Mechanism Regulation Of Bovine Endometrial Epithelial Cells Apoptosis Induced By High NEFA Via FoxO1 Pathway

The occurrence of reproductive disorder diseases is related to the level of nutritional metabolism in periparturient dairy cows,and past studies had confirmed that high blood nonesterified fatty acids(NEFA)Which caused by negative energy balance(NEB)can increase the incidence of retained fetal membranes(RFM),but the specific regulatory mechanism was unclear.Previous studies had shown that the occurrence of retained fetal membranes in periparturient dairy cows would not only cause a decrease in milk production,reduce the quality of dairy products,reduce production performance,but also affect the body’s immune function.The occurrence of RFM in dairy cows is significantly related to the degree of apoptosis of endometrial epithelial cells,which has been demonstrated by our team’s laboratory.FoxO1 is a longevity transcription factor that is involved in cell proliferation,metabolism,differentiation,tumorigenesis and immunity,which is regulated by SIRT1 and plays an important role in the process of apoptosis through post-translational modification.In order to explore whether highly non-esterified fatty acids affect the occurrence of retained fetal membranes through the FoxO1 pathway,this study,explored the inhibitor(EX527)and accelerator(Resveratrol)of SIRT1 from the perspective of apoptosis in endometrial epithelial cells,and investigated the regulatory effect on NEFA-induced apoptosis of bovine endometrial epithelial cells,so as to elucidate the regulatory mechanism between high NEFA and the occurrence of retained fetal membranes in dairy cows.It provides a theoretical basis to reduce the occurrence of reproductive disorders from the perspective of nutritional regulation in the future,and provides an experimental basis for the formulation of reasonable prevention and control measures.This study mainly includes the following three contents.First of all,the optimal addition concentration and action time were screened by adding 0 mmol/L NEFA(control group),0.2mmol/L NEFA group(low concentration group),and 1.2 mmol/L NEFA group(high concentration group)NEFA.Flow cytometry was used to determine the change of apoptosis and cell cycle arrest induced by different concentrations of NEFA.The expression levels of the key factors in apoptotic pathway and FoxO1 pathway were detected with qRT-PCR and Western blotting techniques.The activation of FoxO1 was detected with Laser confocal technology.To determine the effect of different concentrations of NEFA on apoptosis of endometrial epithelial cells and expression of key factors in the FoxO1 pathway.And then,0 mmol/L NEFA,1.2mmol/L NEFA,10 μmol/L EX527,and 1.2 mmol/L NEFA+10 μmol/L EX527 were added to the culture solution.Finally,0 mmol/L NEFA,1.2 mmol/L NEFA,50 μmol/L Resveratrol,and 1.2mmol/L NEFA+50 μmol/L Resveratrol were added to the culture solution.The expression levels of the key factors in apoptotic pathway and FoxO1 pathway were detected with Western blotting techniques.The activation of FoxO1 was detected with Laser confocal technology.This elucidates the regulatory mechanism of NEFA on apoptosis of endometrial epithelial cells in dairy cows through the FoxO1 pathway.Results: Compared with the control group,There was no significant change on the apoptosis rate and cell cycle of endometrial epithelial cells(P > 0.05)in the low concentration group,but the apoptosis rate was very significantly increased in the high concentration group(P < 0.01),and endometrial epithelial cell blockade was in the G1 phase(P < 0.01).The results of qRT-PCR and Western blotting showed that apoptotic protein and mRNA expression had no significant change in the low concentration group(P > 0.05),but Caspase-3,Bax,Bax/Bcl-2 were significantly increased(P < 0.01),and Bcl-2 and Bcl-xl(P < 0.01)were significantly decreased in the high concentration group.Compared with the control group.The proteins expression levels of IGF-1,p-AKT,AKT,FoxO1,p-FoxO1,Ac-FoxO1,SIRT1,AKT/p-AKT were not significantly change(P > 0.05).The activity level of Foxo1 had not significantly change in the low concentration group(P > 0.05),but the proteins expression levels of AKT,FoxO1,p-FoxO1AKT/p-AKT and Ac-FoxO1 were significantly increased(P < 0.01),IGF-1,SIRT1 and p-AKT expression levels were significantly decreased(P < 0.01)in the high concentration group.The activity level of Foxo1 was significantly promoted(P < 0.01)in the high concentration group.Compared with the control group,after adding EX527.The protein and mRNA expression levels of SIRT1 were significantly decreased(P < 0.01).The protein levels of IGF-1,p-FoxO1,FoxO1/p-FoxO1,Caspase-3,Bax/Bcl-2 and Bcl-xl had no significant change(P > 0.05),but the expression levels of Ac-FoxO1 and FoxO1 were significantly increased(P < 0.05).The protein expression level of Bcl-2(P < 0.05)was significantly decreased and the protein expression level of Bax was significantly decreased(P < 0.01).Compared with NEFA stimulation group.The protein expression levels of AKT/p-AKT,Ac-FoxO1,FoxO1/p-FoxO1,Caspase-3,Bax,Bax/Bcl-2(P < 0.01)were significantly increased,but IGF-1,AKT,p-AKT,SIRT1,FoxO1,pFoxO1,Bcl,Bcl-xl were significantly decreased(P < 0.01)in the NEFA+EX527 stimulation group.Compared with the control group,after adding Resveratrol,the protein and mRNA expression levels of SIRT1 were significantly increased(P < 0.01),and the protein expression levels of FoxO1,p-FoxO1,FoxO1/p-FoxO1,IGF-1 and Caspase-3 had no significant change(P > 0.05),but the protein expression level of Ac-FoxO1(P < 0.01)was significantly decreased.The protein expression levels of Bax and Bax/Bcl-2 were significantly decreased(P < 0.05),and the protein expression levels of Bcl-2 and Bcl-xl were significantly increased(P < 0.01)in the Resveratrol group.Compared with the NEFA group,the protein expression levels of IGF-1,pAKT,SIRT1,p-FoxO1,Bcl-2 and Bcl-xl were significantly increased(P < 0.01),and the protein expression levels of AKT,AKT/p-AKT,Ac-FoxO1,FoxO1,FoxO1/p-FoxO1,Caspase-3,Bax and Bax/Bcl-2 were significantly decreased(P < 0.01)in the NEFA+Resveratrol stimulation group.The results of laser confocal showed that the activity of FoxO1 was significantly promoted(P < 0.01)in the NEFA+EX527 group.The activity of FoxO1 was significantly inhibited(P < 0.01)in the NEFA+Resveratrol group.Conclusion: In summary,high NEFA can cause apoptosis of endometrial epithelial cells through the FoxO1 pathway,and the phosphorylation and acetylation levels of FoxO1 directly determine the degree of apoptosis,and then participates in the occurrence of RFM.