【作者】 李明；

【导师】 赵跃然；

【作者基本信息】 山东大学 ， 临床检验诊断学， 2024， 博士

【摘要】 研究背景围绝经期综合征又称更年期综合征(Menopausal syndrome,MPS),指妇女绝经前后出现性激素波动或减少所致的一系列以自主神经系统功能紊乱为主,伴有神经心理症状的一组症候群。MPS常表现为潮热、出汗、睡眠障碍、记忆力减退和抑郁症等。出现MPS的主要原因是卵巢功能衰退引起的体内雌激素(Estradiol)水平下降。因此,雌激素替代疗法(Estrogen Replacement Therapy,ERT)被认为是治疗MPS的重要方法。雌激素是一类甾体激素,属于性类固醇激素,主要由雌性动物的卵巢分泌。生理性雌激素主要包括雌酮(E1)、雌二醇(E2)和雌三醇(E3)三种形式,其中E2活性最强,且在绝经过渡期迅速下降。除了在生殖系统中具有重要作用外,雌激素在中枢神经系统的活动及发育中也必不可少,与记忆、学习及行为活动密切相关。一般来说,雌激素对神经系统有保护作用。然而,多项研究表明,临床开展的基于生理剂量的ERT(维持外周血中E2含量为绝经前卵泡早期水平)并不能有效改善MPS的相关症状。有研究表明,超生理剂量的雌二醇(supraphysiological doses of estrogen,sE2)补充可以促进脑卒中后运动感觉功能恢复、病毒性肺炎后免疫系统功能恢复等,同时生理剂量的ERT则没有以上治疗效果。目前,临床上有时也会开展sE2补充疗法,主要应用于绝经后骨质疏松患者。然而,sE2在MPS治疗中的作用及其对更年期相关神经退行性病变进程的影响尚不清楚。研究目的在本研究中,我们通过卵巢切除术(ovariectomy,OVX)建立女性更年期小鼠模型,结合行为学测试、神经病理学染色、蛋白质组学分析、非靶向代谢组学分析和体外实验,深入探索外源性sE2补充对更年期小鼠认知和抑郁样行为的影响及分子机制,为sE2的临床应用提供理论依据和参考。研究方法1.卵巢切除术 手术摘除雌性C57/BL6J小鼠双重卵巢建立更年期小鼠模型,假手术组作为对照。手术后一周开展进一步实验。2.药物处理 根据预实验的结果,选择0.5mg/kg/天的作为sE2的使用剂量,对小鼠进行进行腹腔注射,持续5周。用药结束后检测小鼠血清E2浓度、体重、抓力和脑指数(脑重/体重)。3.行为学测试 利用Y迷宫自发交替实验检测小鼠短期认知能力,利用旷场实验、强迫游泳实验和蔗糖偏好实验检测小鼠抑郁样行为。4.免疫组织化学染色(Immunohistochemistry,IHC)通过IHC的方法,标记脑切片中Iba1阳性的小胶质细胞,观察并分析小胶质细胞形态变化(细胞体积和分支节点数量)。5.组织免疫荧光染色 利用Iba1、CD86、IL-1β、IL-6和TNF-α单克隆抗体进行组织免疫荧光染色标记小鼠脑中的促炎型小胶质细胞并计数;利用COX-1单克隆抗体检测小鼠脑中神经元细胞的代谢状态;定位小胶质细胞表面和细胞核中的雌激素受体(ERa、ERB和 GPER)。6.苏木素-伊红(hematoxylin-eosin,HE)染色利用HE染色检测小鼠大脑海马和皮层细胞形态变化。7.尼氏染色 利用尼氏染色法定量小鼠大脑中受损神经元细胞的数量。8.实时荧光定量PCR(Quantitative Real-time PCR,qPCR)提取脑组织(海马和大脑皮层)和细胞中的RNA后,利用qPCR检测促炎性细胞因子(IL-1β、IL-6和TNF-α)和抗炎性细胞因子(IL-4、IL-10和TGF-β)的表达水平;细胞转染后利用qPCR检测目的基因转染效率(ERa、ERB和GPER);检测小鼠大脑中大麻素受体1(Cannabinoid receptor 1,CB1)表达情况。9.蛋白质组学分析 对经过或未经过sE2处理的更年期小鼠进行蛋白质组学分析,筛选差异表达蛋白和富集通路。10.非靶向代谢组学分析 对经过或未经过sE2处理的更年期小鼠进行非靶向学分析,筛选差异代谢分子及相应代谢通路。11.细胞培养和药物处理 使用不同浓度的E2(200-3200 nM/L)处理小鼠小胶质细胞系BV2,神经元细胞系HT22以及原代小胶质细胞和神经元,并建立小胶质细胞和神经元细胞共培养模型,在体外试验中探索E2对小胶质细胞活化和神经元细胞活性的影响;在小胶质细胞培养液中加入脂多糖(LPS)处理后,再加入低浓度的E2(200 nM/L)或高浓度的E2(800 nM/L),检测促炎性分化后的小胶质细胞对不同浓度E2刺激的反应;使用NF-κB抑制剂QNZ处理BV2或原代小胶质细胞,探索NF-κB信号通路在高浓度E2激活小胶质细胞中的作用。12.细胞转染 使用siRNA构建雌激素受体(ERa、ERB和GPER)缺陷的BV2和原代小胶质细胞,敲低目的基因后再检测E2对小胶质细胞活化的影响,确定高浓度E2激活小胶质细胞时的目标受体。13.细胞免疫荧光 利用Iba1和CD86单克隆抗体进行免疫荧光染色分析小胶质细胞激活和促炎性极化状态;利用NF-κB、IL-1和TNF-α相应抗体进行免疫荧光染色探索高浓度E2对小胶质细胞NF-κB信号通路激活的情况;定位小胶质细胞表面和细胞核中的雌激素受体(ERa、ERB和GPER);分析不同浓度E2对小胶质细胞大麻素受体CB1表达的影响。14.蛋白质印迹(Western blotting)定量分析不同浓度的E2及雌激素受体表达缺陷或加NF-κB抑制剂QNZ处理后的原代小胶质细胞核内NF-κB及促炎型小胶质细胞标志物CD86的表达水平。15.CCK-8实验 在体外利用CCK-8实验检测不同浓度E2对小胶质细胞及神经元细胞细胞活性的影响。研究结果1.sE2补充加重了更年期小鼠的抑郁样行为。与假手术组相比,更年期小鼠(OVX组)出现外周血E2减少,体重降低,抓力和脑指数没有显著变化,Y迷宫中自发交替次数显著降低,强迫游泳实验中静止时间增加,旷场试验中移动距离和在中心区域的时间降低,蔗糖偏好试验中蔗糖偏好比降低;sE2补充显著提高了更年期小鼠外周血E2水平,对体重、抓力、脑指数和Y迷宫中自发交替次数没有显著影响,但是显著延长了其在强迫游泳实验中的静止时间,增加了其在旷场实验中的移动距离和在中心区域的时间,进一步降低了更年期小鼠的蔗糖偏好比。2.sE2补充加重了更年期小鼠脑小胶质细胞激活和促炎性表型。与假手术组相比,OVX组小鼠脑内小胶细胞胞体体积显著增加,突起的分支节点减少,促炎型小胶质细胞数量显著增加,海马和皮层内促炎性细胞因子(IL-1β、IL-6和TNF-α)表达水平显著提升,抗炎性细胞因子(IL-4、IL-10和TGF-β)显著减少;sE2处理进一步加重了更年期小鼠以上小胶质细胞激活和促炎性表型。3.高浓度E2可导致小胶质细胞激活和促炎性表型。在体外试验中,低浓度E2(200 nM/L)不能激活小胶质细胞,但是随着E2浓度升高,小胶质细胞被显著激活,且分化为促炎型小胶质细胞;加入LPS以后,静息状态的小胶质细胞分化为促炎型小胶质细胞(Iba1和CD86表达增多,促炎性细胞因子表达增多,抗炎性细胞因子表达降低),低浓度E2可抑制LPS诱导的小胶质细胞促炎性分化,而高浓度的E2(800 nM/L)则显著促进小胶质细胞的促炎性分化。4.高浓度E2通过ERa激活小胶质细胞。在体外试验中,利用siRNA敲减小胶质细胞的雌激素受体(ERa、ERB和GPER)后,只有缺乏ERa的小胶质细胞不被高浓度E2激活。5.高浓度E2通过NF-κB信号通路激活小胶质细胞。蛋白质组学的结果表明,与OVX组相比,经过sE2处理的更年期小鼠大脑内与NF-κB信号通路及神经炎症相关的蛋白高度富集;在体外试验中,高浓度E2可显著激活小胶质细胞的NF-κB信号通路。加入NF-κB抑制剂后,小胶质细胞则不能被高浓度E2激活,相关促炎性表型也被抑制。6.sE2补充加重更年期小鼠脑神经元损伤。HE和尼氏染色的结果表明,与假手术组相比,OVX组小鼠大脑皮层和海马区神经元数量显著减少,受损神经元数量显著增多;sE2处理进一步加重了更年期小鼠以上小脑神经元受损表型。7.高浓度E2通过激活小胶质细胞引起神经元损伤体外试验中,不同浓度的E2(200-1600 nM/L)可显著提高神经元细胞系HT22的细胞活性,但是高浓度(3200nM/L)无效;对于原代神经元细胞,不同浓度的E2(200-1600 nM/L)对细胞活性没有显著影响,但是高浓度(3200nM/L)的E2可降低细胞活性;E2在不同浓度和作用时间下都可提升BV2和原代小胶质细胞的细胞活性;在神经元和小胶质细胞共培养条件下,加入E2(800nM/L)可显著降低神经元的细胞活性;组织免疫荧光的结果表明,经过sE2治疗的更年期小鼠脑小胶质细胞可释放更多促神经元死亡的细胞因子。8.sE2补充引起更年期小鼠脑甘油磷脂代谢紊乱及逆行内源性大麻素信号抑制。与OVX组相比,经过sE2治疗的更年期小鼠大脑皮层和海马区神经元细胞COX1表达显著升高;非靶向代谢组学分析的结果表明,经过sE2治疗的更年期小鼠大脑出现严重的代谢紊乱,其中包括甘油磷脂代谢紊乱和逆行内源性大麻素信号通路的异常;在体外实验中,高浓度E2可显著抑制CB1;sE2治疗的小鼠大脑皮层和海马中CB1表达显著降低。结论外源性sE2补充会加重更年期小鼠的抑郁样行为,这可能与其通过促进ERa/NF-κB信号通路激活导致大脑小胶质细胞激活及促炎型分化介导的神经炎症,和神经元细胞甘油磷脂代谢紊乱及逆行内源性大麻素信号抑制有关。这项研究揭示了在女性更年期时开展外源性sE2补充的危害和潜在机制。更多还原

【Abstract】 BackgroudPerimenopausal syndrome,also known as Menopausal syndrome(MPS),refers to a series of neuropsychological symptoms accompanied by autonomic nervous system dysfunction caused by fluctuations or decreases in sex hormones before and after menopause,and is often manifested by hot flashes,sweating,sleep disorders,memory loss and depression.The main cause of MPS is a decrease in estrogen(Estradiol)levels due to ovarian failure.Therefore,Estrogen Replacement Therapy(ERT)is considered an important treatment for MPS.Estrogens are a class of steroid hormones,which are sex steroid hormones and are mainly secreted by the ovaries of female animals.Physiological estrogens mainly include three forms of estrone(E1),estradiol(E2),and estriol(E3),of which E2 is the most active and declines rapidly during the menopausal transition.In addition to its important role in the reproductive system,estrogen is essential in the activity and development of the central nervous system and is closely related to memory,learning and behavioural activities.In general,oestrogen has a protective effect on the nervous system.However,several studies have shown that clinically conducted ERT based on physiological doses(maintaining E2 levels in peripheral blood at early premenopausal follicular levels)is not effective in ameliorating the symptoms associated with MPS.It has been shown that supraphysiological doses of estrogen treatment(sE2)can promote the recovery of motor-sensory function after stroke and the recovery of immune system function after viral pneumonia,while physiological doses of ERT do not have the above therapeutic effects.At present,sE2 supplementation therapy is sometimes used in clinical practice,mainly for postmenopausal osteoporosis patients.However,the role of sE2 in MPS treatment and its effect on the progression of menopause-associated neurodegenerative diseases is unknown.PurposeIn this study,we established a female menopausal mouse model by ovariectomy,combined with behavioural tests,neuropathological staining,proteomic analysis,non-targeted metabolomics analysis and in vitro experiments,to explore in depth the effects and molecular mechanisms of sE2 on cognitive and depressive-like behaviours in menopausal mice,and to provide theoretical basis and references for the clinical application of sE2.Methods1.Ovariectomy Surgical removal of the double ovaries of female C57/BL6J mice to establish a menopausal mouse model,with the Sham-operated group as a control.Further experiments were carried out one week after surgery.2.Drug treatment According to the results of the pre-test,0.5mg/kg/day was selected as the dose of sE2,which was injected intraperitoneally into the mice for 5 weeks.The serum E2 concentration,body weight,grip strength and brain index(brain weight/body weight)of the mice were measured at the end of the drug administration.3.Behavioural tests Short-term cognitive ability of mice was detected using the Y-maze spontaneous alternation test,and depression-like behaviour was detected using the open field test,forced swimming test and sucrose preference test.4.Immunohistochemistry(IHC)By IHC method,Ibal-positive microglia were labelled in brain sections,and the morphological changes of microglia(cell volume and number of branching nodes)were observed and analysed.5.Tissue immunofluorescence staining Tissue immunofluorescence staining using monoclonal antibodies to Ibal,CD86,IL-1β,IL-6 and TNF-α to label pro-inflammatory microglia in mouse brain and count them;using COX-1 monoclonal antibody to detect the metabolic status of neuronal cells in mouse brain;locating estrogen receptors on the surface of microglia and in the nucleus of the cells(ERa,ERB and GPER).6.Hematoxylin-eosin(HE)staining Use HE staining to detect morphological changes in hippocampus and cortical cells of mouse brain.7.Nissen staining Quantification of the number of damaged neuronal cells in the mouse brain by Nissen staining.8.Quantitative Real-time PCR(qPCR)After extracting RNA from brain tissues(hippocampus and cerebral cortex)and cells,qPCR was used to detect the expression levels of pro-inflammatory cytokines(IL-1β,IL-6 and TNF-α)and anti-inflammatory cytokines(IL-4,IL-10 and TGF-β).After cell transfection,qPCR was used to detect the transfection efficiency of target genes(ERa,ERB and GPER);and the expression of cannabinoid receptor 1(CB1)was detected in the brains of mice.9.Proteomic analysis Proteomic analysis of menopausal mice with or without sE2 treatment to screen for differentially expressed proteins and enriched pathways.10.Non-targeted metabolomic analysis Non-targeted metabolomic analysis of menopausal mice with or without sE2 treatment to screen for differentially expressed proteins and enriched pathways.11.Cellular drug treatment The mouse microglia cell line BV2,neuronal cell line HT22 and primary microglia and neurons were treated with different concentrations of E2(200-3200 nM/L),and microglia and neuronal cell co-culture models were established,to explore the effects of E2 on microglia activation and neuronal cell activity in vitro assays;lipopolysaccharide(LPS)was added into the microglia culture medium.treated with lipopolysaccharide(LPS)followed by low concentration of E2(200 nM/L)or high concentration of E2(800 nM/L),to detect the response of pro-inflammatory differentiated microglia to stimulation with different concentrations of E2;and treating BV2 or primary microglia with the NF-κB inhibitor QNZ to explore the NF-κB signalling pathway in the activation of microglia at high concentrations of E2.Role.12.Cell transfection Construct siRNA plasmids targeting estrogen receptors(ERa,ERB and GPER)in BV2 and primary microglial cells,knock down the target genes,and then test the effect of E2 on microglia activation,and identify the target receptors activated by high concentration of E2.13.Cellular immunofluorescence Immunofluorescence staining with monoclonal antibodies against Ibal and CD86 was used to analyse microglia activation and pro-inflammatory polarisation;the activation of the NF-κB signalling pathway in microglia by high concentrations of E2 was analysed by using the corresponding antibodies against NF-κB,IL-1 and TNF-α;the oestrogen receptors on the surface of microglia and in the nucleus(ERa,ERB and GPER)were located;the effect of different concentrations of E2 on microglia activation was analysed;and the target receptors at different concentrations of E2 were identified.GPER);analyse the effect of different concentrations of E2 on CB1 expression in microglia.14.Western blotting Quantitatively analyse the expression levels of NF-κB and CD86 in the nucleus of primary microglia treated with different concentrations of E2,estrogen receptor gene knockdown and the NF-κB inhibitor QNZ.15.CCK-8 assay The effects of different concentrations of E2 on the cellular activity of microglia and neuronal cells were examined in vitro using the CCK-8 assay.Results1.sE2 treatment aggravated the depression-like behaviour of menopausal mice.Compared with the Sham-operated group,menopausal mice(OVX group)showed a decrease in peripheral blood E2,a decrease in body weight,no significant changes in grip strength and cerebral index,a significant decrease in the number of spontaneous alternations in the Y maze,an increase in the resting time in the forced swimming test,a decrease in the distance moved and the time spent in the central area in the open-field test,and a decrease in the sucrose preference ratio in the sucrose preference test.sE2 treatment significantly increased peripheral blood E2 levels,had no significant effect on body weight,grip strength,brain index and the number of spontaneous alternations in the Y maze,but significantly prolonged their resting time in the forced play experiment,increased their moving distance and time in the centre area in the open field experiment,and further decreased the sucrose preference ratio in menopausal mice.2.sE2 treatment exacerbated brain microglia activation and pro-inflammatory phenotype in menopausal mice.Compared with the Sham-operated group,microglia cytosolic volume in the brain of mice in the OVX group was significantly increased,branching nodes of protrusions were reduced,the number of pro-inflammatory microglia was significantly increased,the expression levels of pro-inflammatory cytokines(IL-1β,IL-6,and TNF-α)were significantly elevated in the hippocampus and cortex,and anti-inflammatory cytokines(IL-4,IL-10,and TGF-β)were significantly reduced;sE2 treatment further exacerbated microglia activation and pro-inflammatory phenotype above menopausal mice.3.High concentrations of E2 resulted in microglia activation and pro-inflammatory phenotype.In the in vitro assay,low concentration of E2(200 nM/L)could not activate microglia,but with the increase of E2 concentration,microglia were significantly activated and differentiated into pro-inflammatory microglia;after the addition of LPS,the resting microglia were differentiated into pro-inflammatory microglia(with the increase of the expression of Ibal and CD86,the increase of the expression of pro-inflammatory cytokines and the decrease of the expression of anti-inflammatory cytokines),and low concentration of E2 could further aggravate the activation and pro-inflammatory phenotype of the microglia above menopause.decreased),low concentration of E2 inhibited LPS-induced pro-inflammatory differentiation of microglia,while high concentration of E2(800 nM/L)significantly promoted pro-inflammatory differentiation of microglia.4.High concentration of E2 activated microglia through ERa.In an in vitro assay,after knockdown of estrogen receptors(ERa,ERB,and GPER)in microglia using siRNA,only microglia lacking ERa were not activated by high concentration of E2.5.High concentration of E2 activated microglia through NF-κB signalling pathway.The results of proteomics showed that proteins related to NF-κB signalling pathway and neuroinflammation were highly enriched in the brains of sE2-treated menopausal mice compared with the OVX group;in in vitro assays,high concentrations of E2 significantly activated the NF-κB signalling pathway in microglia.With the addition of NF-κB inhibitors,microglia could not be activated by high concentrations of E2,and the associated pro-inflammatory phenotype was suppressed.6.sE2 treatment exacerbated brain neuronal damage in menopausal mice.The results of HE and Nysted staining showed that compared with the Sham-operated group,the number of neurons in the cerebral cortex and hippocampal region of mice in the OVX group was significantly reduced,and the number of damaged neurons was significantly increased;the sE2 treatment further exacerbated the damaged cerebellar neuron phenotype of the above cerebellar neurons in menopausal mice.7.High concentration of E2 induces neuronal damage by activating microglial cellsIn in vitro assays,different concentrations of E2(200-1600 nM/L)significantly increased the cell activity of the neuronal cell line HT22,but the high concentration(3200 nM/L)was ineffective;for primary neuronal cells,different concentrations of E2(200-1600 nM/L)had no significant effect on the cell activity,but the high concentration(3200 nM/L)of E2 decreased the cell activity;E2 elevated the cell activity of BV2 and primary microglia at different concentrations and duration of action;under the co-culture conditions of neurons and microglia,the addition of E2(800 nM/L)significantly reduced the cell activity of neurons;the results of tissue immunofluorescence showed that the brain microglia of menopausal mice treated with sE2 released more pro-neuronal death cytokines The results of tissue immunofluorescence showed that sE2-treated microglia in menopausal mice released more pro-neuronal death cytokines.8.sE2 supplementation causes brain glycerophospholipid metabolism disorder and retrograde endocannabinoid signaling inhibition in climacteric mice.Compared with OVX group,the expression of COX1 in the cerebral cortex and hippocampal neurons of menopausal mice treated with sE2 was significantly increased.The results of untargeted metabolomics analysis showed that sE2-treated menopausal mice developed severe metabolic disorders in the brain,including abnormal glycerophospholipid metabolism and retrograde endocannabinoid signaling pathway;In vitro,CB1 was significantly inhibited by high concentration of E2.CB1 expression was significantly reduced in the cerebral cortex and hippocampus of sE2-treated mice.ConclusionsE2 can aggravate the depression-like behavior of postmenopausal mice,and its mechanism may be related to the promotion of proinflammatory differentiation of microglia by ERa/NF-κB signaling pathway,disorder of glycerophospholipid metabolism in neurons,and inhibition of retrograde endocannabinoid signaling.This study sheds light on the deleterious effects and underlying mechanisms of sE2 on the brain in women during menopause.更多还原