The Molecular Mechanism Of Apoptosis In Bovine Mammary Epithelial Cells Induced By LPS

Mastitis is one of the important multiple diseases,which restricts the development of dairy industry,causes the great loss of dairy industry,and endangers human health.In dairy cow,the main cause of mastitis is the invasion of pathogen,and Escherichia coli(E.coli)are one of the most common environmental pathogens for mastitis.Asgram-negative bacteria,E.coli has Lipopolysaccharide(LPS)in the extracellular membrane.During the course of mastitis,LPS will enter into the blood through the diseased breast tissue,leading to systemic inflammation and septicopyemia in dairy cow.As an important part of the innate immune system of mammary gland,the bovine mammary epithelial cells play a key role in resisting pathogen.Apoptosis of mammary epithelial cells is closely related to the immune defense about mastitis.Studying the molecular mechanism of apoptosis of mammary epithelial cells induced by LPS in dairy cow will laid a foundation for the prevention and treatment of mastitis.In present study,firstly,the bovine mammary epithelial cells(BMECs)were isolated by enzyme digestionin vitro,then were purified and identified.Secondly,using the BMECs as a cell model,the effects of LPS on cell proliferation,inflammatory factor secretion and apoptosis were measured.The results shown that LPS could inhibit the cell proliferation and promoted the secretion of TNF-α and IL-18 in time-dependence manners.LPS could induce the apoptosis of BMECs in time-and concentration-dependent manners.These data indicated that LPS may induce the apoptosis of the BMECs through promoting the secretion of inflammatory factor,and then inhibite the proliferation of the BMECs.Thirdly,the pathway of apoptosis in the BMECs were measured using Molecular Cytology.The results shown that LPS improved the levels of TLR-4,MyD88,and FADD proteins,but decreased the level of Caspase-8 precursor protein.The secretion of TNF-α induced by LPS was significantly decreased after the silence of MyD88 while there was no significant difference aboutthe silence of MyD88 compared to control.These results demonstrated that LPS was recognized by membrane receptor TLR-4,and induced the apoptosis of BMECs via MyD88-dependent signaling pathway and extracellular death receptor pathway.Lastly,to study the role of mTORC1 pathway in LPS inducing cell apoptosis,the BMECs were treated by Rapamycin,(a specific inhibitor of mTORC1 signaling pathway).The results shown that LPS could activate the mTORC1 pathway,however,this activation was inhibited by Rapamycin.In addition,Rapamycin suppressed the degradation of pro-Caspase-3 and,the expression of MyD88 or FADD gene and the phosphorylation of STAT1 induced by LPS.These results suggested that mTORC1 signaling pathway perhaps regulatedthe expression of apoptosis-related proteins,such as MyD88,FADD,and Caspase-3,through regulating the level of phosphorylation of transcription factor STAT1,leading to mediating the LPS-induced apoptosis of the BMECs.In conclusion,LPS could induce the apoptosis of the BMECs.LPS was recognized by membrane receptor TLR-4 and aggregated the adapter protein MyD88,constituting the LPS/TLR4/MyD88 signaling pathway.Accordingly,the LPS/TLR4/MyD88 signaling pathway activated the transcription factor STAT1 and promoted the secretion of TNF-α in BMECs.Moreover,when the BMECs were treated by LPS,thelevel of FADD increased and the level of pro-Caspase-8 decreased,indicating that LPS may induce the apoptosis of BMECs through regulating the classical death receptor pathway.The mTORC1 signaling pathway regulates the expression of apoptosis-related proteins such as MyD88,FADD,and Caspase-3,which may be achieved by regulating the transcription factor STAT1 activity.