N-氨甲酰谷氨酸干预二硫化碳致孕鼠胚胎植入障碍及分子机制的研究

The intervention effect of N-carbamoyl glutamic acid on embryo implantation disorder induced by carbon disulfide and its possible molecular mechanism

目的探讨胚胎植入期膳食补充N-氨甲酰谷氨酸(NCG)在二硫化碳(CS2)致小鼠胚胎植入障碍中的预防作用及可能的分子机制。方法分别于清洁级昆明种小鼠受孕后第3、4、5天单次腹腔染毒CS2构建胚胎植入障碍模型,并平行设围植入期增补NCG组和溶剂对照组,每组的每个观察终点有12只孕鼠。各组染毒CS2后24 h收取子宫内膜组织进行Western-blot和免疫组织化学染色分析,检测子宫内膜组织中AKT/AMPK及其磷酸化形式的蛋白表达水平,孕9 d观察胚胎植入数目。结果与单纯CS2染毒组比较,孕4d NCG+CS2组孕胚胎植入数目增加24.0%,差异有统计学意义(P<0.05)。NCG+CS2组pAKT蛋白表达量增加了46.5%,差异有统计学意义(P<0.05);NCG+CS2组pAMPK蛋白表达水平降低28.0%,差异有统计学意义(P<0.05)。免疫组织化学定位结果显示,pAKT、pAMPK、AKT、AMPK蛋白主要表达于子宫内膜的腔上皮细胞、腺上皮细胞和基质细胞;与单纯CS2染毒组比较,孕4 d NCG+CS2组pAKT蛋白染色加深(蛋白表达水平明显增加),pAMPK蛋白染色变浅(蛋白表达水平明显降低)。结论膳食补充NCG可能通过改变磷酸化AKT/AMPK分子总量干预CS2染毒导致的胚胎丢失作用。

Objective To explore the preventive effect and possible molecular mechanism of dietary supplementation of N-carbamylglutamate(NCG)in the implantation of carbon disulfide(CS2)into embryo implantation disorders.Methods embryo implantation disorder model was established by single intraperitoneal exposure to CS2 on the 3rd,4th,and 5th days after pregnancy.Endometrial tissues were collected for 24h after exposure to CS2 for western-blot and immunohistochemical staining.Results The number of embryo implantation was increased in NCG+CS2 group,compared with CS2 alone group.Day 4 of pregnancy when CS2-exposed after 24 h,the expression of pAKT protein in NCG+CS2 group was significantly increased(P<0.05),the expression level of pAMPK protein in NCG+CS2 group was significantly decreased,compared with CS2 alone group,respectively.Immunohistochemical results showed that pAKT,pAMPK,AKT and AMPK proteins were expressed in luminal epithelial cells,glandular epithelial cells and stromal cells of endometrium;Day 4 of pregnancy when CS2-exposed after 24 h,deep staining of ATK and pAKT protein in NCG+CS2 group,the AMPK and pAMPK protein staining became lighter.Conclusion Dietary supplementation of NCG can interfere with the embryo loss induced by CS2 by altering the total amount of AKT/AMPK molecules.