孕期慢性应激对子代雌性大鼠抑郁样行为及印记基因IGF-2/H19甲基化的影响

Effects of chronic stress during pregnancy on depression-like behavior and methylation of imprinted gene IGF-2/H19 in female offspring rats

目的探究孕期慢性应激对子代雌性大鼠抑郁行为及海马组织印记基因胰岛素样生长因子-2(insulin-like growth factor-2,IGF-2)/长链非编码RNA(lncRNA)H19甲基化的影响。方法32只SPF级雌性SD大鼠按照随机数字表法分为模型组和对照组,模型组大鼠采用慢性不可预知性温和应激(chronic unpredictable mild stress model,CUMS)方法建立抑郁模型,对照组正常饲养。应激刺激第7天时,雌雄鼠合笼交配。在应激刺激前1 d和应激刺激第1、7、14、21、28天对两组母鼠行内眦静脉采血,测定血浆皮质酮浓度;在雌性仔鼠出生后第28天和出生后第42天时,每组随机抽取8只,行内眦静脉采血后测定血浆皮质酮浓度;在出生后第42天时,分别通过糖水偏爱实验、悬尾实验和强迫游泳实验测定两组雌性仔鼠的抑郁样行为。采用RT-PCR和Western blot法检测仔鼠海马组织中IGF-2/H19及相关转移酶表达情况。利用Methyl Target技术,对IGF-2差异性甲基化区域(differentially methylated region,DMR)片段2的19个CpG位点,H19印记控制区(imprinting control region,ICR)片段1中8个CpG位点和H19-ICR片段2的15个CpG位点,同时捕获测序,计算每个CpG位点甲基化水平。采用SPSS 26.0,采用重复测量方差分析、t检验和非参数检验对相关数据进行统计分析。结果(1)重复测量方差分析结果显示,母鼠血浆皮质酮的时间与组别的交互效应不显著(F=2.997,P=0.066),时间主效应显著(F=4.44,P=0.010),组别主效应显著(F=41.40,P=0.001)。组间因素的单独效应分析,在应激第14、21、28天,模型组母鼠血浆皮质酮浓度均高于对照组母鼠(均P<0.001)。(2)在糖水偏爱实验中,模型组雌性仔鼠的液体总消耗[11.10(10.38,11.58)mL,13.55(12.00,15.77)mL,Z=-3.055,P=0.002]、1%蔗糖水消耗[(5.50±1.30)mL,(8.56±2.04)mL,t=-3.582,P=0.003]及1%蔗糖偏爱百分比[(51.35±8.69)%,(62.11±8.05)%,t=-2.576,P=0.022]均低于对照组。(3)模型组雌性仔鼠悬尾实验中静止持续时间[(126.95±39.89)s,(54.30±25.00)s,t=4.375,P=0.001]和强迫游泳实验中持续不动时间[(7.97±6.66)s,(1.85±2.12)s,t=2.478,P=0.037]均长于对照组。(4)模型组雌性仔鼠海马组织IGF-2 mRNA[(0.46±0.24),(1.00±0.00),t=3.821,P=0.019]、H19 mRNA[(0.60±0.25),(1.00±0.00),t=3.574,P=0.007]表达均低于对照组;模型组雌性仔鼠IGF-2蛋白相对表达低于对照组[(0.77±0.04),(1.00±0.00);t=9.876,P=0.01);CCTC结合因子(CCTC-binding factor,CTCF)的mRNA[(1.29±0.12),(1.00±0.00),t=-4.850,P=0.003]和蛋白相对表达水平[(1.90±0.28),(1.00±0.00),t=-5.513,P=0.005]均高于对照组。(5)模型组雌性仔鼠海马组织IGF-2 DMR区域3个CpG位点甲基化水平均低于对照组(t=-3.21,-3.00,-3.34,均P<0.05),分别位于chr1215831028、chr1215831055和chr1215831205;IGF-2 DMR片段甲基化水平低于对照组(t=-3.453,P=0.048);模型组雌性仔鼠甲基转移酶DNMT3A mRNA(t=5.102,P=0.002)、DNMT3A(t=10.213,P<0.001)和DNMT3B(t=4.169,P=0.014)蛋白相对表达水平均低于对照组。结论孕期慢性应激致雌性仔鼠出现抑郁、绝望状态,其机制可能与甲基化转移酶表达降低引起印记基因IGF-2 DMR甲基化水平降低有关。

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.