Effect Of Escherichia Coli And Staphylococcus Aureus Infection On The Expression Of Apoptosis-related Factors In BEECs

Endometritis of dairy cows(Bostaurus)will have a significant impact on the fertility of dairy cows and restrict the development of the dairy industry.Postpartum bacterial infections are the leading cause of endometritis.Studies have shown that bacterial infections can cause apoptosis(Apoptosis),thereby affecting the development of the disease.In order to study the effect of Escherichia coli(E.coli)and Staphylococcus aureus(S.aureus)infection on bovine Endometrial epithelial cells(BEECs)of cows on related apoptosis factors Bax,Caspase-3 and Bcl-2,Mainly through the following experiments:(1)Isolation and culture of bovine endometrial epithelial cells:BEECs were obtained by two-step enzymatic digestion,cells were purified by differential adherence method for 30 mins,and cells containing 10%FBS were used.The culture medium was cultured and then subjected to immunocytochemical identification.(2)The BEECs after purification for 6th generation were treated with different concentrations of E.coli and S.aureus(0,10~3,10~4,10~5,10~6,10~7 CFU/mL).After 12hours of infection,the TUNEL kit was used to detect apoptosis of cells after bacterial infection.(3)After the purified 6th generation of BEECs were cultured until the cell monolayer was filled with the bottom of the well,different concentrations of E.coli or S.aureus were added for 12 h,real-time quantitative PCR(Quantitative Real-time PCR,qRT-PCR).The mRNA levels of Bax,Caspase-3 and Bcl-2 in BEECs were detected.(4)After the purification of 6th generation of BEECs,different concentrations of E.coli and S.aureus(0,10~3,10~4,10~5,10~6,10~7 CFU/mL)were infected for 12 h,Western blotting(Western-blotting,WB)assays for protein expression of Bax,Caspase-3 and Bcl-2.research shows:(1)Endometrial epithelial cells with morphology and growth pattern conforming to epithelial cells were obtained by two-step enzymatic digestion,and immuno cytochemical identification showed that the cultured cells were cow endometrial epithelial cells.(2)The apoptosis of cells was detected by Tunel.The results showed that the apoptosis rate was the highest at the concentration of 10~7CFU/mL in different concentrations of E.coli and S.aureus infection groups,and the apoptosis rate was significantly higher than other groups.Increased(P<0.05).(3)qRT-PCR results showed that Bax had the highest expression at 10~6 CFU/mL at different concentrations of E.coli infection,which was significantly increased compared with other groups(P<0.05).Caspase-3 had the highest expression at E.coli concentration of 10~7 CFU/mL,which was significantly increased compared with the other groups(P<0.05).The expression of Bcl-2 was the lowest at E.coli concentration of 10~7CFU/mL,which was significantly lower than that of other groups(P<0.05).The expression of Bax was the highest at concentrations of 10~6 CFU/mL under different concentrations of S.aureus infection.Significantly increased compared with the other groups(P<0.05).Caspase-3 had the highest expression at the concentration of S.aureus 10~7 CFU/mL,which was significantly increased compared with the other groups(P<0.05).The expression of Bcl-2 was the lowest at the concentration of S.aureus at 10~7 CFU/mL,which was significantly lower than that of the other groups(P<0.05).(4)WB results showed that the expression of Bax protein was highest at the concentration of 10~6CFU/mL under different concentrations of E.coli infection,and significantly increased compared with other groups(P<0.05).The expression level of Caspase-3 protein was the highest at E.coli concentration of 10~7 CFU/mL,which was significantly increased compared with the other groups(P<0.05).The expression of Bcl-2 protein was the lowest at E.coli concentration of 10~3CFU/mL,which was significantly lower than that of other groups(P<0.05).The expression of Bax protein at different concentrations of S.aureus infection was at 10~6 CFU/mL.The highest was significantly increased compared with the other groups(P<0.05).The expression level of Caspase-3 protein was highest at the concentration of S.aureus 10~7 CFU/mL,which was significantly increased compared with the other groups(P<0.05).The expression of Bcl-2 protein was the highest at the concentration of S.aureus 10~6 CFU/mL,which was significantly increased compared with the other groups(P<0.05).As the bacterial concentration continued to increase,the protein of Bcl-2 expression decreased.In vitro cultured BEECs in different concentrations of E.coli and S.aureus infection,with the increase of liquid concentration,BEECs apoptosis rate will increase,the amount of mRNA expression of Bax and Caspase-3 to a rise in the trend,the amount of mRNA expression of Bcl-2 to a downward trend,and the expression of Bax and Bcl-2 situation reflects the two mutual antagonism effect of apoptosis factors.The protein expression of Bax,and caspase-3 also showed the same trend as their mRNA expression,The expression level of Bcl-2 protein showed a low expression status as a whole,and its changes showed a certain correlation with the expression levels of Bax and Caspase-3.The results of this study showed that E.coli and S.aureus could induce the apoptosis of endometrial epithelial cells,which could provide reference for the study of the related mechanism of endometritis in cows.