| Zearalenone（ZEA）a mycotoxin it extensively exposed in a variety of grains and feed,and fed the diets containing ZEA in gilts could induce uterine hypertrophy.ZEA could induce uterine hypertrophy via multiple pathways in gilts,whereas the key molecular mechanism of ZEA induced uterine hypertrophy is still unclear.The first part:Screening of signaling pathway for ZEA induced porcine uterine hypertrophy based on proteomics and metabolomics.A total of 16 healthy weaned gilts（Duroc×Landrace×Yorkshire）with average body wight of 12.45±0.19 kg were randomly divided into 2 treatments,8 replicates per treatments.Control and ZEA3.0 group were fed the basal diet and basal diet supplement with the 3.0mg/kg of ZEA,respectively.And the experiment contains 7 d adaptation period and 32 d experiment period.At end of the feeding experiment,blood and uterine samples were collected for further study.The results showed that:（1）Uterine morphology and uterine index tests revealed that the thickness of lamina propria and myometrium and the number of uterine glands of the ZEA 3.0 group were significantly increased（P<0.05）compared with the control group.And the contents of zearalenone and its metabolites were increased（P<0.05）in ZEA 3.0 group.（2）Through the detection of antioxidant enzymes,malondialdehyde（MDA）,and8-hydroxy-2 deoxyguanosine（8-OHDG）in the uterus of piglets,it was found that the activity of total superoxide dismutase（T-SOD）,glutathione peroxidase（GSH-Px）,and catalase（CAT）were decreased（P<0.05）compared with the control,whereas contents of the MDA and8-OHDG were increased（P<0.05）in the uterine sample.（3）Metabolomics analysis revealed a total of 698 metabolites between the control group and the ZEA3.0 group.Further screening revealed that there was a total of 20 differentially expressed metabolites（FC>1.2 or FC<0.83,VIP>1,P<0.05）,with 13 upregulated and 7downregulated differentially expressed metabolites,respectively.Metabolites related to energy metabolism such as Adenosine monophosphate,Inosine 5’-monophosphate（IMP）,Adenylosuccinate,and Adenosine 3’-monophosphate level were increased（P<0.05）in ZEA3.0 group,compared with control.KEGG function analysis showed that energy metabolism related pathways including phosphoinositide 3-kinase/protein kinase B（PI3K/Akt）signaling pathway,ABC transporters signaling pathway,AMPK signaling pathway,and PPAR signaling pathway were changed（P<0.05）.In addition,the differentially expressed metabolites were significantly enriched（P<0.05）in nucleotide metabolism,lipid metabolism,and amino acid metabolism pathways.（4）Proteomic analysis showed that a total of 423 differential proteins were identified between the control group and the ZEA3.0 group,of which 291 were upregulated and 132were downregulated（FC>1.2 or FC<0.83,P<0.05）.Compared with control group,the energy metabolism-related proteins such as Q5XLD3（CKM）,cell growth and motility proteins A0A5G2R354（SAXO2）,cell proliferation and differentiation such as A0A480UJS2and A0A480Q1D2 were up-regulated（P<0.05）in ZEA 3.0 group,and tumor suppressor-related proteins A0A286ZLG5（NBL1）,A0A4X1V1P1（GPC6）,A0A4X1T3Y9,and A0A481D9A4,and apoptosis proteins such as A0A480UYE2,A0A5G2RN58（ALDH1A2）,and A5A8Y8（EGFL8）were down-regulated（P<0.05）.The KEGG function analysis showed that the PI3K/Akt signaling pathway,the extracellular matrix（ECM）-receptor interaction,focal adhesion,Protein digestion and absorption,Amoebiasis,and Renin-angiotensin system were changed（P<0.01）in ZEA3.0 group.Moreover,compared to other signaling pathways,the PI3K/Akt signaling pathway has the most differential proteins,totaling 37.（5）The correlation analysis of differential metabolites and PI3K/Akt signaling pathway differential proteins showed that PI3K/Akt signaling pathway related proteins were significantly positively correlated with the energy metabolism differential metabolites adenosine monophosphate succinate,adenosine monophosphate,and 3’-adenosine monophosphate,respectively（P<0.05）.Moreover,the relative protein expressions of PI3K,and Akt were increased（P<0.05）in ZEA3.0 group compared with the control group.It is speculated that ZEA induced uterine hypertrophy through the activation of PI3K/Akt signaling pathway.The second part:Study on the Molecular Mechanism of Zearalenone Induced Uterine Development.In order to further investigate the effect of ZEA on uterine hypertrophy of pigs.Using porcine endometrial epithelial cells（PEECs）as materials.After PEECs by treated with different concentrations of ZEA andβ-Estradiol（E2）for 24 h,it was found that 10μmol/L ZEA and 10 nmol/L E2could increase the cell viability of PEECs（P<0.05）.Cell proliferation determination showed that 10μmol/L ZEA and 10 nmol/L E2could induce the proliferation of PEECs（P<0.05）.The relative m RNA and protein expressions of the PI3K,Akt,and cyclin-dependent kinase 7（CDK7）were increased（P<0.05）,while,cyclin dependent kinase Inhibitor 1B（p27）were decreased（P<0.05）in the ZEA and E2groups,compared with control group.Further addition of PI3K specific inhibitor Wortmannin（W,1μmol/L）to the ZEA and E2groups for 24 h,the activity and proliferation of PEECs significantly decreased（P<0.05）.In addition,compared with ZEA group,the relative m RNA and protein expressions of PI3K,Akt,and CDK7 were decreased（P<0.05）,and the relative m RNA and protein expressions of p27 were increased（P<0.05）after addition of PI3K inhibitor.Overall,（1）ZEA could increase the uterine development and induce oxidative stress of uterus,ZEA and metabolites were accumulated in uterus of weaned gilts;（2）The differentially expressed metabolites mainly involved in energy metabolism,nucleotide metabolism,amid acid metabolism,and lipid metabolism;（3）The differentially expressed proteins mainly involved in PI3K/Akt signaling pathway,ECM-receptor interaction,and focal adhesion;（4）ZEA could regulate cell cycle to induce the uterine development of gilt via PI3K/Akt signaling pathway. |
