【作者】 王芳；

【导师】 邓启红；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2025， 硕士

【摘要】 全球气候变暖加剧孕期高温暴露风险,其子代神经发育毒性机制亟待阐明。尽管研究表明产前不良环境应激可诱发子代焦虑样行为,但子代受孕期高温影响的跨代路径、孕期高温暴露敏感窗口期尚未明确。目的本研究旨在阐明孕期高温暴露对子代焦虑行为的影响及其分子机制,聚焦不同妊娠阶段(孕早、中、晚期及全孕期)的差异效应,为环境应激致神经发育障碍的预防与干预提供理论依据。方法将SPF级SD雌性大鼠随机分为5组,每组16只,合笼受孕成功后分为正常饲养(control,CON组)和实验组:孕早期高温暴露组(early-stage heat exposure in pregnancy,EHP组)、孕中期高温暴露组(middle-stage heat exposure in pregnancy,MHP组)、孕晚期高温暴露组(late-stage heat exposure in pregnancy,LHP组)和全孕期高温暴露组(whole-stage heat exposure in pregnancy,WHP组)。各实验组分别于孕早、中、晚期和全孕期将母鼠暴露于35℃高温环境8h。每组各取8只于分娩前取胎盘和胚胎,记录每窝胎数和平均重量。苏木精-伊红染色法(Hematoxylin-Eosin Staining,HE)观察胎盘迷路区形态,采用免疫组织化学技术(Immunohistochemistry,IHC)对胎盘中的白细胞介素-1β(Interleukin-1β,IL-1β)、肿瘤坏死因子-α(Tumor Necrosis Factor-α,TNF-α)和白细胞介素-6(Interleukin-6,IL-6)进行半定量和定位分析。HE染色和Nissl染色用于观察胚胎脑组织前额叶皮质(prefrontal cortex,PFC)的神经细胞形态和发育情况,免疫荧光Iba-1标记胚胎PFC小胶质细胞观察活化状态。另外8只孕鼠用于繁殖子代。子代4周龄时,通过旷场实验、高架十字迷宫和强迫游泳实验评估焦虑样行为。眼球采血取上清,取子代脑组织PFC部位进行病理学检测:HE染色观察神经元形态,Nissl染色评估尼氏体完整性,免疫荧光检测Iba-1标记的小胶质细胞活化状态和突触素(Synaptophysin,SYP)的表达水平,透射电镜观察神经细胞和突触超微结构。取PFC进行转录组测序,筛选差异表达基因并进行KEGG富集,筛选相关信号通路。采用酶联免疫吸附试验(Enzyme-Linked Immunosorbent Assay,ELISA)检测血清和PFC中IL-1β、TNF-α和IL-6的水平。采用Western Blot检测丝裂原活化蛋白激酶/细胞外信号调节激酶(Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase,MAPK/ERK)的磷酸化状态(p-ERK Thr202/Tyr204)、磷脂酰肌醇3激酶-蛋白激酶B(Phosphatidylinositol 3-Kinase-Protein Kinase B,PI3K-AKT)的磷酸化水平(p-AKT Ser473)、脑源性神经营养因子(Brain-Derived Neurotrophic Factor,BDNF)及其受体酪氨酸激酶受体B(Tyrosine Kinase Receptor B,Trk B)的磷酸化状态p-Trk B(Tyr706/707),以及c AMP反应元件结合蛋白(c AMP Response Element-Binding Protein,CREB)的磷酸化状态p-CREB(Ser133)的蛋白表达。最后将胎盘炎性指标与胚胎Iba-1、子代BDNF、SYP表达水平及行为学指标进行相关性分析。数据以Mean±SD表示,Graph Pad Prism 9.3.0进行单因素方差分析(ANOVA)及统计图绘制,显著性阈值设为P<0.05。Image J 4.0软件对蛋白条带灰度值以及荧光强度进行分析。结果1.EHP组和WHP组胎鼠显著减少(P<0.05),胎盘、胎儿平均重量和子代雌雄比例无显著差异。EHP组胎盘迷路区面积占比显著小于CON组(P<0.05)。胎盘HE染色显示,高温暴露后迷路区结构紊乱,滋养层细胞核形态不规则,母体血窦狭小,血管间膜增厚。IHC结果显示,与对照组相比,WHP组IL-1β、TNF-α和IL-6表达水平均升高(P<0.05),MHP组IL-1β和TNF-α表达水平均升高(P<0.05),LHP组IL-6表达水平升高(P<0.05)。胚胎脑组织皮质板神经细胞分布稀疏或堆积,细胞核形态不规则,出现核固缩,细胞质深染,可见空泡化。神经元胞质内可见尼氏体颗粒减少或消失,胞体形态不规则,染色变浅。孕期各阶段高温暴露增加了PFC的小胶质细胞Iba-1的荧光强度(P<0.05)。2.子代经孕期高温暴露后显示出不同程度的焦虑样行为,具体表现在:旷场实验中,LHP组和WHP组的大鼠运动总路程显著性减少(P<0.01);EHP组、LHP组和WHP组的大鼠的中央区域路程显著性减少(P<0.01);LHP组和WHP组的大鼠进入中央区域次数显著少于CON组(P<0.01)。高架十字迷宫实验中,MHP组和WHP组大鼠进入开臂次数显著性减少(P<0.05);EHP组、MHP组、LHP组和WHP组的大鼠进入开臂的次数均显著少于对照组(P<0.05)。强迫游泳实验中,MHP组和WHP组大鼠在水中不动时间明显增多。WHP组的子代血清和脑组织PFC中的IL-1β、TNF-α和IL-6均显著高于CON组(P<0.05)。孕早、中、晚期高温暴露不同程度地增加了炎性因子水平,总体来说,MHP组血清中炎性因子水平较EHP组和LHP组增加更显著,而PFC中未见明显此现象。高温暴露后,各实验组均显示出不同程度神经细胞分布稀疏,细胞核形态不规则,边界模糊,出现核固缩,尼氏体颗粒减少或消失,胞体形态不规则,染色变浅,还可见细胞核偏位和核皱缩。EHP组、MHP组和WHP组的Iba-1标记的小胶质细胞荧光强度显著增强(P<0.05)。电镜下可见神经细胞核形态不规则,核膜边界模糊不清,线粒体结构异常,突触囊泡数量减少,突触后致密区缩小。且孕期各阶段高温暴露后SYP表达水平均显著性降低(P<0.05)。3.高温暴露后,子代脑组织PFC转录组测序显示,与CON组相比,EHP组、MHP组、LHP组和WHP组均有基因差异表达,差异基因KEGG富集到两条共有通路:MAPK与PI3K-AKT信号通路。子代脑组织PFC中MAPK/ERK(p-ERK)、PI3K和p-AKT蛋白表达水平均显著下降(P均<0.05),WHP组下降最多,其次为MHP组。上下游调节因子BDNF、p-Trk B、p-CREB蛋白表达水平均显著下降(P均<0.05),WHP组下降最多,其次为MHP组。将胎盘炎症指标与胚胎Iba-1、子代BDNF、SYP表达水平及行为学指标进行相关性分析,结果提示上述指标的表达水平均与胎盘炎症因子表达显著相关。以上结果表明孕期高温暴露可通过胎盘炎症,引起子代小胶质细胞活化以及BDNF/Trk B轴的抑制,进而抑制MAPK和PI3K/AKT通路和下游转录因子CREB的激活,从而削弱突触可塑性,引起子代大鼠表现出焦虑样行为。结论孕期高温暴露可通过“胎盘炎症→子代脑小胶质细胞活化与BDNF/Trk B-CREB轴抑制→MAPK/PI3K-AKT通路活性降低→突触损伤”的级联机制,显著增加子代大鼠焦虑样行为风险,并提示孕中期可能是孕期高温暴露引起子代焦虑样行为的敏感窗口期。更多还原

【Abstract】 The exacerbation of global climate warming has heightened the risk of high-temperature exposure during pregnancy.The mechanisms underlying the neurodevelopmental toxicity in offspring are yet to be fully elucidated.Although evidence indicates that prenatal adverse environmental stress can induce anxiety-like behaviors in offspring,the transgenerational pathways influenced by high-temperature exposure during pregnancy and the sensitive window period of such exposure remain to be clarified.ObjectiveThis study aimed to investigate the impact of prenatal heat exposure on offspring anxiety-like behaviors and its molecular mechanisms,with a focus on differential effects across gestational stages(early,middle,late,and whole pregnancy).The findings provide a theoretical basis for preventing and intervening in neurodevelopmental disorders induced by environmental stressors.MethodsSpecific pathogen-free(SPF)Sprague-Dawley(SD)female rats were randomly divided into five groups(n=16/group):a control group(CON)and experimental groups exposed to 35°C for 8 hours during specific gestational windows—early-stage heat exposure(EHP),middle-stage(MHP),late-stage(LHP),and whole-stage(WHP).Placental and embryonic tissues were collected from 8 dams per group pre-delivery for analysis.Hematoxylin-Eosin(HE)staining evaluated placental labyrinth morphology,while immunohistochemistry(IHC)semi-quantified placental interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6).Embryonic prefrontal cortex(PFC)neuronal morphology and microglial activation(Iba-1 immunofluorescence)were assessed.The remaining 8 dams per group delivered offspring subjected to behavioral tests(open field,elevated plus maze,forced swim)at 4 weeks.The PFC region of the offspring’s brain was harvested for pathological examination:HE staining was used to observe neuronal morphology,Nissl staining to evaluate the integrity of Nissl bodies,and immunofluorescence to detect the activation state of microglia labeled with Iba-1and the expression levels of Synaptophysin(SYP).Transmission electron microscopy was employed to observe the ultrastructure of neurons and synapses.ELISA was employed to measure the levels of IL-1β,TNF-α,and IL-6 in serum and the prefrontal cortex(PFC).The PFC was harvested for transcriptome sequencing to identify differentially expressed genes,which were then subjected to KEGG enrichment analysis to screen for relevant signaling pathways.Western Blot was used to detect the phosphorylation status of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase(MAPK/ERK)(p-ERK Thr202/Tyr204),Phosphatidylinositol 3-Kinase-Protein Kinase B(PI3K-AKT)(p-AKT Ser473),Brain-Derived Neurotrophic Factor(BDNF),its receptor Tyrosine Kinase Receptor B(Trk B)(p-Trk B Tyr706/707),and c AMP Response Element-Binding Protein(CREB)(p-CREB Ser133)in the PFC.Finally,the placental inflammatory indicators were correlated with the expression levels of Iba-1 in the embryo,BDNF and SYP in the offspring,as well as behavioral indices.Data are presented as Mean±SD.Graph Pad Prism 9.3.0 was used for one-way ANOVA and statistical graph plotting,with a significance threshold set at P<0.05.Image J 4.0 software was used to analyze the gray values of protein bands and fluorescence intensity.Results1.The number of fetuses in the EHP and WHP groups was significantly reduced(P<0.05),while there were no significant differences in the average weight of placentas and fetuses or the sex ratio of offspring.The area proportion of the placental labyrinth zone in the EHP group was significantly smaller than that in the CON group(P<0.05).Hematoxylin-Eosin(HE)staining of the placenta revealed disordered labyrinth zone structure,irregular trophoblast nuclei,narrow maternal blood sinuses,and thickened intervascular membranes following heat exposure.Immunohistochemistry(IHC)results showed that compared to the control group,the expression levels of IL-1β,TNF-α,and IL-6 were elevated in the WHP group(P<0.05),IL-1βand TNF-αwere elevated in the MHP group(P<0.05),and IL-6 was elevated in the LHP group(P<0.05).In the embryonic brain tissue,neurons in the cortical plate were sparsely distributed or clustered,with irregular nuclei,condensed nuclei,deeply stained cytoplasm,and vacuolization.Nissl bodies in the neuronal cytoplasm were reduced or absent,and the cell bodies were irregularly shaped and lightly stained.Increased fluorescence intensity of Iba-1-marked microglia in the prefrontal cortex(PFC)was observed in all experimental groups(P<0.05).2.Offspring exposed to prenatal heat exposure exhibited varying degrees of anxiety-like behaviors.In the open-field test,the total distance in the LHP and WHP groups was significantly reduced(P<0.01),and the distance traveled in the central area was significantly reduced in the EHP,LHP,and WHP groups(P<0.01).The number of entries into the central area was significantly lower in the LHP and WHP groups compared to the CON group(P<0.01).In the elevated plus maze test,the number of entries into the open arms was significantly reduced in the MHP and WHP groups(P<0.05),and all experimental groups had fewer entries into the open arms than the control group(P<0.05).In the forced swim test,the immobility time in water was significantly increased in the MHP and WHP groups.Serum and PFC levels of IL-1β,TNF-α,and IL-6 in the WHP group were significantly higher than those in the CON group(P<0.05).Prenatal heat exposure at different stages of pregnancy differentially increased inflammatory cytokine levels,with the MHP group showing more significant increases in serum cytokines compared to the EHP and LHP groups,while no such pattern was observed in the PFC.Histological examination revealed sparse neuronal distribution,irregular nuclear morphology,blurred boundaries,condensed nuclei,reduced or absent Nissl bodies,irregular cell body shape,and lighter staining in the PFC.Nuclear displacement and nuclear wrinkling were also observed.Increased fluorescence intensity of Iba-1-marked microglia was observed in the EHP,MHP,and WHP groups(P<0.05).Electron microscopy revealed irregular nuclear morphology,blurred nuclear membranes,abnormal mitochondrial structure,reduced synaptic vesicles,and narrowed postsynaptic density zones in neurons.After exposure to high temperatures during various stages of pregnancy,the expression levels of SYP were significantly decreased(P<0.05).3.Transcriptome sequencing of the PFC in offspring’s brain tissue showed differential gene expression in all experimental groups compared to the CON group,with KEGG enrichment in two common pathways:MAPK and PI3K-Akt signaling pathways.Protein expression levels of MAPK/ERK(p-ERK),PI3K,and p-AKT in the PFC were significantly decreased in all experimental groups(P<0.05),with the most significant reduction in the WHP group,followed by the MHP group.The expression levels of upstream and downstream regulatory factors BDNF,p-Trk B,and p-CREB were also significantly decreased(P<0.05),with the most significant reduction in the WHP group,followed by the MHP group.Correlation analyses were performed between placental inflammatory markers and the expression levels of embryonic Iba-1,offspring BDNF,and SYP,as well as behavioral indices.The results indicated a significant correlation between the expression levels of these indices and placental inflammatory factors.These findings suggested that prenatal heat exposure can induce anxiety-like behaviors in offspring rats through placental inflammation,which in turn activates microglia(as indicated by Iba-1 expression)and suppresses the BDNF/Trk B axis.This suppression further inhibits the activation of downstream signaling pathways,including MAPK and PI3K/AKT,and the transcription factor CREB,thereby compromising synaptic plasticity.Consequently,this leads to the manifestation of anxiety-like behaviors in the offspring rats.ConclusionPrenatal heat exposure significantly increases the risk of anxiety-like behaviors in offspring through a cascade mechanism involving“placental inflammation→activation of microglia in the offspring’s brain and inhibition of the BDNF/Trk B-CREB axis→reduced activity of the MAPK/PI3K-AKT pathways→synaptic plasticity damage.”Additionally,these findings suggested that the mid-gestation period may be a sensitive window for prenatal heat exposure to induce anxiety-like behaviors in offspring.更多还原