孕鼠DEHP暴露对子代神经行为的影响及其机制

Effect of DEHP exposure in pregnant mice on neurobehavior of offsprings and its mechanism

目的:探讨围生期邻苯二甲酸二(2-乙基己基)酯(DEHP)暴露对小鼠神经行为的影响,阐明DEHP影响神经行为的可能机制。方法:选用受孕的ICR小鼠32只,随机分为3个不同剂量DEHP和玉米油对照组,于怀孕第3天起至孕17 d止,每天分别给予低剂量(10 mg∙kg-1)、中剂量(50 mg∙kg-1)、高剂量(250 mg∙kg-1)DEHP和玉米油灌胃,在仔鼠出生后观察其神经发育学指标,并在7周龄后进行神经行为学检测;利用Western blotting法检测仔鼠大脑前额叶皮层组织中丝切蛋白(cofilin)和P21蛋白激活激酶1(Pak1)蛋白表达水平。将体外培养的Neuro-2A细胞分为不同剂量DEHP组和二甲基亚砜(DMSO)对照组,分别用1 nmol∙L-1、10 nmol∙L-1、1μmol∙L-1 DEHP和DMSO孵育,在0、6、12、24和48 h利用细胞划痕实验观察细胞迁移能力,在相差显微镜下拍照观察细胞突起生长的情况。结果:与玉米油对照组比较,不同剂量DEHP组新生仔鼠神经发育学指标差异无统计学意义(P>0.05);与玉米油对照组比较,中剂量DEHP组雌性仔鼠自由探索新环境的能力降低(P<0.01);与玉米油对照组比较,中剂量DEHP组仔鼠前额叶皮层组织中cofilin蛋白表达水平降低(P<0.05),但Pak1蛋白表达水平差异无统计学意义(P>0.05)。细胞划痕实验,与DMSO对照组比较,不同剂量DEHP组细胞孵育12 h后,细胞迁移速率均降低(P<0.05);1μmol∙L-1 DEHP组孵育6 h后细胞平均突起长度均缩短(P<0.05),孵育48 h后有突起细胞百分比降低(P<0.05)。结论:ICR小鼠在孕期暴露于50 mg∙kg-1及以上剂量的DEHP可导致仔鼠神经行为异常,1μmol∙L-1 DEHP孵育Neuro-2A细胞12 h后明显抑制细胞迁移和突起生长能力,其机制可能与DEHP抑制细胞骨架调控蛋白cofilin的表达有关。

Objective:To investigate the effect of bis(2-ethylhexyl)phthalate(DEHP)exposure during the perinatal period on the neurobehaviors of the mice,and to clarify the possible mechanism of DEHP’s influence in the neurobehaviors.Methods:A total of 32 pregnant ICR mice were selected and randomly divided into different doses of DEHP groups and corn oil control group.From the 3rd day of pregnancy to the 17 th day of pregnancy,the mice were given by gavage with low dose(10 mg·kg-1)of DEHP,medium dose(50 mg·kg-1)of DEHP,high dose(250 mg·kg-1)of DEHP and corn oil,respectively.The neurodevelopmental indicators were observed after birth,and neurobehavioral tests were performed after 7 weeks of age;the expression levels of cofilin and Pak1 proteins in the prefrontal cortex tissue of the offsprings were detected by Western blotting method.The in vitro cultured Neuro-2 A cells were divided into different doses of DEHP groups and DMSO control group;the Neuro-2 A cells were incubated respectively with 1 nmol·L-1,10 nmol·L-1,1μmol·L-1 DEHP and DMSO.After 0,6,12,24,and 48 h of incubation,cell scratch test was used to observe the migration abilities of cells,and the neurite outgrowth of cells were observed under phase-contrast microscope.Results:Compared with corn oil control group,the neurodevelopmental indicators of the newborn mice in different doses of DEHP groups had no significant differences(P>0.05),and the ability to freely explore new environments of the female offsprings in medium dose of DEHP exposure group was significantly decreased(P<0.01).The Western blotting results showed that compared with corn oil control group,the expression level of cofilin protein in prefrontal cortex tissue of the offsprings in medium dose of DEHP group was significantly decreased(P<0.05),but the expression level of Pak1 had no significant difference(P>0.05).The results of cell scratch test showed that compared with DMSO control group,the cell migration rates in different doses of DEHP groups were reduced after 12 h of incubation(P<0.05),the mean neurite lengths were also shortened after 6 h of incubation in 1μmol·L-1 DEHP group(P<0.05),and the percentage of neurite-bearing cells was decreased after 48 h of incubation in 1μmol·L-1 DEHP group(P<0.05).Conclusion:Exposure of the ICR mice to DEHP at a dose of 50 mg·kg-1 or more during pregnancy can lead to the abnormal neurobehaviors of the offsprings;to incubate the Neuro-2 A cells with 1μmol·L-1 DEHP for 12 h can significantly inhibit the cell migration and neurite growth;its mechanism may be related to inhibiting the expression of cytoskeleton regulatory protein cofilin.