Study On EPO Regulation Autophagy And Apoptosis Of Bovine Mammary Epithelial Cells

Mastitis is a disease that is the most common,the most difficult to prevent and treat,and the most expensive in dairy farming.It seriously restricts the advancement of dairy industry.Mastitis not only reduces milk production and milk quality in dairy cows,but also potentially harms human health.Erythropoietin(EPO)is a pleiotropic growth factor that is involved in the regulation of oxidative responses,apoptosis,inflammation,and autophagy in a variety of tissues and cells.Mastitis easily leads to damage and inflammation of the mammary gland tissue of dairy cows,and there are few reports on EPO and dairy cow mastitis.In this paper,from the perspective that EPO can participate in the regulation of autophagy and apoptosis,based on the signaling pathway PI3K/Akt/m TOR of autophagy and apoptosis,the effect of EPO on breast inflammation and injury was studied.We took cow mammary epithelial(MACT)cells as the research object,and used lipopolysaccharide(LPS)to construct an inflammatory mammary epithelial cell model,combined with exogenous EPO to treat the cells.We used MTT,CCK-8,Western blot,RT-PCR,ELISA,Annexin V FITC/PI,transmission electron microscopy and other methods to evaluate the effects of EPO on cell proliferation,inflammatory response,apoptosis and autophagy activities;Combined with the treatment of PI3 K or m TOR inhibitor,the molecular mechanism of EPO regulating autophagy and apoptosis of cow mammary epithelial cells was revealed.The result is as follows:Effects of EPO on MAC-T cells proliferation and inflammatory response: The results of MTT and CCK-8 methods showed that 100 U/m L EPO treatment of MAC-T cells for 12 h could significantly improve cells viability and promote cells proliferation.100 U/m L EPO inhibited LPS up-regulation of IL-6,IL-8,TNF-α m RNA and protein expression in MAC-T cells.These results indicated that EPO could promote cells proliferation and reduce the inflammatory response of LPS-induced MAC-T cells.In the follow-up experiments,100 U/m L EPO was selected to treat MAC-T cells for 12 h.Effects of EPO on autophagy and apoptosis of MAC-T cells: Western blot and RTPCR results showed that EPO decreased LC3 B,Beclin1,Bax and Caspase-3 m RNA levels,and increased p62 and Bcl-2 m RNA levels in LPS-treated MAC-T cells,while LC3II/LC3 I Ratio,Beclin1,Bax,Caspase-3 protein expression decreased,p62 and Bcl-2 protein expression increased in LPS-treated MAC-T cells;Annexin V FITC/PI results showed that EPO inhibited the number of apoptotic cells in LPS-treated MAC-T cells;Transmission electron microscopy showed that the number of autophagy-lysosomes in cells was significantly reduced after co-treatment with EPO and LPS.These results indicated that EPO was able to inhibit autophagy and apoptosis in LPS-induced MACT cells.PI3K/Akt/m TOR signaling mediates EPO to regulate autophagy and apoptosis in MAC-T cells: The detection results of p-Akt,p-m TOR,p-4E-BP1 and p-70S6 K protein showed that LPS could inhibit the activity of PI3K/Akt/m TOR signaling pathway;EPO can activate PI3K/Akt/m TOR signaling pathway and reverse the effect of PI3K/Akt/m TOR signaling pathway blocked by LPS;Blocking PI3 K or m TOR signaling,EPO could not inhibit LPS-induced IL-6,IL-8,TNF-α expression in MACT cells;The results of autophagy and apoptosis-related molecular detection showed that after blocking the PI3K/Akt/m TOR signaling pathway,EPO’s anti-apoptosis(inhibiting the expression of Bax and Caspase-3 m RNA and protein,and promoting the expression of Bcl-2 m RNA and protein)and anti-autophagy(inhibiting the expression of LC3 B and Beclin1 m RNA and protein,and promoting the expression of p62 m RNA and protein)weakened.Flow cytometry results showed that PI3 K or m TOR inhibitor significantly altered the anti-apoptotic effect of EPO.These results suggest that PI3K/Akt/m TOR signaling is involved the regulation of autophagy and apoptosis by EPO in LPS-induced MAC-T cells.In conclusion,EPO promotes cell proliferation and reduces the inflammatory response of LPS-induced MAC-T cells;EPO inhibits LPS-induced autophagy and apoptosis of MAC-T cells through PI3K/Akt/m TOR signaling.