The Regulatory Effects Of Cortisol On LPS-induced Inflammatory Injury In The BEECs And RAW264.7 Cells

Cortisol,as an endogenous glucocorticoid,plays an important role in regulating normal physiological functions of body.During the perinatal period,the concentration of cortisol elevates significantly because dairy cattle undergoes multiple stresses,such as pregnancy stress,labor stress,lactation stress,metabolic stress,etc.And in this period,cattle is susceptible to many reproductive tract diseases.Bovine endometritis is the main cause of infertility after parturition,and also leads to huge economic losses in dairy industry.After calving,cervix is opened,and the cattle commonly challenges ascending bacterial contamination of the female genital tract.Escherichia coli is the most commonly isolated pathogenic bacteria from cases of uterine disease in cattle.Now although there are various of methods for preventing and treating the bovine endometritis,the standard treatment is not existed and the prognosis is not good,which lead to high elimination rate.Glucocorticoids have been at the front of therapies to control inflammatory diseases,and it is very effective in suppressing the inflammation caused by various pathogens.However,fewer studies have shown the effect of physiological level of cortisol on endometritis.In this study,the primary bovine endometrial epithelial cell(BEECs)and macrophage cell line(RAW264.7)were used as models,which were treated with LPS and different concentrations of cortisol.The effects of cortisol on related cytokines and key proteins in LPS-induced BEECs and RAW264.7 cells were investigated by CCK-8,qRT-PCR,Western Blot and immunofluorescence.These will clarify the regulatory mechanism of cortisol on LPS-induced inflammatory injury,which would provide scientific theoretical basis for preventing and curing bovine endometritis.1.The effects of LPS on NF-?B and MAPK signaling pathways in BEECs and RAW264.7 cellsThe primary BEECs were cultured by enzyme digestion,and the enzyme was protease from Streptomyces griseus.Then the epithelial cells were identified by immunohistochemistry.The BEECs and RAW264.7 cells were treated with LPS for different time points(0 min?15 min?30 min?45 min),after treatment,cells were collected to extract protein.The key proteins of NF-?B and MAPK signaling pathways were detected by Western Blot.The results showed that primary BEECs were successfully cultured.LPS promoted the phosphorylation levels of p65 and I?B as well as ERK1/2,JNK and p38 in BEECs and RAW264.7 cells(p<0.05),and the phosphorylation levels reached the peak at 30 min(p<0.01).The results demonstrated that LPS could induce the activation of NF-?B and MAPK signaling pathways in BEECs and RAW264.7 cells.2.The effects of cortisol on iNOS and COX-2 in LPS-induced BEECs and RAW264.7 cellsThe BEECs and RAW264.7 cells were treated with different concentration of cortisol(5 ng/mL?10ng/mL?15 ng/mL?20ng/mL?30ng/mL?60 ng/mL)for 24 h,and then the cell viability was detected by CCK-8.The BEECs and RAW264.7 cells were treated with 1 ?g/mL LPS and different concentration of cortisol(5 ng/mL,15 ng/mL and 30 ng/mL)for different time points,then mRNA and protein expression levels of iNOS and COX-2 were respectively detected by qRT-PCR and Western Blot.The concentration of inflammatory mediator PGE2 in supernatant was measured by ELISA.As the results,various concentrations of cortisol(5 ng/mL?10 ng/mL?15 ng/mL?20 ng/mL?30 ng/mL?60 ng/mL)did not have toxic effect on BEECs and RAW264.7 cells.The mRNA and protein expression levels of iNOS and COX-2 were increased in the LPS group compared with the control group,and the concentration of PGE2 was also higher in the supernatant of RAW264.7 cells(p<0.05).However,cortisol could inhibit the mRNA and protein expression levels of iNOS and COX-2,and reduce the concentration of PGE2 compared with the LPS group(p<0.05).The results showed that cortisol could inhibit the production of inflammatory mediator and protect cells from inflammatory injury by reducing the expressions of iNOS and COX-2.3.The effects of cortisol on NF-?B and MAPK signaling pathways in LPS-induced BEECs and RAW264.7 cellsThe BEECs and RAW264.7 cells were treated with l?g/mL LPS and different concentration of cortisol(5ng/mL,15ng/mL and 30ng/mL)for different time points.The mRNA expression levels of inflammatory factors(IL-1??IL-6?TNF-? and IL-8)were detected by qRT-PCR,the concentrations of inflammatory factors(IL-1??IL-6 and TNF-?)in supernatant were measured by ELISA,and the key proteins of NF-?B and MAPK signaling pathways were detected by Western Blot and immunofluorescence.The concentrations of IL-1??IL-6 and TNF-? in supernatant and the mRNA expression levels of IL-1??IL-6?TNF-? and IL-8 in the LPS group were higher in the LPS group compared with the control group(p<0.01).However cortisol could inhibit the mRNA expression levels of IL-1??IL-6?TNF-? and IL-8,and reduce the concentration of IL-1? in supernatant compared with the LPS group(p<0.05).In the signaling pathways,cortisol could inhibit the phosphorylation levels of p65 and I?B,as well as inhibit the phosphorylation levels of ERK1/2,JNK and p38 in LPS-induced BEECs and RAW264.7 cells compared with the LPS group(p<0.05).What is more,the inhibitory effect reached peak at 30 ng/mL cortisol(p<0.01).The results demonstrated that cortisol could reduce the expression of inflammatory cytokines induced by LPS,which may be achieved by inhibiting the activities of NF-?B and MAPK pathways.4.The effect of cortisol on repair mechanisms in LPS-induced BEECsThe BEECs were treated with 1?g/mL LPS and different concentration of cortisol(5 ng/mL,15 ng/mL and 30 ng/mL)for different time points.The mRNA expression levels of growth factors(TGF-?1?TGF-?3?CTGF and VEGF)were detected by qRT-PCR,and the key proteins of proliferative pathways(Wnt/?-catenin and PI3K/AKT)were detected by Western Blot and immunofluorescence.Compared with the control group,the mRNA expression levels of TGF-?1 and TGF-?3 were higher(p<0.01),the level of CTGF was lower at 3 h and 12 h,and was higher at 18 h(p<0.01),while there was no significant effect on VEGF.Compared with the LPS group,cortisol could inhibit the mRNA expression levels of TGF-?1,TGF-?3 and CTGF,but promote the mRNAlevel of VEGF(p<0.05).In the signaling pathways,LPS promoted the expression levels of ?-catenin,C-Myc and CyclinD1,as well as increased the phosphorylation levels of PI3K and AKT(p<0.05).However,cortisol could significantly reduce the expression levels of ?-catenin,C-Myc and CyclinDl(p<0.05),and inhibit the phosphorylation levels of PI3K and AKT(p<0.01).The results showed that cortisol could inhibit the activation of Wnt/?-catenin and PBK/AKT signaling pathways induced by LPS.In summary,cortisol can inhibit the activation of NF-?B and MAPK pathways as well as the expression of inflammatory mediators and cytokines in LPS-induced BEECs and RAW264.7 cells,which reduced the damage caused by inflammation.Furthemore,cortisol suppressed the activation of Wnt/?-catenin and PI3K/AKT pathways as well as the expression of related growth factors in LPS-induced BEECs,which would help to reduce collagen deposition and hyperplasia.