The Effects Of Cortisol On The Innate Immunity And Proliferation Of Inflammatory Endometrium In Dairy Cows

The concentration of cortisol elevates in response to various stresses in postpartum dairy cows,and this increases the risk of uterine infection and delays uterine involution.The endometrium is mainly composed of epithelial cells and stroma cells,which promote the repair of endometrium synergistically.Bovine endometrial stromal cells(BESCs)expose to pathogenic microorganisms due to the damage of the epithelial layer during delivery.It is important to clarify the effects of cortisol on innate immunity and proliferation of BESCs.In this study,the primary BESCs was used as model and treated with LPS and concentrations of cortisol.We used CCK-8,RT-qPCR,Western blot,flow cytometry and immunofluorescence to clarify the effects of cortisol on LPS-induced innate immune response and proliferation and the underlying mechanism.Goats were used as models with 3%acetic acid and E.coli infused into the uterine and cortisol injected into muscles.The effect of cortisol on the proliferation and repair of inflammatory endometrium was investigated using paraffin section,RT-qPCR and immunohistochemistry.This study provides a theoretical basis for understanding the pathogenesis of endometritis in dairy cows under stress.1.The inhibitory effects of cortisol on innate immunity/inflammatory response induced by LPS in BESCsThe isolation and cultivation of BESCs was successfully established using collagenase Ⅱ.The effects of concentrations of cortisol(5,15,and 30 ng/mL)on the inflammatory response in LPS-induced BESCs was detected using RT-qPCR,Western blot and immunofluorescence.The mRNA expression levels of cytokines(IL-1β,IL-6,IL-8,TNF-α,COX-2,and iNOS)was elevated by LPS(p<0.01).Concentrations of cortisol significantly suppressed the mRNA expression levels of cytokine in LPSinduced BESCs(p<0.05,p<0.01).Cortisol significantly suppressed the expression of COX-2 and iNOS protein in LPS-induced BESCs(p<0.01).The phosphorylation of IκBα,p65,ERK,JNK,and p38 was significantly increased by LPS compared with control group(p<0.05,p<0.01).Cortisol downregulated the phosphorylation of IκBα,p65,ERK,JNK,and p38 induced by LPS(p<0.05,p<0.01).The translocation of p65 into nuclear induced by LPS was inhibited by cortisol(p<0.01).These results revealed that cortisol inhibited inflammatory response induced by LPS by downregulating the activation of the NF-κB and MAPK signaling pathways in BESCs.2.The inhibitory effects of cortisol on proliferation in LPS-induced BESCsThe LPS-induced BESCs was used to investigate the effects of cortisol on the proliferation of BESCs using CCK-8 assay,flow cytometry,RT-qPCR,and Western blot.Treatment of BESCs with LPS promoted the expression of PCNA protein(p<0.05),decreased the number of cells in G0/G1 phase(p<0.01),and increased the number of cells in S phase significantly(p<0.01).LPS promoted the migration and proliferation significantly in BESCs(p<0.01).Compared with LPS group,the expression of PCNA protein was significantly decreased(p<0.05,p<0.01),and the number of cells in S phase was significantly decreased after co-treatment with different concentrations of cortisol and LPS(p<0.05,p<0.01).Cortisol significantly inhibited the migration and proliferation in LPS-induced BESCs(p<0.05,p<0.01).The phosphorylation of PI3K,AKT,and GSK3β protein and the expression of β-Catenin,c-Myc,and Cyclin D1 protein were increased significantly in LPS-induced BESCs(p<0.05,p<0.01),and these were suppressed by cortisol significantly(p<0.05,p<0.01).Cortisol downregulated the protein level of β-Catenin in the nuclear significantly in LPS-induced BESCs(p<0.05,p<0.01).Moreover,the mRNA expression levels of growth factors(TGF-β1,TGF-β3,VEGF,FGF-2,and MMP-9)was elevated by LPS significantly in BESCs(p<0.05,p<0.01).Cortisol significantly suppressed the mRNA expression levels of growth factors significantly in LPS-induced BESCs(p<0.01).These results showed that cortisol could inhibit proliferation of BESCs via suppressing the activation of the PI3K/AKT/GSK3β and Wnt/β-Catenin signaling pathways and downregulating of mRNA expression levels of growth factors in LPS-induced BESCs.3.Blocking GR reversed the effects of cortisol on proliferation in LPS-induced BESCsGR antagonist,AL082D06(D06)was used to investigate the role of GR in cortisol inhibited proliferation of LPS-treated BESCs.Compared with LPS and cortisol cotreatment group,D06 significantly increased the expression of PCNA protein,decreased the cell number in the G0/G1 phase,increased the cell number in the G0/G1 phase,promoted the migration of LPS-induced BESCs(p<0.05).Compared with the LPS and cortisol co-treatment group,the phosphorylation levels of PI3K,AKT and GSK3β and the expressions of β-Catenin,c-Myc and CyclinDl proteins were significantly increased by D06(p<0.05,p<0.01),The protein level of β-Catenin in the nucleus was significantly enhanced by D06(p<0.01).Compared with the LPS and cortisol cotreatment group,the mRNA expression levels of TGF-β1,TGF-β3,FGF-2,VEGF and MMP-9 were significantly increased after blocking GR(p<0.05,p<0.01).These results indicated that blocking the GR could reverse the inhibitory effect of cortisol on the proliferation of BESCs.AL082D06 significantly reserved the cell viability,PCNA expression,migration and cell cycle phase inhibited by cortisol in BESCs(p<0.05).The phosphorylation of PI3K,AKT,and GSK3β induced by LPS was downregulated cortisol significantly(p<0.05,p<0.01),while these was increased by AL082D06 significantly in BESCs(p<0.05,p<0.01).The expression of β-Catenin,cMyc,and Cyclin D1 induced by LPS was suppressed by cortisol significantly(p<0.01),and was increased by AL082D06 significantly in BESCs(p<0.05,p<0.01).Moreover,the translocation of β-Catenin was suppressed by cortisol in LPS-induced BESCs(p<0.01),and was accelerated by AL082D06(p<0.01).The expression levels of growth factors were inhibited by cortisol in LPS-induced BESCs(p<0.01),which was elevated by AL082D06(p<0.05,p<0.01).These results demonstrated that the inhibitory effect of cortisol on cell proliferation may be mediated through GR affecting the activation of the PI3K/AKT/GSK3β and Wnt/β-Catenin signaling pathways and the expression levels of growth factors in BESCs.4.The effects of cortisol on histopathology and proliferation-related factors of endometrium in bacterial-infected goatsThe dairy goats were used to establish endometrial infection model by uterine perfusion of E.coli after uterine injury with 3%glacial acetic acid.The stressful state(high levels of cortisol)is achieved by intramuscular injection of cortisol.The changes in endometrial proliferation and repair were observed.The trials were divided into control group,glacial acetic acid group,glacial acetic acid+E.coli group,glacial acetic acid+E.coli+cortisol group,and glacial acetic acid+ cortisol group.The results showed that the respiratory rate,heart rate and body temperature of goats increased significantly after 12 h of E.coli infection compared with glacial acetic acid group(p<0.05).After E.coli infection,a large number of polymorphonuclear neutrophils were observed in the uterine secretions.The endometrial epithelial layer was significantly damaged and shed,a large number of inflammatory cells were infiltrated in the lamina propria,and part of the uterine glands were destroyed.The above pathological changes were significantly reduced in goats of high cortisol group.Compared with the glacial acetic acid group,the endometrial thickness,the number of glands,and the number of PCNA-positive cells in goats of the glacial acetic acid+ E.coli group increased significantly(p<0.01),and the mRN A expression levels of TGF-β1,VEGF,FGF-2,and PCNA was also significantly increased(p<0.05,p<0.01).The above changes were significantly inhibited in goats of the glacial acetic acid+E.coli group(p<0.05,p<0.01)compared with the glacial acetic acid +E.coli group.The above results showed that cortisol could inhibit the innate immunity response as well as the proliferation and repair of the endometrium after E.coli infection.In summary,cortisol inhibits the innate immune response/inflammatory response and cell proliferation of BESCs,and this inhibitory effect of cortisol is mediated by GR.High levels of cortisol inhibited the proliferation and repair of inflammatory endometrium after E.coli infection.