| The opening of the birth canal often leads to the infection of the uterus by pathogenic bacteria and causes diseases such as metritis or endometritis in postpartum dairy cows,which results in the decline in the breeding performance and huge losses to the breeding industry.Escherichia coli(E.coli)is a common pathogenic microorganism that induces endometritis in cattle.Its endotoxin lipopolysaccharides(LPS)can activate nuclear factor kappa B(NF-κB)signaling pathway by combining toll-like receptor 4(TLR4),which induces the expression and release of downstream proinflammatory cytokines and causes the inflammatory response.Excessive inflammation can cause the uterine tissue damage,resulting in delayed uterine involution.Beta-endorphin(BEP)is an endogenous opioid peptide that acts by binding to the opioid receptors on cell membrane.Evidence has shown that plasma BEP concentrations in dairy cows elevated during the metritis.BEP has also been found to inhibit inflammation through the NF-κB signaling pathway.However,the effect of BEP on the inflammatory response of the bovine endometrium has not been clarified.The aim of this study was to explore the effects of BEP on the inflammatory response and NF-κB pathway in bovine endometrial epithelial cell(BEEC).The primary BEEC was used and stimulated with LPS.The opioid antagonists,were used to clarify the potential mechanism.1.The effect of BEP on LPS-induced NF-κB activation and gene expression of downstream inflammatory cytokines in BEECIn order to explore the influence of BEP on NF κB pathway and the gene expression of downstream inflammatory cytokines in BEEC,the cells were treated with various concentrations of BEP(2,20,200,2000,20000 pg/mL or 20,80,200 pg/mL).The cell viability was detected by CCK-8 assay.The gene expressions of TLR4,myeloid differential primary response gene 88(MyD88)and the downstream proinflammatory factors including interleukin 1β(IL1B),interleukin 6(IL6),C-X-C motif chemokine ligand 8(CXCL8),tumor necrosis factor α(TNF)and prostaglandin-endoperoxide synthase 2(PTGS2)were detected by quantitative RT-PCR.The key protein levels in NF-κB pathway such as MyD88,the inhibitor of NF-κB α(IκBα)and NF-κB subunit p65 were detected by western blot.As a result,the cells treated with 2,20,200,2000 and 20000 pg/mL BEP or co-treated with LPS and BEP for 24 h showed no change in the cell viability(P>0.05).The expressions of TLR4,MYD88,IL1B,IL6,CXCL8,TNF and PTGS2 were not influenced by 20,80,or 200 pg/mL BEP at observed time point(P>0.05).After treated with 200 pg/mL BEP for 30 min,the protein levels of MyD88,pκBα and p-p65 showed no change(P>0.05).These results indicated that BEP alone had no effect on BEEC cell viability,NF-κB pathway and the expression of downstream inflammatory cytokines.To investigate the effects of BEP on the NF-κB pathway and cell inflammatory response in LPS-stimulated BEEC,the mRNA abundance of TLR4,MyD88,and downstream inflammatory factors were detected by quantitative TR-PCR.The NF-κB pathway and MyD88 protein level were detected by western blot.As a result,compared with blank control,the mRNA abundance of TLR4,MYD88,IL1B,IL6,CXCL8,TNF and PTGS2,and the protein level of MyD88,p-IκBα and p-p65 increased after LPS treatment(P<0.05).Compared with LPS group,the mRNA abundance of inflammatory factors and the key protein level of NF-κB pathway decreased(P<0.05)in LPS and BEP(20,80,and 200 pg/mL)co-treatment groups.These results indicated that BEP inhibited the activation of NF-κB pathway and the gene expressions of IL1B,IL6,CXCL8,TNFs and PTGS2 in LPS-stimulated BEEC.2.The effects of opioid receptor antagonists on LPS-induced BEEC inflammatory responseTo explore the mechanism of BEP’s regulation on the inflammatory response in BEEC,the cells were co-treated with opioid receptor antagonists,LPS,and BEP.The opioid antagonists used in this study included nonselective opioid antagonist naloxone(Nx),the δopioid receptor antagonist ICI 154129,the μ opioid receptor antagonist CTAP,The groups were as follows:the blank control group,the LPS treatment group,the LPS and BEP(200 pg/mL)co-treatment group,the LPS and antagonist(20 pg/mL Nx,40 pg/mL ICI 154129,or 60 pg/mL CTAP)co-treatment group,the BEP and antagonist co-treatment group,and the LPS,BEP(200 pg/mL),and antagonist co-treatment group.In this study,the changes in the cell viability,the mRNA abundance of TLR4,MyD88,and downstream inflammatory factors,the protein levels of MyD88 and key proteins of NF-κB pathway were detected.As a result,the cell viability unchanged(P>0.05)in BEEC treated with different concentrations of Nx(0,2,20,200,2000 pg/mL),ICI 154129(0,4,40,400,4000 pg/mL)and CTAP(0,6,60,600,6000 pg/mL)or co-treated with LPS and antagonists for 24 h.Compared with the blank control,the mRNA abundance of TLR4,MYD88,IL1B,IL6,CXCL8,TNF and PTGS2,and the protein levels of MyD88,p-IκBα and p-p65 increased(P<0.05)in LPS treatment group,unchanged(P>0.05)in BEP and antagonists co-treatment group.Compared with the LPS group,the gene expression of the inflammatory factors and the key protein level of NF-κB pathway decreased(P<0.05)in LPS and BEP co-treatment group and,the LPS and Nx co-treatment group.No change(P>0.05)was found in the ICI 154129 and LPS co-treatment group or the CTAP and LPS co-treatment group as compared the the LPS group.Compared the the LPS and BEP co-treatment group,the gene expression of the inflammatory factors and the key protein level of NF-κB pathway increased(P<0.05)in the LPS,BEP and Nx co-treatment group and the LPS,BEP and ICI154129 co-treatment group,and unchanged(P>0.05)in the LPS,BEP and CTAP group.These results indicated that Nx and ICI 154129 reversed the anti-inflammatory effect of BEP in BEEC,whaereas CTAP did not.In conclusion,BEP alone had no effect on BEEC NF-κB pathway.BEP alleviated the LPS-induced inflammatory response in BEEC by inhibiting the NF-κB signaling pathway and the mRNA level of downstream inflammatory factors.This anti-inflammatory effect of BEP was mediated by δ opioid receptor,rather than μ opioid receptors in BEEC. |
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