The Effects Of Cortisol On The Changes Of NF-?B And MAPK Signaling Pathways In Bovine Endometrial Epithelial Cells Induced By Escherichia Coli

Endometritis usually occurs in postpartum dairy cows,resulting from the bacterial infections.It causes many reproductive problems of dairy cows and leads to great economic loss.Increased level of endogenic cortisol(COR)was observed during parturition.The high cortisol level is associated with the endometrial innate immunity against pathogen like Escherichia coli(E.coli),although mechanism details underlying how it regulates E.coli-induced inflammatory responses remain elusive.In this study,we used E.coli-induced bovine endometrial epithelial cells(BEECs)as the inflammatory model,and investigated the effect of cortisol on the inflammatory response of BEECs,providing theoretical foundation for the prevention and treatment of bovine endometritis.1?The effect of cortisol on pro-inflammatory gene expressions in LPS-,heat-killed E.coli-or live E.coli-induced BEECsThe cells were treated with 1 ?tg/mL LPS,1 × 108 CFU/mL heat-killed E.coli or 1 × 106 CFU/mL live E.coli as inflammatory model groups.In test groups,the cells were co-treated with 5,15 or 30 ng/mL COR.After treated for different time,the gene expressions of IL-1?,IL-6,IL-8,TNF-? and TLR-4 mRNA were measured by qPCR.The results showed that,as compared with control group,TLR-4 mRNA expressions increased(p<0.01)only at 6 h,and IL-1?,IL-6,IL-8 and TNF-? mRNA increased(p<0.01)at 2,6 or 18 h after LPS challenged.Compared with LPS group,COR decreased(p<0.05)the pro-inflammatory gene expressions at most observed time points.Exposure of cells to heat-killed E.coli for 6,18 and 48 h upregulated(p<0.01)the mRNA expressions of IL-1?,IL-6,IL-8,TNF-? and TLR-4 genes.Compared with heat-killed E.coli group,COR generally downregulated(p<0.05)these gene expressions.After treated by live E.coli,the mRNA expressions of IL-1?,IL-6,IL-8 and TNF-? increased(p<0.01)at 2,4 and 6 h,the TLR-4 mRNA decreased(p<0.01)at 4 and 6 h.Compared with live E.coli group,COR generally increased(p<0.05)these mRNA expressions in most time points.These results indicated that cortisol had different effects on inflammatory gene expressions in LPS-,heat-killed E.coli-or live E.coli-induced BEECs.2?The effects of cortisol on changes of NF-?B and MAPK signaling pathways in heat-killed E.coli-or live E.coli-induced BEECsThe cells were treated with 1 × 108 CFU/mL heat-killed E.coli or 1 × 106 CFU/mL live E.coli as inflammatory model groups.In test groups,the cells were co-treated with 5,15 or 30 ng/mL COR.After heat-killed E.coli treated for 120 or live E.coli treated for 30 min,the protein expressions of NF-?B and MAPK signaling pathways were measured by Western blot,and the activation of p65 was visualized by immunofluorescence.The results showed that,the protein expressions of P-p65,P-I?B?,P-p38 and P-JNK in heat-killed E.coli group were higher than those in control group(p<0.01).COR decreased(p<0.05)the expressions of these proteins compared with heat-killed E.coli group.There was no significant change in the protein level of p-ERK in different groups.After challenged with live E.coli,the phosphorylation levels of p65,I?B?,p38,JNK and ERK were increased(p<0.05).Compared with live E.coli group,COR upregulated(p<0.05)the phosphorylation levels.The results of immunofluorescence indicated that the level of p65 in nucleus increased in both model groups.Nuclear p65 in COR co-treated group was less than that in heat-killed E.coli group.Compared with live coli group,COR co-treated with live E.coli increased the p65 level in nucleus.These results indicated that cortisol exerted different effects on protein expressions of NF-?B and MAPK signaling pathways in heat-killed E.coli-or live E.coli-induced BEECs.3?The effects of cortisol and RU486 on inflammatory response in heat-killed E.coli-induced BEECsIn order to investigate whether the regulation of cortisol on the inflammatory response of heat-killed E.coli-induced BEECs depends on the glucocorticoid receptor,we used 1 × 108 CFU/mL heat-killed E.coli treated BEECs as inflammatory model groups.Cells that co-treated with E.coli and 30 ng/mL COR was COR group,co-treated with E.coli and 35 ng/mL RU486 was RU486 groups,co-treated with E.coli,COR and RU486 was co-treated group.The gene expressions of inflammatory facters were detected at 18 h,the protein expressions in MAPK and NF-?B signaling pathways were detected at 120 h.As the results showed,the mRNA expressions of IL-1?,IL-6,IL-8,TNF-? and TLR-4,and the protein expressions of P-p65,P-I?B?,P-p38,P-JNK and P-ERK in the model group was higher than control group(p<0.01).In the CORgroup,the expressions of genes and proteins were lower(p<0.01)than model group.There was no significant change in the expressions of genes and proteins between RU486 group and model group.Compared with COR co-treated group,the level of genes and proteins increased(p<0.01)in the co-treated group.The results of immunofluorescence indicated that the level of p65 in nucleus increased in model group.Nuclear p65 in COR group was less than that in model group and co-treated group There was no significant difference between RU486 group and model group.The present study demonstrated that the effect of cortisol on the expressions of inflammatory genes and NF-?B and MAPK signaling pathways in heat-killed E.coli-induced BEECs may exerted through the glucocorticoid receptor.