased the level of GAT3.The adjustment of GABAergic transmission in AMY after OVX was suggested by increased calretinin and decreased calbindin.6.Emodin improved depressive behavior and cognitive impairment in OVX rats 7 weeks after operation,the percentage of sugar consumption in OVX rats was 60.62%,which was significantly lower than that in SHAM group(77.25%),and that in Emodin group(75.28%)and Estradiol group(72.46%)were significantly higher than that in the OVX group.In OFT,total distance of OVX group was 2087.30 cm,which was significantly lower than that of SHAM group(3027.60 cm),and that in Emodin group(2637.00 cm)and Estradiol group(2912.50 cm)were significantly higher than that in OVX group.The number of zone crossing within 5 mins,OVX group was 56.94,which was significantly lower than SHAM group(82.19),and that in Emodin group(76.44)and Estradiol(91.75)were significantly higher than that in OVX group.The data of FST showed that immobility time within 5 mins of OVX rats(158.25 s)was obviously longer than that of SHAM rats(86.49 s),and that of Emodin rats(55.28 s)and Estradiol rats(69.44s)were remarkably shorter than that of OVX rats.Those results showed that emodin or estrodial improved the depressive behavior of OVX rats.In the MWMT,in the learning phase,OVX rats took longer latencies to find the platform than SHAM rats,and Emodin rats and the Estradiol rats were significantly shorter than OVX rats.In the memory test,the latency of the first platform acrossing of OVX rats was significantly longer than SHAM rats,and mean annulus crossings within 60 s of OVX rats was significantly reduced.The latency of Emodin rats and the Estradiol rats was significantly shorter than that of OVX rats,and mean annulus crossings of Emodin rats and the Estradiol rats was significantly higher than that of OVX rats.Those results suggest that emodin or estradiol significantly improved cognitive impairment of OVX rats.Nissl staining showed that,compared with SHAM group,the number of neurons in CA1 and CA3 area of HIP,HYP and AMY and PFC of OVX group significantly reduced by 10.23%,12.03%,9.49%,5.53%,and 8.70%,and compared with OVX group,that of Emodin group significantly increased by 14.13%,26.17%,9.53%,10.64%,and 10.26%,and that of Estradiol group significantly increased by 17.12%,17.58%,12.84%,10.94%,and 9.42% respectively.Those results showed that emodin or estradiol improved the numbers of neurons decreasing in the brain of OVX rats.The data of Golgi staining showed that,compared with SHAM rats,the densities of dendritic spines of neurons in the PFC,HIP,HYP and AMY of OVX group significantly reduced by 25.76%,53.06%,45.10%,and 31.75% respectively,and compared with OVX group,that of Emodin group significantly increased by 40.82%,91.31%,75.00%,and 55.81% respectively,and that of Estradiol group significantly increased by 53.06%,108.70%,89.28%,and 46.51% respectively.Compared with SHAM rats,the proportion of mushroom dendritic spines of neurons in the PFC,HIP,HYP and AMY of OVX rats significantly decreased by 37%,56%,42%,and 43%,and compared with OVX rats,that of Emodin rats significantly increased by 61%,104%,80%,and 78% respectively,and that of Estradiol rats significantly increased by 65%,106%,63% and 68% respectively.Those results suggested that emodin or estrodial significantly increased the densities of dendritic spines and the numbers of mushroom dendritic spines in OVX rats.In order to further study the mechanism of emodin or estrogen in improving depressive behavior and cognitive abilities and dendritic spine loss of OVX rats,the data of western blotting showed that emodin or estrogen significantly upregulated the levels of synapsinrelated proteins(synapsin1,PSD95,p-NR2 B and p-Glu R1)in HIP of OVX rats.Meanwhile,emodin or estrogen significantly upregulated the levels of Ca2+-mediated signaling pathway related proteins(STIM2,p-Ca MKIIα and TRPC1).In addition,it was also observed that the inhibition of ERK pathway in OVX rats was improved by emodin or estrogen.7.Emodin reduced body weight of OVX rats The food intake in the 2th week after operation and cumulative food intake in 2 weeks of OVX group,Emodin group and Estradiol group were remarkably higher than the SHAM group respectively.Then from the 3rd to the 8th week after operation,OVX rats were treated with emodin and estradiol.Food intake in the 8th week and total cumulative food intake in 8 weeks of OVX rats were conspicuously more than that of SHAM rats,and that of Emodin rats and Estradiol rats were remarkably lower than OVX rats respectively.8 weeks after operation,the body weights of Emodin group were remarkably lower than that of OVX group.The abdominal circumferences of OVX rats were obviously more bulky than that of SHAM rats,and that of Emodin rats and Estradiol rats were remarkably lower than OVX group.The data of Micro-CT showed that the ratio object volume to total volume of abdominal fat of SHAM group,OVX group,Emodin group and Estradiol group,was 15.77%,30.50%,14.97%,and 16.19% respectively.That of OVX group was remarkably higher than that of SHAM group,and that of Emodin group or Estradiol group were significantly lower than that of OVX group.Those results suggested that emodin or estradiol obviously reduced abdominal adipose tissue of OVX rat.Compared with SHAM rats,the levels of POMC(recognized by ACTH)in HYP of OVX rats significantly reduced by 49%,and compared with OVX group,and that of Emodin group and Estradiol group significantly increased by 82% and 79% respectively.Those results showed that the levels of POMC in OVX group decreased,and the feed intake of OVX rats increased.Estradiol or emodin improved the levels of POMC and inhibited the appetite.8.Effects of emodin on the levels of estradiol,GLP-1 and their receptors in OVX rats 8 weeks after operation,compared with SHAM group and Estradiol group,the levels of ERα in the PFC,HIP,HYP and AMY of OVX group and Emodin group were reduced significantly.The plasma levels of 17β-estradiol of OVX group and Emodin group was significantly lower than that of SHAM group and Estradiol group.Those results showed that estrodial significantly improved the levels of ERα in the brain and the plasma levels of 17β-estradiol of OVX rats,but emodin did not.The plasma levels of active GLP-1 in OVX rats were obviously lower than that in SHAM rats,and compared with OVX rats,and that in Emodin rats and Estradiol rats were conspicuously increased.The result suggested that emodin or estrogen could reverse the plasma levels of activity GLP-1 in OVX rats.The data of IHC showed that,compared with SHAM group,the elevels of GLP-1R in CA3 region of OVX group obviously reduced by 23%,and compared with OVX group,that of Emodin group and Estradiol group obviously increased by 43% and 48% respectively.The data of western blotting and ICH showed that the levels of GLP-1R in PFC,HYP and AMY of OVX group was significantly lower than that of SHAM group,and compared with OVX group,that of Emodin group and Estradiol group significantly increased.Those results showed that the levels of GLP-1R in the brain of OVX rats were down regulated,and emodin or estradiol reversed the levels of GLP-1R.9.Effects of emodin on intestinal function in OVX rats The intestinal contraction and diastolic function were measured in vitro perfusion,and 5μΜ,and 10μΜ 17β-estradiol was given in Kreb’s bathing solution.It was observed that estradiol decreased the intestinal systolic amplitude of normal control(NC)rats and showed dose-dependent,while inhibition was gradually increased from the proximal end to the distal end.Importantly,the sensitivity of the intestine of OVX rats to estradiol was decreased.1μΜ,5μΜ,10μΜ,20μΜ,and 50μΜ emodin were given in Kreb’s bathing solution.It was observed that emodin decreased the intestinal systolic amplitude of NC rats and showed dose-dependent,while inhibition was gradually increased from the proximal end to the distal end.Compared with NC rats,inhibition of emodin on the ileum and colon of OVX rats significantly enhanced.The data of western bloting showed that,compared with SHAM group,the levels of ERβ in ileum and colon of OVX group obviously decreased by 41.97% and 51.71%,and the level of TRPC1 obviously decreased by 54.38% and 74.72% in OVX group.Conclusion: In this research,we reproduced a surgical menopause rat model with estrogen deprivation by bilaterally OVX,which showed depressive and dementing behaviors.The numbers of microglia in HIP and AMY and the astrocytes in PFC,HYP and AMY were significantly increased after OVX,while neuron loss presented in PFC,HIP,HYP and AMY.By mass spectrometric analysis and Western blotting,it was supported that ER stress and mitochondrial dysfunction triggered apoptosis involved in OVX induced neuron loss.Synaptic impairments,cytoskeletal abnormalities,and the injury of myelin and dysfunction of oligodendrocytes were also presented in OVX rats.The observed dysregulations of excitatory and inhibitory neurotransmissions in AMY might be responsible for the OVX induced depressive behaviors.Collectively,our study presented the variabilities related to depression and dementia in different brain regions after OVX,which is benefit for understanding the pathophysiologic mechanism in menopause related brain injuries and searching potential targets for early intervention.Emodin improved depressive and dementing behaviors in OVX rats.Emodin upregulated the plasma levels of GLP-1 and the levels GLP-1R in the brain of OVX rats.Emodin upregulated STIM2-TRPC1-Ca MKIIα pathway to improve neurons and spines in the HIP of OVX rats via GLP-1-GLP-1R pathway.Emodin significantly increased the level of POMC of HYP,and inhibited food intake,and reduced abdominal fat in OVX rats.Emodin inhibited intestinal contraction in OVX rats and may promote intestinal secretion of GLP-1. |
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