The Effect Of ?-endorphin On Lipopolysaccharide-induced NF-?B Signaling Pathway In Bovine Endometrial Stromal Cells

Postpartum endometritis in cows is an acute inflammation of the endometrium.If not treated in time,endometritis can easily cause chronic inflammation and lead to long-term infertility.Endometritis causes damage to the endometrial glands,delayed endometrial regeneration,and the internal environmental changes of fallopian tubes,resulting in reduced milk production,prolonged calving interval,increased elimination rate,and huge economic losses.One of the main pathogenic bacteria causing endometritis is E.coli.LPS is the main component of E.coli cell wall involved in the adhesion and infection of E.coli in the process of infecting the host,and causes the immune response of the tissue.The NF-?B signaling pathway plays an important role in immune regulation and inflammation.LPS can act on the cell TLR4,participate in the activation of nuclear factor ?B signaling pathway,and lead to the synthesis and release of downstream inflammatory factors.Beta-endorphin is a naturally-occurring opioid peptide in mammals,and ?-EP and its receptors have been confirmed to be presented in the endometrium stroma and epithelial cells.Studies have shown that ?-EP can inhibit inflammation through the NF-?B signaling pathway,but no research on innate immunity of the endometrium has been reported yet.By using the bovine endometrial stromal cell,the present study explored the effect of ?-EP on NF-?B signaling pathway.1.Effects of ?-EP on NF-?B signaling pathway in bESC cells and its downstream inflammatory factorsCells were treated with different concentrations of ?-EP alone.The changes of cell activity were tested by CCK-8 method.The expression changes of inflammatory related genes TLR4,MyD88,IL-1?,TNF-?,IL-6,IL-8,COX-2 and iNOS were detected by fluorescence quantitative PCR.Western blot was used to test the changes of NF-?B pathway key proteins MyD88,P-p65,p65,P-I?B? and I-?B?.0?2.5 × 105 pg/mL ?-EP showed no effect(p>0.05)on cell viability.Beta-EP(0,20,80,200 pg/mL)showed no effect(p>0.05)on the gene expressions of TLR4,MyD88 and inflammatory factors at 4,12,and 24 h,or the expressions(p>0.05)of NF-?B key proteins at 30 min.2.Effects of ?-EP on NF-?B signaling pathway induced by LPS in bESC cells and its downstream inflammatory factors.The experiment was divided into blank group,LPS group(1 ?g/mL),and LPS plus?-EP(20,80,200 pg/mL)co-treatment group.Fluorescence quantitative PCR was used to detect the expression changes of cell inflammatory related genes,and Western blot was used to detect the changes of NF-?B pathway key proteins.Compared with the blank group,the inflammatory factor gene expressions in the LPS group increased(p<0.05)at 4,12,and 24 h.The levels of key proteins of NF-?B signaling pathway increased(p<0.05)at 30 min.Compared with the LPS group,the gene expressions of TLR4,MyD88 and inflammatory factors in the co-treatment group of ?-EP(20,80,200 pg/mL)and LPS at 4,12,and 24 h,and the protein levels of NF-?B key proteins at 30 min were down-regulated(p<0.05).3.Effects of opioid receptor antagonist on ?-EP-regulated NF-?B signaling pathway and its downstream inflammatory factorsThis experiment was divided into blank group,LPS group(1?g/mL),LPS and opioid receptor antagonist co-treatment group,LPS and ?-EP co-treatment group,and LPS,?-EP plus opioid receptor antagonist co-treatment group.The changes of cell activity were tested by CCK-8 method.Fluorescence quantitative PCR was used to detect the expression changes of cell inflammatory related genes,and Western blot was used to detect the changes of NF-?B pathway key proteins.There were no effect(p>0.05)on cell viability by 0?2.5 × 105 pg/mL naloxone,ICI 154129,or CTAP.Compared with the co-treatment group of LPS and ?-EP,the gene expressions of TLR4,IL-8,and iNOS increased(p<0.05)at 24 h,the protein level of MyD88 and the ratio of P-p65/p65 increased(p<0.05)at 30 min in the co-treatment group of LPS,?-EP and naloxone.No change in P-I?B?/I?B? ratio was detected(p>0.05).Compared with the co-treatment group of LPS and ?-EP,all gene expressions of inflammatory factors,except the TNF-?,increased(p<0.05)at 24 h,the ratios of P-p65/p65 and P-I?B?/I?B? increased(p<0.05)at 30 min in the co-treatment group of LPS,?-EP and ICI 154129.No change(p>0.05)was observed in the MyD88 protein level.There was no change(p>0.05)in gene expression of inflammatory factor in the co-treatment group of LPS,?-EP and CTAP.In conclusion,?-EP suppressed the LPS-induced cell inflammation,reduced the expressions of inflammatory factors,and inhibited the activation of NF-?B signaling pathway.This inhibitory effect can be partially reversed by the non-selective opioid receptor antagonists naloxone and the ? receptor antagonist ICI 154129.It suggested that?-EP may inhibit the LPS-induced cell inflammatory responses through ? opioid receptors in bESC.