Zearalenone Promotes Porcine Uterine Hypertrophy Through Autophagy Mediated By AMPK/mTOR Pathway

Zearalenone is a nonsteroidal oestrogenic mycotoxin produced by fusarium.ZEA and its metabolites mainly contaminate corn,barley,wheat and sorghum.The estrogen-like effects of ZEA can disrupt oestrogenic levels in farm animals and affect the reproductive ability of animals,especially pigs.It has caused huge losses to agricultural production by abortion,stillbirth and malformation of sows fed dietary ZEA.The first part: ZEA promoted porcine uterine hypertrophy through autophagy mediated by AMPK/mTOR pathway.A total 32 healthy female weaned gilts（Landrace×Yorkshire×Duroc）,with a mean body weight of 12.39 ± 0.24 kg,were randomly allocated to four treatments,with eight gilts per treatment.The test diets in the present study were basal diets supplemented with 0（Control）,0.15（ZEA0.15）,1.5（ZEA1.5）,or 3.0（ZEA3.0）mg/kg ZEA,respectively.Gilts were fed for a32-d experiment period after 7-d adjustment.During the experiment period,the mid-term body weight was measured on the 16 th day.On the last day of the test,all weaned gilts were slaughtered,uterine samples were collected and organ weights were recorded to calculate organ index,zearalenone and zearalenone metabolites,uterine histomorphology,granulosa cell ultrastructure,and the expression of AMPK/mTOR signaling pathway related genes,autophagy related genes,and proliferation and apoptosis related genes in the uterine were also examined.Results indicated that: With increasing ZEA levels,the uterine length,uterine weight,uterine weight/uterine length and uterine weight/final weight increased linearly（P < 0.05）;Uterine ZEA,α-Zearalenol and β-Zearalenol increased linearly（P < 0.05）;Myometrium and endometrium increased linearly（P < 0.05）;The quantity of mitochondria in 1.5 and 3 mg/kg ZEA treatments increased,and endoplasmic reticulum vacuolized;Phospho-AMP-activated protein kinase（p-AMPK）,Phospho-mammalian target of rapamycin,p-mTOR（p-mTOR）,Microtubule associated protein 1 light chain 3 alpha（LC3）,Beclin1 and Proliferating cell nuclear antigen（PCNA）were mainly localized in the endometrium,uterine gland and lamina propria,the immunoreactivity of p-AMPK,LC3,Beclin1 and PCNA enhanced,however,the immunoreactivity of p-mTOR declined with ZEA levels increasing;The relative m RNA and protein expressions of Beclin1,LC3,Autophagy related 5（ATG5）,ATG7,ATG9,PCNA,and B-cell lymphoma-2（BCL2）increased linearly（P < 0.05）,the relative m RNA and protein expressions of Bcl-2-associated x protein（BAX）decreased linearly（P < 0.05）,the relative protein expressions of p-AMPK/AMPK increased linearly（P < 0.05）,the relative protein expressions of p-mTOR/mTOR decreased linearly（P < 0.05）with increasing ZEA levels.Research on the correlation of zearalenone and zearalenone metabolites with uterine index and relative m RNA and protein expressions: The uterine ZEA,α-ZOL andβ-ZOL were positively correlated（P < 0.01）with uterine length,uterine weight,uterine weight/uterine length and uterine weight/final weight,relative m RNA and protein expressions of autophagy genes（Beclin1,LC3,ATG5,ATG7 and ATG9）and proliferation genes（PCNA and BCL2）and relative protein expressions of p-AMPK/AMPK,however,the uterine ZEA,α-ZOL and β-ZOL were negatively correlated（P < 0.01）with relative m RNA and protein expressions of apoptosis gene BAX and relative protein expressions of p-mTOR/mTOR.Research on the correlation of relative m RNA expressions: Proliferation gene PCNA and BCL2 were positively correlated（P < 0.01）with autophagy genes Beclin1,LC3,ATG5,ATG7 and ATG9,however,apoptosis gene BAX was negatively correlated（P < 0.01）with autophagy genes Beclin1,LC3,ATG5,ATG7 and ATG9.Research on the correlation of relative protein expressions: The p-AMPK/AMPK was positively correlated（P < 0.01）with autophagy genes（Beclin1,LC3Ⅱ/Ⅰ,ATG5,ATG7 and ATG9）,however,negatively correlated（P < 0.01）with apoptosis gene BAX;pmTOR/mTOR was negatively correlated（P < 0.01）with autophagy genes（Beclin1,LC3Ⅱ/Ⅰ,ATG5,ATG7 and ATG9）,positively correlated（P < 0.01）with apoptosis gene BAX;Autophagy genes（Beclin1,LC3Ⅱ/Ⅰ,ATG5,ATG7 and ATG9）were positively correlated（P <0.01）with proliferation genes（PCNA and BCL2）,negatively correlated（P < 0.01）with apoptosis gene BAX.The second part: the effects of ZEA promoted porcine uterine endometrial epithelium proliferation through autophagy mediated by AMPK/mTOR pathway.Porcine endometrial epithelial cells were resuscitated and cultured,and ZEA dose of 0（Control）,5 μmol/L（ZEA 5）,20 μmol/L（ZEA 20）and 40 μmol/L（ZEA 40）were added for continuous culture.All cells were collected after 24 h to examine cell viability,transcriptomics,and the expression of AMPK/mTOR pathway related genes,autophagy related genes,and proliferation and apoptosis related genes.Results indicated that: The 5 μmol/L ZEA treatment for 24 h significantly increased cell viability,the transcriptome m RNA and protein relative expression of PCNA,and transcriptome m RNA relative expression of BCL2（P < 0.05）,however,significantly decreased the transcriptome m RNA relative expression of BAX（P < 0.05）.20 and 40 μmol/L ZEA treatment for 24 h significantly decreased cell viability,the transcriptome m RNA and protein relative expression of PCNA,and transcriptome m RNA relative expression of BCL2（P < 0.05）,however,significantly increased the transcriptome m RNA relative expression of BAX（P < 0.05）.The transcriptome m RNA relative expressions of ULK1,TSC2,Beclin1,LC3,ATG5,ATG7 and ATG9 and the protein relative expression of p-AMPK/AMPK and Beclin1 increased linearly（P < 0.05）,whereas the transcriptome m RNA relative expression of Rheb and the relative protein expression of p-mTOR/mTOR decreased linearly（P < 0.05）with increasing ZEA levels.Overall conclusion:（1）ZEA（0-3 mg/kg）could affect the morphological results of the uterus of weaned piglets,promote abnormal uterine development.（2）ZEA could activate the uterine AMPK/mTOR signaling pathway,promote uterine autophagy and proliferation,down regulate the expression of apoptosis genes BAX and up regulate proliferation genes（PCNA and BCL2）,so as to promoting the proliferation of endometrium and uterine hypertrophy.（3）Low does ZEA（0-5 μmol/L）promote the proliferation of endometrial epithelial cells,however,high does ZEA（20-40 μmol/L）promote the apoptosis of endometrial epithelial cells.（4）ZEA could activate the the phosphorylation of AMPK in endometrial epithelial cells,affect the phosphorylation level of mTOR through TSC2 and Rheb,promote uterine autophagy and proliferation of endometrial epithelial cells,up regulate proliferation genes（PCNA and BCL2）and down regulate the expression of apoptosis genes BAX,so as to promoting the proliferation of endometrial epithelial cells.