Establishment Of A New Model Of Menopausal Depression And Preliminary Exploration Of Pathologic Mechanism

Depression is an emotional disorder,characterized by low mood,cognitive impairment,slow thought,and decreased willpower symptoms.Depression affects about 12.8 % of the world’s population,and the world health organization predicts that by 2020,depression will be the second disease in the world.It is noteworthy that a large number of studies have shown that women are more than twice as likely as men to suffer from depression,and are characterized by longer duration and higher recurrence rates.Women who were in menopause were more likely to suffer from depression than other age groups in the life cycle,and the perimenopausal period had a higher prevalence rate than the postmenopausal period.The pathogenesis of menopausal depression is unclear,and lacking of objective index for diagnosis and effective treatment methods.Moreover,hormone treatment methods have many disadvantages in menopausal depression treatment,such as poor curative effect and side effects,and there is an urgent need for researching the mechanism、new therapeutic targets and treatments of menopausal depression.The traditional bilateral ovariectomy plus chronic mild stress(CMS)stimulation animal model produces a postmenopausal depressive-like state but the transition from perimenopausal period to postmenopausal period was ignored.Thus we established and estimated a novel animal model,in which the mice were stimulated by CMS for three months and removed the ovaries by two-step operation,and used as the basic for the continue research.Micro RNA(miRNA)is a class of non-coding single stranded RNA molecules encoded by endogenous genes with a length of about 22 nucleotides,and is involved in the post-transcriptional regulation process of gene expression.Mi RNA widely exists in the nervous system,involved in regulating the biosynthesis and release of neurotransmitters,neural circuits formation and emotion regulation process,also involved in the depression.We analyzed micro RNAs expression profile of menopausal depression mice and found that the expression of mi R-99 a significantly changed,and mi R-99 a target genes FKBP51 involved in the regulation of synaptic plasticity,it may be mediated the occurrence and development process of menopausal depression.Therefore,this article will explore the correlation between mi R-99 a and menopausal depression.In the first part of this work,we removed the ovaries by two-step operation and combined with CMS,established a new animal model.Moreover,we evaluated the behavior,neurotransmitter and hormone levels,etc,compared the superiority of the new model with the traditional animal model.In the second part,mi R-99 a was used as the research object.We analyzed the miRNA expressions、downstream FKBP51 expressions and impact on synaptic plasticity in the hypothalamus,confirmed the correlation between mi R-99 a and menopausal depression,offers a new way for the treatment of menopausal depression.Part 1: Establishment and Evaluation of a Novel Mouse Model of Peri/postmenopausal DepressionAIM: Establish a novel animal model which has more similarities to perimenopausal and postmenopausal depression by two-step ovarietomy plus three-month-CMS,and evaluate the model feasibility,confirmed that it can be used as the basis of menopausal depression mechanism research.METHODS: Seven to 8-week-old female C57BL/6 mice were randomly divided into 5 groups: 1)sham group;2)chronic mild stress(CMS)with sham surgery;3)bilateral ovariectomy group(CMS+BO)with exposure to chronic mild stress;4)unilateral ovariectomy group(CMS+UO)with exposure to chronic mild stress;and 5)the ovaries were removed in the first and second month(CMS+UO+UO)with exposure to chronic mild stress.After modeling stress for 12 weeks,mice were sacrificed and their serum,brain,uterus and tibiae were collected for test:(1)The depression status of each group was assessed by behavioral experiments such as sucrose preference test(SPT),tail suspension test(TST),forced swimming test(FST),etc.;(2)The concentration of corticosterone(CORT),estradiol(E2),and brain derived neurotrophic factor(BDNF)was determined by enzyme-linked immunosorbent assay(ELISA);(3)Application q RT-PCR to detect the pro-inflammatory cytokines IL-1β,TNFα,IL-6,the expressions of 5-HT receptors in hippocampus was also detected;(4)The application of high performance liquid chromatography(HPLC)method to analyzed norepinephrine(NE),dopamine(DA),3,4-2 hydroxy benzene acetic acid(DOPAC)and homovanillic acid(HVA),serotonin(5-HT)and 5-hydroxyindoleacetic acid(5-HIAA)and glutamate(Glu)and aspartate(Asp),γ-aminobutyric acid(GABA),taurine(Tau),glycine(Gly),serine(Ser)and glutamine(Gln)in the hippocampus;(5)Western blot was used to observe the expression of estrogen receptor(ERβ)in hippocampus;(6)The bone morphology of the tibia was observed by micro-CT,and the osteoporosis status was observed in the menopausal mice.RESULTS:(1)Behavioral tests showed stress groups,compared with the sham group,the percent of sucrose preference and the weight gain of mice significantly decreased,and the immobility time in TST and FST significantly increased after three months of modeling;(2)Menopausal depression evaluated the expression of pro-inflammatory cytokines IL-1β,TNFα,IL-6 and CORT significantly,and HPA axis appeared disorder phenomenon.Moreover,CMS+UO+UO group showed a more stable increase tendency;(3)The secretion of neurotransmitters in CMS+UO and CMS+UO+UO groups were disordered,menopausal depression also caused the 5-HT2CR、5-HT2 AR increased and SERT decreased expression,and CMS+UO+UO group showed more similarities to clinical phenomenon;(4)After removal of the ovaries,the concentration of E2 in the serum of CMS+BO and CMS+UO+UO was significantly decreased compared with other groups and the expression of ERβ in the CMS + UO + UO group showed a more stable downward tendency.Moreover,BDNF concentration decreased significantly especially in CMS+UO+UO group;(5)There was a more serious decline in bone mass、trabecular number and thickness in CMS+UO and CMS+UO+UO group compared with CMS+BO group;(6)Ovariectomy-induced uterine atrophy,and the uterine weight was significantly decreased.CONCLUSION: Two-step ovariectomy operation plus CMS induces a stabilized and gradual transition to peri/postmenopausal depressive status,which has more similarities to clinical manifestation of peri/postmenopausal depression.This novel model could be used as a useful model for simulating peri/postmenopausal status.Part 2: Preliminary Exploration of Menopausal Depression Pathologic MechanismAIM: We use the new menopausal depression animal model,researching the pathologic mechanism of menopause depression;use the gene chip screening and investigating the miRNA-99 a relevance to menopausal depression,revealing its role in the menopausal depression,and providing experimental basis for clarify the new pathological mechanism of menopausal depression.METHODS:(1)Use ELISA to analyze the expression of a variety of related hormones and factors in mice blood,including progestin,aldosterone,T3,insulin,S-100,IL-1β,TNF-α and TGF-β;(2)miRNA gene chip technology was used to analyze miRNA expression profiles of mice hypothalamus,and the significantly changed miRNAs were selected for cluster analysis.The expressions of mi R-99 a in the hypothalamus,hippocampus,prefrontal cortex and striatum were analyzed by q RT-PCR;(3)Western blot was applied to observe the expression of FKBP51,PR(progesterone receptor,PR),PSD-95(postsynaptic density protein 95,PSD-95)and SYN(synaptophysin,SYN)in hypothalamus;(4)Immunofluorescence method was used to observe the expression of FKBP51,PSD-95 in hypothalamus PVN(paraventricular nucleus,PVN);(5)The morphological changes of synapses were observed by transmission electron microscopy;(6)The function changes of synapses were analyzed by electrophysiological examinationRESULTS:(1)ELISA results showed that there was a significant decrease in the levels of progestin,insulin and s-100,and a significant increase in aldosterone,IL-1β and TNF-α in menopausal depression,and the endocrine and neurological functions in menopausal depression were in disorder status;(2)Gene chip screen out a series of miRNAs which expression changed in menopausal depression,including the expression of mi R-99 a.q RT-PCR results show that the expression of mi R-99 a in the hypothalamus,hippocampus,striatum and prefrontal cortex significantly changed,especially in the hypothalamus;(3)Western blot and immunofluorescence experiments demonstrated that the lower expression of mi R-99 a cause the increase expression of FKBP51.Moreover,the increase expression of FKBP51 reduced PR nuclear transfer process;(4)Western blot and immunofluorescence results show that synaptic related protein PSD-95 and SYN expression decreased in hypothalamus.The results of electron microscopy showed that the structure of synaptic membrane in the hypothalamus was damaged,and the density of the synapses decreased.In the electrophysiological test,menopausal depression impaired the LTP induction,and the slope of f EPSP was significantly lower than that of Sham group.CONCLUSION: Menopausal depression is under a state of endocrine,immune,and neurological function disorder;The expression of mi R-99 a in the hypothalamus decreased significantly,and may negative regulate the expression of the FKBP51,thus further influence the synaptic plasticity in the brain,resulting in the brain function disorder of menopausal depression.INNOVATION POINTS OF PAPER:1.Established and evaluated the new animal model of menopausal depression,which provided the basis for studying the mechanism of menopausal depression;2.Clarify the correlation between mi R-99 a and of menopausal depression,providing the foundation of further exploring the new pathogenesis of the menopausal depression.