生命早期6:2 Cl-PFESA暴露对子代小鼠海马AMPA受体基因表达的影响

Effects of early-life 6:2 Cl-PFESA exposure on hippocampal AMPA receptor gene expression in offspring mice

[背景]6:2氯代多氟醚基磺酸(6:2 Cl-PFESA)具有十分强大的生物蓄积性和胎盘屏障穿透力,还可穿过血脑屏障。然而,其生命早期暴露对子代产生神经发育毒性的机制尚为未知。[目的]通过建立6:2 Cl-PFESA暴露动物模型,探究生命早期6:2 Cl-PFESA暴露对仔鼠生长发育及海马α-氨基-3-羟基-5-甲基-4-异恶唑丙酸(AMPA)受体基因表达的影响。[方法]将30只昆明种孕鼠随机分为对照组以及2、10、50和250μg·L^(-1)6:2 Cl-PFESA暴露组,从受孕第1天开始以自由饮水方式暴露相应剂量的6:2 Cl-PFESA,至泌乳结束。仔鼠于出生后(PND)第21天断乳,通过自由饮水方式继续进行6:2 Cl-PFESA暴露。记录仔鼠出生体重和体长作为本次实验基础数据。各组仔鼠于PND7、PND21和PND35分别麻醉后处死,取脑组织并剥离海马,透射电镜观察神经元突触超微结构,实时荧光逆转录聚合酶链式反应法检测AMPA受体GluR1、GluR2和GluR3的基因表达变化情况。PND35仔鼠于处死前进行Morris水迷宫实验观察仔鼠空间学习记忆能力情况。[结果]10、50、250μg·L^(-1)6:2 Cl-PFESA暴露组仔鼠的出生体重和体长分别为(2.23±0.36)、(1.92±0.20)、(1.88±0.31)g和(33.73±0.98)、(32.91±1.30)、(32.52±2.07)mm,均低于对照组(2.78±0.35)g和(36.46±2.34)mm(P<0.05)。PND35仔鼠Morris水迷宫结果显示:定位航行实验第4天的250μg·L^(-1)6:2 Cl-PFESA暴露组、第5天的10、50以及250μg·L^(-1)6:2 Cl-PFESA暴露组仔鼠较比对照组逃避潜伏期延长(P<0.05);空间探索实验中,50和250μg·L^(-1)6:2 ClPFESA暴露组仔鼠的穿越平台次数、250μg·L^(-1)6:2 Cl-PFESA暴露组仔鼠的目标象限停留时间与对照组相比均减少(P<0.05)。经透射电镜观察发现:PND35的250μg·L^(-1)6:2 Cl-PFESA暴露组与对照组相比,仔鼠突触后致密物变薄、突触间隙增宽。不同发育阶段的250μg·L^(-1)6:2Cl-PFESA暴露组仔鼠海马中GluR1、GluR2和GluR3的mRNA表达水平与对照组相比均下降(P<0.05);除了PND7的2μg·L^(-1)6:2 Cl-PFESA暴露组以外,发育早期6:2 Cl-PFESA暴露可以对各阶段仔鼠海马中GluR1、GluR2和GluR3的mRNA表达水平产生不同程度的抑制作用(P<0.05)。其中,发育早期6:2 Cl-PFESA暴露使仔鼠海马GluR1和GluR2 mRNA的表达水平在PND7时下降最多;暴露组仔鼠海马中GluR3 mRNA的表达水平在PND21时呈现最大抑制效应;而暴露组仔鼠海马GluR1、GluR2和GluR3 mRNA的表达水平均在PND35下降最少。[结论]生命早期6:2 Cl-PFESA暴露会影响仔鼠的生长发育,改变仔鼠海马突触结构,降低其学习记忆能力,这可能与其在各发育阶段中对仔鼠海马AMPA受体亚基GluR1、GluR2和GluR3基因表达水平的抑制作用有关。

[Background]The compound 6:2 chlorinated polyfluorinated ether sulfonic acids(6:2 Cl-PFESA)has been demonstrated abilities of strong bioaccumulation and placental barrier penetration,and it can also cross the blood-brain barrier.However,the mechanism of its neurodevelopmental toxicity in offspring induced by early-life exposure is still unknown.[Objective]To explore effects of 6:2 Cl-PFESA on the growth and theα-amino-3-hydroxy-5-methylisoxazole-4-propionic(AMPA)receptor gene expression in the hippocampus of offspring mice by establishing a 6:2 Cl-PFESA exposure animal model.[Methods]Thirty Kunming pregnant mice were randomly divided into five groups:control group,and 2,10,50,and 250μg·L^(-1)6:2 Cl-PFESA exposure groups.The treatment groups were exposed to designed doses of 6:2 Cl-PFESA through drinking water from the first day of gestation until the end of lactation.The pups were weaned on postnatal day(PND)21,and continued to be exposed to 6:2 Cl-PFESA through drinking water.Birth weight and body length of the offspring were recorded.Offspring mice were anesthetized and sacrificed re-spectively on PND7,PND21,and PND35,then their hippocampus was peeled from harvested brain tissue.The ultrastructure of hippocampus was observed via transmission electron microscopy;and the expression of AMPA receptors GluR1,GluR2,and GluR3 in the hippocampus was evaluated by real-time reverse transcription polymerase chain reaction.The learning and memory ability of the PND35 mice was measured by Morris water maze test before they were sacrificed.[Results]The birth weights and the lengths of the pups in the 10,50,and 250μg·L^(-1)6:2 Cl-PFESA exposure groups were(2.23±0.36),(1.92±0.20),(1.88±0.31)g,and(33.73±0.98),(32.91±1.30),(32.52±2.07)mm,respectively,which were lower than those in the control group,(2.78±0.35)g and(36.46±2.34)mm(P<0.05),respectively.The results of Morris water maze showed that the escape latencies in the orientation navigation experiment on the 4th day in the 250μg·L^(-1)6:2 Cl-PFESA exposure group and on the 5th day in the 10,50,and 250μg·L^(-1)6:2 Cl-PFESA exposure groups were longer than those in the control group(P<0.05).In the space exploration experiment,the times of crossing platform in the 50 and 250μg·L^(-1)6:2 Cl-PFESA exposure groups were decreased when compared with the control group(P<0.05),and the time of staying in the target quadrant of the 250μg·L^(-1)6:2 Cl-PFESA exposure groups were also decreased(P<0.05).Via transmission electron microscopy,compared with the control group,the postsynaptic density was decreased and the synaptic cleft width was widened on PND35 in the 250μg·L^(-1)6:2 Cl-PFESA exposure group.The mRNA expression levels of GluR1,GluR2,and GluR3 in the hippocampus of pups exposed to 250μg·L^(-1)6:2 Cl-PFESA during different developmental stages were significantly lower than those in the control group(P<0.05).Except for the 2μg·L^(-1)6:2 Cl-PFESA exposure group on PND7,the 6:2 Cl-PFESA exposure inhibited the mRNA expression levels of GluR1,GluR2,and GluR3 in the hippocampus of pups at different developmental stages(P<0.05).Among them,the 6:2 Cl-PFESA exposure during early development resulted in the highest decrease in the expression levels of GluR1 and GluR2 mRNA in the hippocampus of pups on PND7;GluR3 mRNA expression level in the hippocampus of the exposed pups on PND21 showed the maximum inhibitory effect;the expression levels of GluR1,GluR2,and GluR3 mRNA all showed the least decrease in the hippocampus of the exposure groups on PND35.[Conclusion]Early-life exposure to 6:2 Cl-PFESA may affect the growth and development of offspring mice,alter the hippocampal synaptic structure,and influence the learning and memory abilities,which may be related to their inhibitory effects on the expression levels of AMPA receptor subunits GluR1,GluR2,and GluR3 genes in the hippocampus of offspring mice at various developmental stages.