【作者】 张玲；

【导师】 刘寰忠；

【作者基本信息】 安徽医科大学 ， 精神病与精神卫生学， 2024， 博士

【摘要】 第一部分:临床研究:产后抑郁的发病率及与产前应激的关系背景:产后抑郁(Postpartum depression,PPD)是妇女在生育后出现的一种情绪和心理障碍,通常在分娩后的几周至几个月内发生,但也可能持续更长的时间。产后抑郁的主要表现包括情绪低落、焦虑、失眠、疲劳、注意力不集中、对日常活动失去兴趣、心理痛苦和自我负面评价等症状。产后抑郁的成因涉及多个方面,包括荷尔蒙水平的变化、生活变化、个人和家庭压力以及社会支持不足等。方法:在产前采用妊娠生活事件问卷、自我效能量表、自制人口学问卷评估孕中晚期孕妇的产前应激、自我效能水平和基础人口学资料。在产后约40天采用爱丁堡产后抑郁量表、育儿胜任感问卷评估产妇的产后抑郁水平和育儿胜任感水平。结果:(1)产后抑郁的发病率高达25%,产前应激组产妇产后抑郁量表评分及产后抑郁的发病率均高于无产前应激组。而母婴依恋总分及依恋的质量及互动的愉悦性均低于无产前应激组,两组在育儿胜任感和自我效能得分差异无统计学意义;(2)与无产后抑郁组产妇相比,有产后抑郁组的产妇具有更高的产前应激发生率,且无产后抑郁组的产妇计划妊娠的比例高于有产后抑郁组的产妇。在母婴依恋维度中,产后抑郁组产妇的母婴依恋总分、依恋的质量及敌对的不存在性均低于无产前应激组,差异均具有统计学意义,而在互动的愉悦性得分两组差异无统计学意义;(3)多元Logistic回归研究结果显示,产前应激是产后抑郁的危险因素,而计划妊娠是产后抑郁的保护因素,且母婴依恋质量得分也是产后抑郁的保护因素。结论:产前应激是孕妇产后抑郁的危险因素之一,并影响后续的母婴依恋。第二部分:基础研究:短暂束缚应激基于ERβ/PI3K/AKT通路改善产后抑郁背景:应激也是影响产后抑郁样行为的因素之一,基础研究也有关于使用产前慢性不可预测应激进行产后抑郁模型研究的相关成果。近年来,有研究显示短暂束缚应激被认为是提高应对未来压力的一种技巧。虽然长时间的束缚应激可以导致小鼠情绪异常,但研究证实短暂轻度束缚应激既可以促进正常小鼠的情绪和认知功能的改善,又可以逆转产前应激导致的子代情绪异常。然而,慢性轻度可预测束缚应激对产后抑郁的作用尚未被研究。因此,本研究针对产后抑郁的母鼠采用短暂轻度可预测束缚应激,并探究这种应激方式对母鼠产后抑郁情绪的干预作用及其相关机制。方法:(1)首先将8-9周龄SPF级C57BL/6J雌鼠与雄鼠采用2:1的比例进行合笼,查到阴栓那天记为孕第1天。于孕7-21天对应激组雌鼠采用慢性不可预测轻度应激(Chronic unpredictable mild stress,CUMS)进行产前应激(Prenatal Stress,PS)造模。分娩后第7-21天对PS母鼠予以持续14天的5分钟束缚应激(RS5)进行干预(通过旷场实验、悬尾实验、强迫游泳、蔗糖偏好实验评估干预效果);(2)后采用前额叶mRNA测序找到RS5改善产后抑郁的可能通路;后通过蛋白质印记(Western Blot,WB)和定量聚合酶链反应(Quantitative Polymerase Chain Reaction,qPCR)测量该通路受体和ERβ的表达水平,并用酶联免疫吸附试验(ELISA)测量雌激素(E2)水平和应激相关激素变化;(3)后将母鼠分为Con组、PS组、PS+RS5组、PS+E2组、PS+RS5+PHTPP(ERβ受体阻滞剂)组,并采用旷场实验、悬尾实验、强迫游泳实验、蔗糖偏好实验评估不同组别母鼠行为学变化,并采用WB测量雌激素通路相关蛋白的变化。并采用ELISA试验测量HPA轴相关激素变化。最后,采用苏木素-伊红染色(hematoxylin-eosin staining,HE)和末端脱氧核苷酸末端标记法(TdT-mediated dUTP nick end labeling,Tunel)染色评估前额叶神经元变化;(4)最后通过脑立体定位技术过表达产后抑郁母鼠前额叶ERβ受体水平来评估相应行为学变化及前额叶神经元凋亡情况。结果:(1)RS5产生了改善产后抑郁的结果,表现为与PS组母鼠相比,PS+RS5组母鼠在10分钟内的移动总距离高于PS组,而悬尾实验和强迫游泳的不动时间缩短,蔗糖偏好指数增加。(2)mRNA测序结果显示在PS与PS+RS5组母鼠中存在433个差异基因,其中有意义的通路之一为雌激素信号通路。(3)雌激素及ERβ受体参与RS5改善产后抑郁的机制,即与Con组母鼠相比,PS组母鼠ERβ的mRNA表达水平下降;且与PS组母鼠相比,PS+RS5组母鼠的ERβ的mRNA水平上升。而三组母鼠的ERα的mRNA表达水平的差异无统计学意义。而参与雌激素信号通路发挥机制的关键酶芳香化酶(CYP19)的mRNA表达水平在三组母鼠中存在统计学差异,具体表现为与Con组母鼠及PS+RS5相比,PS组母鼠CYP19的mRNA表达水平均下降;与Con组母鼠相比,PS组母鼠ERβ的蛋白表达水平下降,PS+RS5组母鼠的ERβ蛋白表达较PS组母鼠水平上升。而ERα在Con组、PS组及PS+RS5组的表达水平差异无统计学意义;且ELISA结果显示雌激素在PS组母鼠血液中含量下降,而RS5治疗后母鼠雌激素水平升高;且应激相关激素FK506结合蛋白5(FKBP5)在PS组母鼠中较Con组母鼠含量下降,而核受体亚家族3C组成员1(NR3C1)含量上升,而RS5治疗后组母鼠能逆转FKBP5的下降,且使得NR3C1呈上升趋势。(4)后采用雌激素(E2)灌胃的方法也能模仿RS5治疗产后抑郁的作用,表现PS+E2组母鼠与PS组母鼠相比,旷场实验中的移动总距离增高,强迫游泳不动时间缩短,且蔗糖偏好指数增加;而采用ERβ的抑制剂PHTPP灌胃的方式能逆转PS+RS5组改善产后抑郁的作用,表现为Con组母鼠相比,PS+RS5+PHTPP组母鼠在悬尾实验和强迫游泳实验中的不动时间均延长,而在旷场实验中的平均速度和总距离降低,且蔗糖偏好指数降低;而血液学结果显示五组在HPA轴激素ACTH、CORT和CRH的水平差异也呈统计学意义;PI3K/AKT通路参与RS5改善产后抑郁的机制,表现为与PS组母鼠相比,PS+RS5组母鼠和PS+E2组母鼠的P-AKT和P-PI3K表达上调;PI3K/AkT信号通路下游抗凋亡蛋白Bcl-2,促凋亡蛋白Bax的表达在5组不同母鼠间的表达也存在统计学差异。与PS组母鼠相比,PS+RS5组母鼠和PS+E2组母鼠的促凋亡蛋白Bax表达含量下降,而抗凋亡蛋白Bcl-2表达含量上升。且He染色结果表明PS+RS5组神经元排列较为有序,偶见变性,损伤程度较PS组明显改善;E2组整体情况与PS+RS5组相当,偶见神经元变性;PS+RS5+PHTPP组整体损伤严重,与PS组相当,大量神经元变性,胶质细胞数量增多。而Tunel染色结果显示PS组和PS+RS5+PHTPP组母鼠的凋亡率较Con组母鼠增高,而PS+RS5组母鼠与PS组相比,凋亡率呈下降趋势。(5)前额叶区注射AAV9-ERβ过表达病毒组能改善母鼠产后抑郁样行为,表现为AAV9-ERβ过表达病毒组产后母鼠在旷场实验中的总距离和平均速度较产后抑郁母鼠空载对照病毒处理组升高,悬尾实验和强迫游泳实验中不动时间显著降低,而蔗糖偏好指数显著升高且ERβ表达含量增加,神经元凋亡减少。结论:(1)产前CUMS造模能够诱导母鼠产生产后抑郁样行为;而持续14天,每天5分钟短暂轻度可预测束缚应激(RS5)能够改善产后抑郁样行为,并抑制神经细胞凋亡;(2)RS5改善产后抑郁可能由ERβ/PI3K/AKT通路介导;(3)抑制ERβ受体能逆转RS5改善产后抑郁的作用;(4)过表达前额叶ERβ受体能达到改善产后抑郁的作用。更多还原

【Abstract】 Part one:Clinical research:The prevalence of postpartum depression and its relationship with prenatal stressBackground:Postpartum depression(PPD)is an emotional and psychological disorder that occurs in women after childbirth,typically occurring within weeks to months after delivery,but may also persist for a longer period of time.The main manifestations of postpartum depression include low mood,anxiety,insomnia,fatigue,lack of concentration,loss of interest in daily activities,psychological pain,and negative self-evaluation.The causes of postpartum depression involve multiple aspects,including changes in hormone levels,lifestyle changes,personal and family stress,and insufficient social support.Methods:1.Clinical research,prenatal stress scales,self-efficacy scales,and self-made demographic questionnaires were used to evaluate the prenatal stress,self-efficacy levels,and basic demographic data of pregnant women in the middle or late stages of pregnancy.The Edinburgh Postpartum Depression Scale and Parenting Competency Questionnaire were used to evaluate the levels of postpartum depression and parenting competence in pregnant women approximately 40 days postpartum.Results:(1)the score of postpartum depression scale and the prevalence of postpartum depression in the prenatal stress group were higher than those in the non prenatal stress group.The total score of maternal infant attachment,the quality of attachment,and the pleasure of interaction were lower than those of the non prenatal stress group.There was no statistically significant difference in parenting competence and self-efficacy scores between the two groups;(2)Compared with the group without postpartum depression,the group with postpartum depression has a higher prevalence of prenatal stress,and the proportion of planned pregnancies in the group without postpartum depression is higher than that in the group with postpartum depression.In the dimension of maternal infant attachment,the total score of maternal infant attachment,the quality of attachment,and the absence of hostility in the postpartum depression group were lower than those in the non prenatal stress group,and the differences were statistically significant.However,there was no statistically significant difference in the score of interactive pleasure between the two groups;(3)The results of multiple logistic regression research showed that prenatal stress is a risk factor for postpartum depression,while planned pregnancy is a protective factor for postpartum depression,and the quality and total score of maternal and infant attachment are also protective factors for postpartum depression.Conclusion:Prenatal stress is one of the risk factors for postpartum depression in pregnant women and affects subsequent maternal and child attachment.Part two:Basic research:Transient restraint stress improves postpartum depression based on ERβ/PI3K/AKT pathwayBackground:Stress is also considered to be one of the important factors leading to postpartum depression.Basic research has also yielded relevant results on using chronic unpredictable prenatal stress for postpartum depression models.In recent years,studies have shown that transient restraint stress is considered a technique to improve coping with future stress.In addition,prolonged restraint stress can lead to emotional abnormalities in mice,studies have shown that transient mild restraint stress can promote improvements in the emotional and cognitive functions of normal mice,as well as reverse the emotional abnormalities in offspring caused by prenatal stress.However,the effect of chronic mild predictable restraint stress on postpartum depression has not been studied.Therefore,this study first explores the related effects of prenatal stress on postpartum depression from clinical studies,and then applies transient mild predictable restraint stress for postpartum depression in female mice from basic research.It also explores the intervention effect of this stress method on postpartum depression in female mice and its related mechanisms.Methods:(1)Firstly,8-9 week old SPF grade C57BL/6J female mice and male mice were housed in a 2:1 ratio,and the day of discovery of the Yin plug was recorded as the first day of pregnancy.Chronic unpredictable mild stress(CUMS)was used to create prenatal stress(PS)models in the stress group of female mice from 7 to 21 days of pregnancy.On the 7th to 21st day after delivery,PS female mice were subjected to a continuous 14 day 5-minute restraint stress(RS5)intervention.The intervention effect was evaluated through open field tests,tail suspension tests,forced swimming,and sucrose preference tests.(2)mRNA sequencing of the frontal lobe was used to identify the possible pathway of RS5 in improving postpartum depression;Subsequently,Western blot(WB)and quantitative polymerase chain reaction(qPCR)were used to measure the expression levels of receptors in this pathway and ERβ;the enzyme-linked immunosorbent assay(ELISA)was used to measure the changes in estrogen(E2)levels and stress related hormones;(3)Afterwards,the mother mice were divided into Con group,PS group,PS+RS5 group,PS+E2 group,and PS+RS5+PHTPP((ERβ receptor blocker).The receptor blocker group was evaluated for behavioral changes in different groups of female mice using open field tests,tail suspension tests,forced swimming tests,and sucrose preference tests,and changes in estrogen pathway related proteins were measured using WB.And ELISA assay was used to measure changes in HPA axis related hormones.Subsequently,changes in frontal lobe neurons were evaluated using hematoxylin eosin staining(HE)and TdT mediated dUTP nick end labeling(Tunel)staining;(4)Finally,overexpression of ERβ in the prefrontal lobe of postpartum depressed female mice was achieved through brain stereotactic technology.And then evaluate the corresponding behavioral changes and apoptosis of frontal lobe neurons based on receptor levels.Results:(1)RS5 resulted in an improvement in postpartum depression,as compared to the PS group female mice,the PS+RS5 group female mice had a higher total distance of movement within 10 minutes,while the tail suspension test and forced swimming had shorter immobility time and an increase in sucrose preference index.(2)The mRNA sequencing results showed 433 differentially expressed genes in the PS and PS+RS5 groups of female mice,with one of the significant pathways being the estrogen signaling pathway.(3)Estrogen and ERβ receptors are involved in the mechanism of RS5 improving postpartum depression.Specifically,compared to the Con group,the mRNA expression level of ERβ in mother mice of the PS group decreased;moreover,compared to the PS group,the mRNA level of ERβ in mother mice of the PS+RS5 group increased.The mRNA expression level of aromatase(CYP19),a key enzyme involved in the estrogen signaling pathway,showed statistical differences among the three groups of mother mice.In detail,compared to the Con group and the PS+RS5 group,the mRNA expression level of CYP19 in mother mice of the PS group decreased.Protein expression level of ERβdecreased in mother mice of the PS group compared to the Con group,whereas it increased in mother mice of the PS+RS5 group compared to the PS group.The differences in expression levels of ERα among the Con group,PS group,and PS+RS5 group were not statistically significant.ELISA results revealed a decrease in estrogen levels in the blood of mother mice in the PS group,while after RS5 treatment,estrogen levels increased.Additionally,the stress-related hormone FK506 binding protein 5(FKBP5)decreased in mother mice of the PS group compared to the Con group,and the nuclear receptor subfamily 3C member 1(NR3C1)increased.RS5 treatment reversed the decrease in FKBP5 and led to an upward trend in NR3C1 levels in female mice.(4)The administration of an ERβ inhibitor,PHTPP,by oral gavage,reversed the beneficial effects of the PS+RS5 group in improving postpartum depression.Compared with the PS group,the PS+E2 group female mice showed an increase in total distance of movement,a shorter duration of forced swimming immobility,and an increase in sucrose preference index in the open field test;Oral ERβ inhibitor PHTPP reversed the improvement of postpartum depression in the PS+RS5 group.Compared with the Con group,the PS+RS5+PHTPP group showed longer immobility time in both tail suspension and forced swimming tests,while the average speed and total distance decreased in open field tests,and the sucrose preference index decreased;The hematological results showed that there were statistically significant differences in the levels of HPA axis hormones ACTH,CORT,and CRH among the five groups;The PI3K/AKT pathway is involved in the mechanism of RS5 in improving postpartum depression,as evidenced by upregulation of P-AKT and P-PI3K expression in the PS+RS5 group and PS+E2 group females compared to the PS group females;The expression of anti apoptotic protein Bcl-2 and pro apoptotic protein Bax downstream of the PI3K/AkT signaling pathway also showed statistical differences among five different groups of female mice.Compared with the PS group of female mice,the expression levels of pro apoptotic protein Bax decreased in the PS+RS5 group and PS+E2 group,while the expression levels of anti apoptotic protein Bcl-2 increased.And the He staining results showed that the neurons in the PS+RS5 group were arranged in an orderly manner,with occasional degeneration,and the degree of damage was significantly improved compared to the PS group;The overall situation of E2 group is similar to that of PS+RS5 group,with occasional neuronal degeneration observed;The overall damage in the PS+RS5+PHTPP group was severe,comparable to that in the PS group,with a large number of neuronal degeneration and an increase in the number of glial cells.The Tunel staining results showed that the apoptosis rate of the PS group and the PS+RS5+PHTPP group of female mice was higher than that of the Con group,while the apoptosis rate of the PS+RS5 group of female mice showed a downward trend compared to the PS group.(5)Injecting AAV9-ERβ overexpression virus into the prefrontal cortex area improved postpartum depression-like behavior in mother mice.This improvement was manifested by increased total distance and average speed in the open field test compared to the postpartum depression mother mice treated with control virus.The immobility time in the tail suspension and forced swimming tests significantly decreased,while the sucrose preference index significantly increased along with an increase in ERβexpression levels and a reduction in neuronal apoptosis.Conclusion:(1)Prenatal CUMS modeling can induce postpartum depression like behavior in female mice;And lasting for 14 days,brief mild predictable restraint stress(RS5)for 5 minutes per day can improve postpartum depression like behavior and inhibit neuronal apoptosis;(2)RS5 may improve postpartum depression by ERβ/PI3K/AKT pathway mediated;(3)Suppress ERβ receptors can reverse the effect of RS5 on improving postpartum depression;(4)Overexpression of frontal lobe ERβ receptors can improve postpartum depression.更多还原