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

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