妊娠期糖尿病对C57BL/6J子代成年鼠神经精神功能的影响

doi:10.3969/j.issn.1674-8115.2022.04.004

摘要/Abstract

摘要：

目的·探究妊娠期糖尿病（gestational diabetes mellitus，GDM）对C57BL/6J子代成年鼠神经精神功能的影响。方法·将8周龄C57BL/6J小鼠随机分为GDM组和对照组，采用高脂饮食（high fat diet，HFD）诱导GDM模型，通过检测孕鼠空腹血糖、口服糖耐量实验（oral glucose tolerance test，OGTT）及胰岛素耐量实验（insulin tolerance test，ITT）指标验证模型是否诱导成功。子代正常饮食喂养至18周，进行开放旷场实验（open field test，OFT）、高架十字迷宫实验（elevated plus maze test，EPMT）、高架零迷宫实验（elevated zero maze test，EZMT）、强迫游泳实验（forced swimming test，FST）、悬尾实验（tail suspension test，TST）、糖水偏好实验（sucrose preference test，SPT）检测子代情绪行为。收集小鼠海马组织进行组织学验证。利用苏木精-伊红（hematoxylin-eosin，H-E）染色、镀银染色明确GDM组子代海马组织结构形态，免疫荧光染色检测神经元及星形胶质细胞标志阳性的细胞数量。利用实时定量PCR（real time quantitative PCR，RT-qPCR）检测海马区域多巴胺（dopamine，DA）、5-羟色胺（5-hydroxytryptamine，5-HT）、脑源性神经因子（brain-derived neural factor，BDNF）及cAMP反应元件结合蛋白（cAMP response element-binding protein，CREB）相关基因（Drd1、Htr2a、Bdnf、Creb1）的表达。多组间比较采用单因素方差分析，2组间差异采用独立样本t检验进行分析。结果·HFD诱导的C57BL/6J小鼠GDM模型表现为OGTT各时间点血糖值、曲线下面积（area under curve，AUC）明显升高，胰岛素耐量明显降低，证实GDM模型成功建立。子代OFT、EPMT、EZMT结果显示GDM组与对照组差异无统计学意义；FST、TST及SPT结果显示GDM组不动时间明显上升、糖水偏好百分比明显降低，且F1代雌性小鼠差异更为显著（P=0.000）。RT-qPCR结果显示，与对照组相比，GDM组Drd1、Htr2a、Bdnf表达量下降。H-E及镀银染色结果分析发现，GDM组海马组织结构没有明显变化，但免疫荧光结果提示GDM组神经元、星形胶质细胞数目下降。结论·GDM与C57BL/6J子代成年鼠神经精神障碍相关，其主要表现为子代成年期抑郁症倾向，不表现为焦虑症倾向。

关键词: 妊娠期糖尿病, 子代, 神经系统疾病, 情绪障碍, 抑郁症, 焦虑症

Abstract:

Objective·To investigate the influence of gestational diabetes mellitus (GDM) on the neuropsychiatric function of C57BL/6J adult offspring mice.

Methods·C57BL/6J mice aged 8 weeks were randomly divided into the GDM group and the control group. GDM model was induced by a high-fat diet (HFD). Fasting glucose, oral glucose tolerance test (OGTT), and insulin tolerance test (ITT) of the pregnant mice were tested to verify whether the model was successfully induced. The offspring were fed with a standard diet for 18 weeks, and then underwent open field test (OFT), elevated plus maze (EPMT), elevated zero maze test (EZMT), forced swimming test (FST), tail suspension test (TST), and sucrose preference test (SPT) to test the emotional behaviors of the offspring mice. The hippocampus was collected after the behavioral experiments for histological verification. Hematoxylin-eosin (H-E) staining and silver staining were used to determine the structure and morphology of the offspring hippocampus of the GDM group, and immunofluorescence staining was used to detect the number of neuron marker and astrocyte marker positive cells. Real time quantitative PCR (RT-qPCR) was used to detect the expression of dopamine (DA), 5-hydroxytryptamine (5-HT), and brain-derived neural factor (BDNF), cAMP response element-binding protein (CREB) relative genes (Drd1, Htr2a, Bdnf and Creb1) in the hippocampus. One-way ANOVA was used for comparison among multiple groups, and independent-sample t-test was used to analyze differences between two groups.

Results·The GDM model induced by HFD in C57BL/6J mice showed that blood glucose levels and AUC were significantly increased at each time point of OGTT, and insulin tolerance was decreased, confirming that the GDM model was successfully established. The results of OFT, EPMT and EZMT in the progeny showed no statistical difference between the GDM group and the control group. The FST, TST and SPT displayed that the immobility time was observably increased, and the percentage of sucrose preference was markedly reduced in the GDM group, and the difference in F1 female mice was more significant (P=0.000). The expression of Drd1, Htr2a and Bdnf was reduced in the GDM group compared with the control group. Analysis of H-E and silver staining results showed that there was no difference in the structure of the hippocampus between the two groups, but the number of neurons and astrocytes decreased in the GDM group.

Conclusion·GDM is associated with neuropsychiatric disorders in adult offspring of C57BL/6J mice, which mainly manifest as depression tendency instead of anxiety tendency.

Key words: gestational diabetes mellitus, offspring, neurological disorders, mood disorders, depressive disorders, anxiety disorders

中图分类号:

R394.3

吴侠霏, 方婕, 漆洪波, 余昕烊. 妊娠期糖尿病对C57BL/6J子代成年鼠神经精神功能的影响[J]. 上海交通大学学报（医学版）, 2022, 42(4): 422-432.

WU Xiafei, FANG Jie, QI Hongbo, YU Xinyang. Neuropsychiatric effects of gestational diabetes mellitus in adult offspring in C57BL/6J mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(4): 422-432.

图/表 6

表1 RT-qPCR引物（5′→3′）

Tab 1 Primer sequence for RT-qPCR（5′→3′）

Gene	Forward primer	Reverse primer
β-actin	CACTGTCGAGTCGCGTCC	TCATCCATGGCGAACTGGTG
Drd1	AGGTTGAGCAGGACATACGC	TTGCTTCTGGGCAATCCTGT
Htr2a	CTGATTCCTCTCTGTGCGCT	TCCAGCACGGTTGAAGTCTG
Bdnf	TAAACGTCCACGGACAAGGC	TCGTCAGACCTCTCGAACCT
Creb1	GAAGAGACTTCTGCCCTCCG	GAAGAGACTTCTGCCCTCCC

图1 高脂饮食诱导GDM模型Note： A. Experimental flow graph. B?D. E0.5d maternal weight (B), pup weight (C) and litter size (D) in the high-fat diet (HFD) and low-fat diet (LFD) feeding groups. E. Fasting blood glucose levels at 0.5, 11.5, and 16.5 days of gestation between groups. F?H. Comparison of blood glucose at each time point of OGTT and AUC between the HFD group and the LFD group at E0.5d (F), E11.5d (G), and E16.5d (H). I?K. Comparison of blood glucose at each time point of ITT and AUC between the HFD group and the LFD group at E0.5d (I), E11.5d (J), and E16.5d(K). n=6. ①P=0.015, ②P=0.042, ③P=0.000, ④P=0.004, ⑤P=0.009, ⑥P=0.000, ⑦P=0.007, ⑧P=0.003,⑨P=0.033, ⑩P=0.002, ?P=0.010, ?P=0.026, ?P=0.006, vs the LFD group.

Fig 1 High fat diet induced GDM model

图2 GDM与成年子代鼠焦虑症无关Note： A?C. Open field test. The number of offspring entering the central region (A) and surrounding region (B) in the GDM and control groups (Ctl♀, n=12; Ctl♂, n=12; GDM♀, n=11; GDM♂, n=12). Thermal images of each group (C). D?F. The elevated plus maze test. The locomotor activity (D) and proportion of offspring mice entering open arms (E) in the GDM and control groups (Ctl♀, n=11; Ctl♂, n=11; GDM♀, n=10; GDM♂, n=11). Thermal images of each group (F). G?I. The elevated zero maze test. The locomotor activity (G) and proportion of offspring mice entering open arms (H) in the GDM and control groups (Ctl♀, n=11; Ctl♂, n=12; GDM♀, n=10; GDM♂, n=12). Thermal images of each group (I).

Fig 2 GDM not associated with anxiety disorders in adult offspring mice

图3 GDM与成年子代鼠抑郁症相关Note： A/B. Forced swimming test. Comparison of immobility time (A) and swimming time (B) of offspring mice in the GDM and control groups (Ctl♀, n=12; Ctl♂, n=11; GDM♀, n=7; GDM♂, n=12). C. Tail suspension test. Comparison of immobility time of offspring mice in the GDM and control groups (Ctl♀, n=11; Ctl♂, n=9; GDM♀, n=10; GDM♂, n=11). D. Sucrose preference test. Comparison of preference of 1% sucrose of offspring mice in the GDM and control groups (Ctl♀, n=12; Ctl♂, n=12; GDM♀, n=11; GDM♂, n=12). E?H. The relative expression of Drd1 (E), Htr2α(F), Bdnf(G), and Creb1(H) in the hippocampi of offspring female mice in the two groups (n=6). ①P=0.043, ②P=0.006, ③P=0.031, ④P=0.048, ⑤P=0.041, ⑥P=0.000, ⑦P=0.000, ⑧P=0.001,⑨P=0.020,⑩P=0.002,?P=0.003, vs the control group.

Fig 3 GDM associated with depression disorders of adult offspring mice

图4 GDM不改变子代小鼠海马组织结构Note： A/B. Images of murine hippocampus tissues stained with H-E staining (A) and silver staining (B) (scale bar=100 μm, 50 μm, respectively). The arrows indicating the enlarged areas in the right pictures.

Fig 4 GDM not affecting the hippocampal structure of offspring mice

图5 GDM影响小鼠海马神经元发生Note： A/C. Representative immunofluorescent staining images of NeuN (A) and GFAP (C) positive cells in the hippocampi of the two groups. Scale bar=100 μm. B/D. Statistical analysis of the NeuN positive (B) or GFAP positive (D) cells in the hippocampus regions (n=3). ①P=0.005, ②P=0.030, vs the control group.

Fig 5 GDM affecting hippocampal neurongenesis

参考文献 37

1	魏玉梅, 杨慧霞. 我国妊娠期糖尿病研究的发展与展望[J]. 中华围产医学杂志, 2018, 21(4): 218-220.
	WEI Y M, YANG H X. Development and prospect of research on gestational diabetes mellitus in China[J]. Chin J Perinat Med, 2018, 21(4): 218-220.
2	BOLTON J L, BILBO S D. Developmental programming of brain and behavior by perinatal diet: focus on inflammatory mechanisms[J]. Dialogues Clin Neurosci, 2014, 16(3): 307-320.
3	SOUSA R, TORRES Y S, FIGUEIREDO C P, et al. Consequences of gestational diabetes to the brain and behavior of the offspring[J]. An Acad Bras Cienc, 2018, 90(2 suppl 1): 2279-2291.
4	GAWLIŃSKA K, GAWLIŃSKI D, KOROSTYŃSKI M, et al. Maternal dietary patterns are associated with susceptibility to a depressive-like phenotype in rat offspring[J]. Dev Cogn Neurosci, 2021, 47: 100879.
5	FARAHVAR S, WALFISCH A, SHEINER E. Gestational diabetes risk factors and long-term consequences for both mother and offspring: a literature review[J]. Expert Rev Endocrinol Metab, 2019, 14(1): 63-74.
6	NOMURA Y, MARKS D J, GROSSMAN B, et al. Exposure to gestational diabetes mellitus and low socioeconomic status: effects on neurocognitive development and risk of attention-deficit/hyperactivity disorder in offspring[J]. Arch Pediatr Adolesc Med, 2012, 166(4): 337-343.
7	王晓瑜, 朱岷, 程昕然. 妊娠糖尿病对子代神经发育的研究进展[J]. 四川医学, 2020, 41(10): 1083-1089.
	WANG X Y, ZHU M, CHENG X R. Research progress of gestational diabetes mellitus on offspring neurodevelopment[J]. Sichuan Med J, 2020, 41(10): 1083-1089.
8	VAN LIESHOUT R J, VORUGANTI L P. Diabetes mellitus during pregnancy and increased risk of schizophrenia in offspring: a review of the evidence and putative mechanisms[J]. J Psychiatry Neurosci, 2008, 33(5): 395-404.
9	VUONG B, ODERO G, ROZBACHER S, et al. Exposure to gestational diabetes mellitus induces neuroinflammation, derangement of hippocampal neurons, and cognitive changes in rat offspring[J]. J Neuroinflammation, 2017, 14(1): 80.
10	BORA E, HARRISON B J, DAVEY C G, et al. Meta-analysis of volumetric abnormalities in cortico-striatal-pallidal-thalamic circuits in major depressive disorder[J]. Psychol Med, 2012, 42(4): 671-681.
11	HAYASAKA S, NAKAMURA M, NODA Y, et al. Lateralized hippocampal volume increase following high-frequency left prefrontal repetitive transcranial magnetic stimulation in patients with major depression[J]. Psychiatry Clin Neurosci, 2017, 71(11): 747-758.
12	SEXTON C E, MACKAY C E, EBMEIER K P. A systematic review and meta-analysis of magnetic resonance imaging studies in late-life depression[J]. Am J Geriatr Psychiatry, 2013, 21(2): 184-195.
13	DABELEA D, KNOWLER W C, PETTITT D J. Effect of diabetes in pregnancy on offspring: follow-up research in the Pima Indians[J]. J Matern Fetal Med, 2000, 9(1): 83-88.
14	LAWLOR D A, LICHTENSTEIN P, LÅNGSTRÖM N. Association of maternal diabetes mellitus in pregnancy with offspring adiposity into early adulthood: sibling study in a prospective cohort of 280,866 men from 248,293 families[J]. Circulation, 2011, 123(3): 258-265.
15	CANNON M, JONES P B, MURRAY R M. Obstetric complications and schizophrenia: historical and meta-analytic review[J]. Am J Psychiatry, 2002, 159(7): 1080-1092.
16	PANARIELLO F, FANELLI G, FABBRI C, et al. Epigenetic basis of psychiatric disorders: a narrative review[J]. CNS Neurol Disord Drug Targets, 2022, 21(4): 302-315.
17	KONG L H, CHEN X X, GISSLER M, et al. Relationship of prenatal maternal obesity and diabetes to offspring neurodevelopmental and psychiatric disorders: a narrative review[J]. Int J Obes (Lond), 2020, 44(10): 1981-2000.
18	JING Y H, SONG Y F, YAO Y M, et al. Retardation of fetal dendritic development induced by gestational hyperglycemia is associated with brain insulin/IGF-I signals[J]. Int J Dev Neurosci, 2014, 37: 15-20.
19	RIVERA H M, CHRISTIANSEN K J, SULLIVAN E L. The role of maternal obesity in the risk of neuropsychiatric disorders[J]. Front Neurosci, 2015, 9: 194.
20	PIAZZA F V, SEGABINAZI E, DE MEIRELES A L F, et al. Severe uncontrolled maternal hyperglycemia induces microsomia and neurodevelopment delay accompanied by apoptosis, cellular survival, and neuroinflammatory deregulation in rat offspring hippocampus[J]. Cell Mol Neurobiol, 2019, 39(3): 401-414.
21	KONG L H, NORSTEDT G, SCHALLING M, et al. The risk of offspring psychiatric disorders in the setting of maternal obesity and diabetes[J]. Pediatrics, 2018, 142(3): e20180776.
22	ORNOY A. Growth and neurodevelopmental outcome of children born to mothers with pregestational and gestational diabetes[J]. Pediatr Endocrinol Rev, 2005, 3(2): 104-113.
23	XIANG A H, WANG X H, MARTINEZ MP, et al. Maternal gestational diabetes mellitus, type 1 diabetes, and type 2 diabetes during pregnancy and risk of ADHD in offspring[J]. Diabetes Care, 2018, 41(12): 2502-2508.
24	VAN LIESHOUT R J, VORUGANTI L P. Diabetes mellitus during pregnancy and increased risk of schizophrenia in offspring: a review of the evidence and putative mechanisms[J]. J Psychiatry Neurosci, 2008, 33(5): 395-404.
25	RAMANATHAN M, JAISWAL A K, BHATTACHARYA S K. Hyperglycaemia in pregnancy: effects on the offspring behaviour with special reference to anxiety paradigms[J]. Indian J Exp Biol, 2000, 38(3): 231-236.
26	PENNA E, PIZZELLA A, CIMMINO F, et al. Neurodevelopmental disorders: effect of high-fat diet on synaptic plasticity and mitochondrial functions[J]. Brain Sci, 2020, 10(11): 805.
27	HASEBE K, KENDIG M D, MORRIS M J. Mechanisms underlying the cognitive and behavioural effects of maternal obesity[J]. Nutrients, 2021, 13(1): 240.
28	KINNEY B A, RABE M B, JENSEN R A, et al. Maternal hyperglycemia leads to gender-dependent deficits in learning and memory in offspring[J]. Exp Biol Med (Maywood), 2003, 228(2): 152-159.
29	HUERTA-CERVANTES M, PEÑA-MONTES D J, MONTOYA-PÉREZ R, et al. Gestational diabetes triggers oxidative stress in hippocampus and cerebral cortex and cognitive behavior modifications in rat offspring: age- and sex-dependent effects[J]. Nutrients, 2020, 12(2): 376.
30	THION M S, LOW D, SILVIN A, et al. Microbiome influences prenatal and adult microglia in a sex-specific manner[J]. Cell, 2018, 172(3): 500-516.e16.
31	SALES V M, FERGUSON-SMITH A C, PATTI M E. Epigenetic mechanisms of transmission of metabolic disease across generations[J]. Cell Metab, 2017, 25(3): 559-571.
32	PINNEY S E, SIMMONS R A. Metabolic programming, epigenetics, and gestational diabetes mellitus[J]. Curr Diab Rep, 2012, 12(1): 67-74.
33	HUERTA-CERVANTES M, PEÑA-MONTES D J, LÓPEZ-VÁZQUEZ M Á, et al. Effects of gestational diabetes in cognitive behavior, oxidative stress and metabolism on the second-generation off-spring of rats[J]. Nutrients, 2021, 13(5): 1575.
34	ROOZENDAAL B, HAHN E L, NATHAN S V, et al. Glucocorticoid effects on memory retrieval require concurrent noradrenergic activity in the hippocampus and basolateral amygdala[J]. J Neurosci, 2004, 24(37): 8161-8169.
35	BORA E, FORNITO A, PANTELIS C, et al. Gray matter abnormalities in major depressive disorder: a meta-analysis of voxel based morphometry studies[J]. J Affect Disord, 2012, 138(1/2): 9-18.
36	MENARD C, PFAU M L, HODES G E, et al. Social stress induces neurovascular pathology promoting depression[J]. Nat Neurosci, 2017, 20(12): 1752-1760.
37	HARRISON P J. The neuropathology of primary mood disorder[J]. Brain, 2002, 125(Pt 7): 1428-1449.

编辑推荐 0

Metrics

阅读次数

全文

456

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	20	0	0	436

来源	本网站	其他网站

次数	323	133
比例	71%	29%

摘要

771

最新录用	在线预览	正式出版

0	0	771

来源	本网站	其他网站

次数	679	92
比例	88%	12%

[1]	谷沁, 夏英倩, 朱亦清, 马莉莹, 瞿蕾, 孙文广. 妊娠期糖尿病孕妇一日门诊饮食个体化指导对血糖控制、体质量增速和妊娠结局的影响分析[J]. 上海交通大学学报(医学版), 2022, 42(2): 185-191.
[2]	李欣, 范青. 机器学习在抑郁症患者面部特征研究中的应用进展[J]. 上海交通大学学报(医学版), 2022, 42(1): 124-129.
[3]	沈梦婷, 张选红, 钱禛颖, 李惠, 盛建华, 王继军, 唐莺莹. 精神分裂症与抑郁症失匹配负波异常的对照研究[J]. 上海交通大学学报(医学版), 2021, 41(8): 1041-1045.
[4]	吴丹, 葛莉萍. 妊娠期糖尿病患者基因DNA甲基化的研究进展[J]. 上海交通大学学报(医学版), 2021, 41(8): 1120-1124.
[5]	耿瑞杰, 姚琳, 黄欣欣, 禹顺英, 苑成梅, 洪武, 吕钦谕, 王庆中, 易正辉, 方贻儒. 基于加权基因共表达网络分析识别抑郁症的差异表达基因模块[J]. 上海交通大学学报(医学版), 2021, 41(6): 724-731.
[6]	秦宁馨, 赵文龙, 周志阳, 施炜慧, 吴琰婷, 黄荷凤. 冻融胚胎移植对子代的近、远期影响[J]. 上海交通大学学报(医学版), 2021, 41(5): 653-658.
[7]	刘庆, 蔡雯, 张瑞晴, 卢聪, 张佳荣, 徐先明. 妊娠期脂质耐量试验在妊娠期糖尿病风险预测中的价值[J]. 上海交通大学学报(医学版), 2021, 41(2): 210-216.
[8]	吴静, 李学义, 陈京红, 王泽剑. 抑郁模型小鼠海马中胆汁酸受体变化的研究[J]. 上海交通大学学报(医学版), 2021, 41(12): 1628-1634.
[9]	施波, 陈建民, 赵俊雄, 唐伟, 范卫星, 张程赪, 张晨. CREB1基因与抑郁症和双相Ⅱ型障碍的关联研究[J]. 上海交通大学学报(医学版), 2021, 41(10): 1303-1307.
[10]	袁瑞雪, 傅迎美, 禹顺英. 辅助性T细胞17和调节性T细胞在抑郁症中的作用机制研究进展[J]. 上海交通大学学报(医学版), 2021, 41(10): 1384-1388.
[11]	朱雅洁, 林慧, 陈茜, 赵奕然, 刘欣梅, 黄荷凤. 妊娠期糖尿病母亲所产新生儿脐带血皮质醇水平变化及其对子代健康的影响[J]. 上海交通大学学报(医学版), 2021, 41(1): 49-54.
[12]	张怡静1, 2，徐健1, 2. 妊娠期维生素A缺乏对子代神经发育影响的研究进展[J]. 上海交通大学学报(医学版), 2020, 40(9): 1277-1282.
[13]	林青青，仇剑崟. 抑郁症患者的社会和人际功能障碍[J]. 上海交通大学学报(医学版), 2020, 40(7): 985-989.
[14]	程佳月，李璞玉，顾秋梦，王佩，陈珏，刘强#，王振#. 强迫症患者的强迫症状在归因方式与抑郁症状间的中介作用[J]. 上海交通大学学报(医学版), 2020, 40(6): 785-790.
[15]	郭垒1，胡嫣然1, 2，亢清1，王钰萍1，王振1，陈涵1#，陈珏1#. 神经性厌食患者应激负性感受和体象关注的关系：抑郁症状的中介作用[J]. 上海交通大学学报(医学版), 2020, 40(6): 798-803.