A Preliminary Study On The Effects Of Estradiol/progesterone On The Release Of Neutrophil Extracellular Traps In Swine

During sow breeding,a healthy physiological state is one of the keys to ensuring the economic effects of the pig industry.As an important part of innate immunity,neutrophil extracellular traps(NETs)is a three-dimensional network structure released by neutrophils,which can capture and destroy pathogens.So,the proper release of NETs helps the immune system eliminate pathogens and is beneficial to the body.However,excessive release or untimely degradation of NETs can cause tissue damage and autoimmune diseases.Estradiol(E2)and progesterone(P4)are key signal molecules that regulate sows’ estrus and pregnancy.At the same time,a large number of research reports also confirmed that E2 and P4 also have a regulatory effect on the immune system.However,the effects of E2 and P4 on the formation of porcine neutrophil extracellular trapping nets have not been reported.In this study,different concentrations of E2(5,10,20,40 ng/mL)and P4(5,10,20,40,80 ng/mL)were selected to explore their effects on NETs formation and their potential molecules mechanism.After incubating E2 with neutrophils for 2 h,the formation of NETs was detected by laser confocal microscopy and pico-green quantitative kit.The results showed that E2(10,20,40 ng/mL)could significantly induce the formation of NETs.In specific function inhibitors and western boltting experiments,it was found that E2 induced NETs formation depends on estrogen receptor α(ERα),protein arginine deiminase 4(PAD4),and ERK1/2(extracellular regulated protein kinases)and other signaling pathways.The above results indicate that E2(10,20,40 ng/mL)can induce the formation of NETs and depends on signal pathways such as ERa,PAD4 and ERK1/2.After co-incubating P4 and neutrophils for 2 h,it was observed that P4(40 ng/mL)induced the formation of NETs with a network structure under a laser copolymerization microscope.Quantitative experiment results found that compared with the control group,P4(20,40,80 ng/mL)significantly increased the level of induced release of NETs.Through the use of specific functional inhibitors,Cl-amidine and UO126 could significantly inhibit P4-induced NETs.The above results indicate that P4(20,40,80 ng/mL)can significantly induce the release of NETs and depends on signal pathways such as PAD4 and ERK1/2.In addition,after neutrophils were treated with E2 and P4 in combination for 2 h,the results showed that P4 could attenuate the level of E2 induced NETs,but there was no significant difference compared with the group receiving E2 alone.The above results indicate that there is a mutual regulation effect between E2 and P4,and they participate in the regulation process of NETs release.All in all,this paper confirms that E2(10,20,40 ng/mL)and P4(20,40,80 ng/mL)could induce the formation of NETs,both of which could regulate the release of NETs,and suggestted that the release process of NETs was related to signal pathways such as PAD4 and ERK1/2.The above conclusions can try to further clarify the relationship between reproductive hormones and the immune system during pregnancy and provide new ideas for disease control and prevention in pregnant sows.