Effects of chronic stress during pregnancy on depression-like behavior and methylation of imprinted gene IGF-2/H19 in female offspring rats / 中华行为医学与脑科学杂志

ABSTRACT

Objective:To investigate the effects of chronic stress during pregnancy on depressive behavior and DNA methylation of insulin-like growth factor-2 ( IGF-2 )/long non-coding RNA ( lncRNA ) H19 in hippocampus of female offspring rats.Methods:A total of 32 SPF female SD rats were divided into model group and control group according to the random number table. The rats in the model group were treated with chronic unpredictable mild stress (CUMS) to establish the depression model, and the rats in the control group were fed normally.On the 7th day of stress stimulation, all female rats mated with male rats. One day before stress stimulation and 1, 7, 14, 21, 28 days after stress stimulation, blood samples were collected from the inner canthus vein of the rats to determine the plasma corticosterone concentration. Eight female pups were randomly selected from each group on postnatal day 28(PND28) and postnatal day 42 (PND42). Plasma corticosterone concentration was measured after angular vein blood collection. At PND42, the depression-like behavior of female pups in the two groups was measured by sucrose preference test, tail suspension test and forced swimming test. The expression of IGF-2/H19 and related transferases in hippocampus of offspring rats was detected by RT-PCR and Western blot. Methyl target technology was used to capture and sequence 19 CpG sites of IGF-2 differentially methylated region(DMR) fragment 2, 8 CpG sites in H19 imprinting control region (ICR) fragment 1 and 15 CpG sites in H19-ICR fragment 2, and calculate the methylation level of each CpG site. SPSS 26.0 was used for statistical analysis of relevant data by repeated measurement ANOVA, t test and non-parametric test. Results:(1) The data of plasma corticosterone content of the two groups of female rats at different times were analyzed by repeated measurement variance.The results showed that the the interaction effect between time and group was not significant ( F=2.997, P=0.066), and the main effect of time was significant ( F=4.44, P=0.010). The main effect of group was significant ( F=41.40, P=0.001). According to the independent effect analysis of factors between groups, on the 14th, 21st, and 28th days of stress, the plasma corticosterone concentration of the model group was higher than that of the control group (all P<0.001). (2) In the sucrose preference test, the total liquid consumption (11.10(10.38, 11.58) mL, 13.55(12.00, 15.77) mL, Z=-3.055, P=0.002), 1% sucrose water consumption ((5.50±1.30) mL, (8.56±2.04) mL, t=-3.582, P=0.003) and 1% sucrose preference percentage ( (51.35±8.69) %, (62.11±8.05) %, t=-2.576, P=0.022) of female pups in the model group were significantly lower than those in the control group. (3) The duration of immobility in tail suspension test ((126.95±39.89) s, (54.30±25.00) s, t=4.375, P=0.001) and forced swimming test ((7.97±6.66) s, (1.85±2.12) s, t=2.478, P=0.037) of female offspring in the model group were longer than those in the control group. (4) The expression of IGF-2 mRNA ((0.46±0.24), (1.00±0.00), t=3.821, P=0.019) and H19 mRNA ((0.60±0.25), (1.00±0.00), t=3.574, P=0.007) in hippocampus of female pups in the model group were lower than those of control group. The relative expression of IGF-2 protein in female offspring of model group was lower than that in control group ((0.77±0.04), (1.00±0.00), t=9.876, P=0.01). The relative expression of CCTC-binding factor (CTCF) mRNA ((1.29±0.12), (1.00±0.00), t=-4.850, P=0.003) and protein ((1.90±0.28), (1.00±0.00), t=-5.513, P=0.005) were higher than those in the control group. (5) The methylation levels of three CpG sites in the IGF-2 DMR region of female offspring in the model group were lower than those in the control group ( t=-3.21, -3.00, -3.34, all P<0.05), located at chr1215831028, chr1215831055 and chr1215831205, respectively. The methylation level of IGF-2 DMR fragment was lower than that of the control group ( t=-3.453, P=0.048). The relative expression levels of DNMT3A mRNA ( t=5.102, P=0.002), DNMT3A ( t=10.213, P<0.001) and DNMT3B ( t=4.169, P=0.014) in female offspring of the model group were lower than those in the control group. Conclusion:Chronic stress during pregnancy causes depression and despair in female offspring mice, and the mechanism may be related to the decrease of methylation level of imprinted gene IGF-2 DMR caused by the decrease of methyltransferase expression.