| BackgroundIt was found that AKT/AMPK/mTOR signaling pathway was involved in the whole process of pregnancy.The expression of mTOR molecule protein in the uterus of pregnant mice was time-specific and tissue-specific during embryo implantation.Injection of rapamycin,an inhibitor of mTOR,into the uterus at the critical period of implantation could caused the embryo loss in mice.Recent animal studies have found that AKT inhibitors can significantly reduce the number of mouse embryos implanted,and AMPK antagonists can significantly improve the development retardation and slow cell growth of rabbit blastocysts induced by AMPK agonists.It is suggested that AKT/AMPK/mTOR signaling pathway is essential for successful pregnancy in animals.Other studies have found that N-carbamoyl glutamic acid(NCG)supplementation during pregnancy can enhance the expression of p-PKB(AKT)and p-S6K1(one of the downstream substrates of mTOR molecule)in uterine tissue of pregnant rats,and interfere the embryo loss caused by rapamycin.These results suggest that AKT/AMPK/mTOR signaling pathway involved in the regulation by NCG on the process of embryo implantation.Carbon disulfide(CS2),as a volatile organic solvent and industrial raw material,is often used in the production of viscose fibers and the production line workers are mainly women.Epidemiology studies and animal experiments have found that CS2 has obviously reproductive and developmental toxicity,but the specific mechanism is still unclear.Previously,our group successfully constructed a CS2-induced dynamic sequence pregnant mouse model,which laid a foundation for further study of the mechanism of CS2-induced embryo implantation disorder.This study hypothesizes that a series of cascade reactions caused by CS2 may lead to embryo loss,and the upstream mechanism may be the abnormal regulation of AKT/AMPK/mTOR signaling pathway.In order to investigate the effect of CS2 on the effect of AKT/AMPK/mTOR signaling pathway in uterus of pregnant mouse and the intervention of NCG,we detected the expression level of AKT/AMPK/mTOR signaling pathway-related molecules in uterus of pregnant mouse and detected the expression level of key molecules of AKT/AMPK/mTOR signaling pathway again after supplementation of NCG during periimplantation.Research objectiveTo construct a time-dependent sequence pregnant mouse model of CS2-induced embryo implantation disorder,collected different uterine tissues of pregnant mouse at different exposure time points and observe the expression levels of AKT,AMPK and mTOR molecules,construct an animal model of NCG supplementation and CS2 exposure during peri-implantation period,and detect the recovery of AKT/AMPK/mTOR-related factors in pregnant rat uterine tissues after NCG supplementation.The key role of AKT/AMPK/mTOR expression disorder in CS2-induced embryo implantation dysfunction and the intervention effect and possible mechanism of NCG supplementation were revealed.Research method1.Experimental designThe design of this experiment is divided into two parts:The first part is to construct a time-dependent sequential pregnant mouse model of CS2-induced embryo implantation disorder.Uterine tissues of pregnant mouse were collected at each observation point and endpoint to detect the expression level of key molecules in AKT/AMPK/mTOR signaling pathway.In the second part,a pregnant mouse model of CS2-induced embryo loss was constructed by NCG intervention.Uterine tissues of pregnant mouse were collected at the corresponding observation endpoints to detect 7 the expression level of key molecules in AKT/AMPK/mTOR signaling pathway.2.Animal TreatmentClean Kunming mice were provided by Beijing Weitonglihua Laboratory Animal Science and Technology Co.,Ltd.The first day in gestation pregnant mouse were randomly allocated to each observation endpoint.The number of pregnant mouse in each observation endpoint was equally.The pregnant mouse were exposed to CS2 or olive oil by single intraperitoneal injection(CS2 or olive oil).The exposure dose of CS2 was 0.4LD50(631.4 mg/kg,dissolved in olive oil was 1:9),and the exposure volume was 0.1 mL/10 g body weight.The control group was given olive oil of equal volume.NCG supplementation mode was free dietary intake in mouse,and the amount of NCG additives accounted for 0.1%of the basic diet.The supplementation time was from the beginning of pregnancy to the end of observation.3.Determination of the expression of key molecules in AKT/AMPK/mTOR signaling pathway in uterine tissueThe weight of uterus,ovary,heart,liver,spleen,kidney,lung and embryo tissues of pregnant mouse at the end of GD9 observation were weighed,and the number of embryo implantation was recorded to evaluate maternal and embryonic toxicity of CS2.The uterus tissues of pregnant mouse at the other end of observation were collected for sample preparation and subsequent protein content detection.The expression levels of AKT,AMPK,mTOR and their phosphorylation form pAKT,pAMPK,and p-mTOR proteins in the uterus tissues were determined by twelve alkyl sulfonate polyacrylamide gel electrophoresis(SDS-PAGE),Western blotting and immunohistochemical staining(Immunohistochmeistry,IHC).4.Statistical analysisExcel was used to establish the database and SPSS21.0 software was used for statistical analysis.For the experimental data,the homogeneity test of variance is carried out first.If the variance is homogeneous,two independent samples T test or one-way ANOWA are used,and Bonferroni method is used for comparison;if the variance is not homogeneous,non-parametric statistical method(Kruskal-WallisH method)or Fisher’s exact probability method is used for testing.The experimental data of normal distribution data are expressed as a=0.05.Results1.Maternal toxicity and embryotoxicityIn maternal toxicity,there was no significant difference in maternal weight gain and organ coefficient between the exposed group and the control group(P>0.05)after CS2 exposure during embryo implantation(GD3,GD4,GD5 and GD6).The results showed that CS2 had no maternal toxicity at this dose.In terms of embryotoxicity,GD4 CS2 exposure reduced the number of embryos implanted in pregnant mouse by 34.77%(P<0.05)compared with the control group.2.The effect of CS2 exposure on the expression of mTOR and p-mTOR in uterus of pregnant mouseCompared with the control group,the expression level of mTOR protein in uterus decreased significantly after GD3 exposure at the end points of GD5 and GD6,and after GD4 exposure at the end points of GD5 and GD7(P<0.05).The expression level of mTOR protein decreased by 38.55%,44.91%,49.35%and 32.42%,respectively.Compared with the control group,the expression level of p-mTOR protein in uterus decreased significantly after GD3 exposure,GD5 and GD7 exposure and GD6 exposure(P<0.05),and the expression level of p-mTOR protein decreased by 59.51%,51.44%,38.00%and 35.84%,respectively.Compared with the control group,the mean optical density of mTOR molecule in uterine cavity epithelium and glandular epithelium decreased after GD4 exposure,GD7 observation and immunohistochemical staining(P<0.05),indicating that CS2 exposure reduced the expression level of mTOR molecule protein in uterine tissue.3.The effect of CS2 exposure on the expression of AKT and pAKTprotein in uterus of pregnant mouseCompared with the control group,the expression levels of pAKT protein in uterus decreased significantly after GD3 exposure,GD5 exposure,GD6 exposure and GD7 exposure(P<0.05).The expression levels of pAKT protein decreased by 39.98%,49.05%,11.76%and 20.02%,respectively.Compared with the control group,the expression level of AKT protein in CS2 exposd group was slightly lower,with no significant difference(P>0.05).There was no significant effect of CS2 exposure on the expression level of AKT molecule protein in the uterus of pregnant mouse.Compared with the control group,the average optical density of pAKT molecule in uterine cavity epithelium and glandular epithelium was decreased by immunohistochemical staining(P<0.05),and the expression level of pAKT molecule protein in uterine tissue was decreased by CS2 exposure(P<0.05).4.The effect of CS2 exposure on the expression of AMPK and pAMPK in uterus of pregnant mouseCompared with the control group,the expression level of pAMPK in uterus increased significantly after GD3 exposure,GD5 exposure and GD6 exposure(P<0.05),and the expression level of pAMPK increased by 47.42%,25.51%and 35.19%,respectively.Compared with the control group,the expression level of AMPK protein in CS2 exposure group increased slightly,with no significant difference(P>0.05).There was no significant effect of CS2 exposure on the expression level of AMPK protein in uterus tissue of pregnant mouse.Compared with the control group,the average optical density of pAMPK in uterine lumen epithelium and glandular epithelium was increased by immunohistochemical staining after GD4 exposure(P<0.05).The expression level of pAMPK in uterine tissue was increased by CS2 exposure.5.NCG intervention in CS2-induced embryo loss Compared with the CS2 exposure group,the number of embryos implanted in the CS2 exposure group supplemented with NCG increased significantly(P<0.05),and the number of embryos implanted increased by 24.0%.6.Effect of NCG on expression of AKT/AMPK/mTOR in uterus of pregnant mouse exposed to CS2Compared with CS2 exposure group,the expression level of pAKT protein in uterus increased significantly after GD4 exposure(P<0.05),pAKT protein increased by 46.5%,pAMPK protein expression in uterus decreased significantly(P<0.05),and pAMPK protein decreased by 28.0%.Compared with CS2 exposed group,the immunohistochemical staining results of CS2 exposed group with NCG supplementation showed that the staining of mTOR protein and pAKT protein in uterine tissue was significantly deepened,suggesting that the expression levels of mTOR protein and pAKT protein were significantly increased,and the staining of pAMPK protein was significantly shallower,suggesting that the expression level of pAMPK protein was significantly decreased.Conclusions1.Exposure to CS2 in pregnant mouse can lead to disorder of expression of key molecules of AKT/AMPK/mTOR signaling pathway in uterus tissue,which may be an important molecular mechanism of CS2-induced embryo loss in pregnant mouse.2.Dietary supplementation of NCG may reverse CS2-induced embryo loss in mouse by correcting the expression disorder of key molecules in AKT/AMPK/mTOR signaling pathway in uterus of mouse. |
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