| Deoxynivalenol(DON)is a type B trichothecene mycotoxin which has toxic effects on humans and animals.The endometrial tissue plays a key role in female reproduction system.Disorders in endometrial development can lead to pregnancy failure,improper implantation,and infertility.Although DON has been studied in various cell types for its cytotoxicity,there is litter information about the effects of DON on mouse endometrial stromal cells(ESCs).In this study,we used mouse ESCs as cell model to study its vitro cell toxicology and to explore its potential mechanisms,and provided theory support for revealing the toxicity mechanism of DON on mouse ESCs.The toxicity of DON on mouse ESCs were tested by CCK-8、TUNEL、flow cytometry、real-time PCR、western blot and so on.The main results are as follows:(1)DON significantly inhibited mouse ESCs viability(p<0.05),and cell viability decreased with increasing DON concentrations in a dose-and time-dependent manner.200ng/ml DON began to cause cell shrinkage and little float,when DON concentration was 1600ng/ml,cells almost floated.(2)The results of TUNEL and flow cytometry showed that DON significantly caused apoptosis(p<0.05)in mouse ESCs and apoptosis rate increased with increasing DON concentrations in mouse ESCs.DON caused intracellular oxidative stress,very significantly decreased mitochondrial transmembrane potential(p<0.01)in mouse ESCs.Real-time results showed that DON very significantly increased the expression levels of apoptosis-related gene including Caspase-9、Caspase-3、PARP and the ratio od Bax/Bcl-2(p<0.01),western blot results indicated that DON significantly increased the ratio of Bax/Bcl-2(p<0.05)and very sinificantly increased the protein expression levels of Caspase-3、Caspase-9 and PARP(p<0.01).These results indicate that DON casues apoptosis via mitochondria apoptosis pathway in mouse ESCs.(3)DON induced G2 phase arrest in mouse ESCs,compared with control group,the proportion of G2 phase was significantly increased(p<0.05)with increasing DON concentrations.Western blot results showed that DON did not affact the expression level of p38 protein,but significantly increased the expression level of p-p38 protein(p<0.05),DON significantly decreased the expression levels of G2 phase-related protein(Cdc25C/p-Cdc25C,Cdc2/p-Cdc2 and cyclinB1)(p<0.05).However,the combination of SB203580(p38 specific inhibitor)and DON treatment significantly reversed the protein expression levels of DON-induced(p<0.05).Real-time PCR results showed that DON significantly decreased the mRNA expression levels of G2 phase arrest-related genes(Cdc25C,Cdc2 and cyclinBl)(p<0.05),however,SB203580 significantly reversed the mRNA expression levels of DON-induced.Collectively,these data suggest that p38 MAPK signaling pathway is involved in DON-induced G2 phase arrest by down-regulation of expression levels of Cdc25C/p-Cdc25C、Cdc2/p-Cdc2 and cyclinB1 in mouse ESCs. |
