Study Of Mechanisms Of Neutrophil Extracellular Trap-mediated Endometrial Inflammation And Tissue Damage In Dairy Cows

Endometritis is a pathogen-induced inflammation of the endometrium in dairy cows,commonly associated with infertility and abortion,and causing significant economic losses to the dairy farming industry.Neutrophil extracellular traps（NETs）are an immune defense strategy employed by neutrophils（polymorphonuclear neutrophils,PMN）.However,uncontrolled NETs release can exacerbate tissue inflammation and damage.Although it has been reported that NETs can induce tissue inflammation and damage via pyroptosis,their involvement in the pathogenesis of endometritis in cows remains to be further studied.To investigate the role of NETs in the occurrence and development of dairy cow endometritis,the current study explores the correlation between NETs and tissue inflammation and damage in the cow endometrium.The study demonstrates that NETs induce endometrial epithelial cell pyroptosis,and participate in endometrial inflammation and damage.Additionally,the research sheds light on the mechanism of NETs-mediated endometrial epithelial pyroptosis.The research contents and results are as follows:1.Study on the relationship between NETs and tissue inflammation and tissue damage in cow endometriumThe aim of this study was to investigate the correlation between NETs,endometrial epithelial tissue inflammation,and damage in dairy cows.To this end,endometrial epithelial tissue samples were collected from 14 dairy cows,and were divided into two groups（endometritis group:PMN≥18%,n=10;healthy group:PMN<18%,n=4）.Scanning electron microscopy,immunofluorescence assay（IFA）,Pico Green kit detection,and enzyme-linked immunosorbent assay（ELISA）were used to detect the presence of NETs and inflammatory factors in the collected tissues.Spearman correlation analysis was conducted to examine the correlation between the variables tested.The results showed that NETs were formed in the endometritis group,but not in the healthy group.Moreover,the levels of inflammatory factors and LDH were significantly higher in the endometritis group than in the healthy group,and were positively correlated with NETs in tissues.These findings suggest that NETs are formed during the pathogenesis of cow endometritis,and are associated with inflammation and damage of cow endometrial epithelial tissue.2.Studies on the role of NETs in mediating endometrial inflammation and injury at the cellular and animal levelsThe aim of this study was to investigate the role of NETs-mediated endometrial inflammation and injury from cell and animal models by inhibiting the effect of NETs.Firstly,bovine endometrial epithelial cells（BEEC）were co-incubated with NETs,and DNase I was used to inhibit the function of NETs.Immunofluorescence assay（IFA）results showed that NETs triggered BEEC cell expansion,membrane rupture,and nuclear degradation.Furthermore,IFA and Western blot（WB）results demonstrated that NETs significantly increased the expression levels of BEEC pyroptotic proteins（caspase 1,caspase 4,ACS,GSDMD,and NLRP3）.ELISA assays showed that NETs significantly increased the expression levels of inflammatory cytokines and LDH in BEEC supernatants.Next,NETs lacking high mobility group protein 1（HMGB1）were generated through pretreatment of PMN with glycyrrhetinic acid and co-incubation with BEEC.ELISA results showed that this approach led to a significant reduction in inflammatory factors and LDH levels in BEEC.Finally,Cl-amidine was used to inhibit the formation of NETs induced by lipopolysaccharides（LPS）in rat uterus.Through Western blotting,histopathological observation,and ELISA experiments,the results showed that Cl-amidine inhibited the formation of NETs in rat uterine tissue,reduced LPS-induced rat uterine congestion and swelling,endometrial epithelial cell necrosis,and PMN infiltration,and significantly reduced inflammatory cytokines in the tissue.The findings suggest that NETs mediate BEEC pyroptosis leading to cellular inflammation and injury,and that NETs lacking HMGB1 are less capable of inducing BEEC inflammation and injury.In addition,in vivo experiments in rats showed that inhibition of NETs formation could effectively alleviate endometrial inflammation and injury.3.Study of the signaling pathway mediating BEEC pyroptosis by HMGB1 released by NETsPrevious studies have established the important role of HMGB1 in NETs in inducing BEEC pyroptosis,but the mechanisms are not yet fully understood.In this study,TLR2^-/-,TLR4^-/-,and RAGE^-/-BEEC cell lines were generated through si RNA-mediated knockdown of HMGB1-related receptors.The interaction between BEEC and HMGB1 was explored by co-incubating HMGB1-EGFP（HMGB1-enhanced green fluorescent protein）with BEEC.The results of the immunofluorescence assay（IFA）revealed that HMGB1 enters BEECs through interaction with RAGE receptors.Subsequently,the results of IFA,Western blotting（WB）,and enzyme-linked immunosorbent assay（ELISA）demonstrated that HMGB1 enters the cell through the RAGE receptor,enters the lysosome,and induces lysosome rupture.The resulting lysosomal damage activates cat B,NLRP3 inflammasome,caspase 1,and ultimately leads to pyroptosis,as well as the release of inflammatory cytokines and LDH.In RAGE^-/-BEEC cell lines,HMGB1-induced lysosomal rupture and cat B activation were markedly inhibited,thereby further inhibiting NLRP3 inflammasome activation and pyroptosis of BEEC.Dynasore was used to control the entry of HMGB1 into lysosomes by regulating the dynamin protein.After dynasore treatment,the activation of cat B was inhibited,which significantly attenuated the activation of NLRP3 inflammasome.CA-074me,MCC950,and AC-YVAD-CMK were used to inhibit cat B activation,which significantly reduced the activation of NLRP3 and caspase 1 induced by NETs,and consequently reduced the NETs-induced BEEC pyroptosis.Finally,antibodies were used to neutralize HMGB1 in NETs,and the levels of cascade proteins in pyroptosis-related signaling pathways activated by NETs were measured.IFA results showed that NETs-induced lysosomal rupture,cat B activation,and NLRP3inflammasome expression levels and pyroptotic activation were significantly inhibited after neutralizing HMGB1.The neutralization of HMGB1 also significantly inhibited lysosomal rupture and cat B activation,which prevented the activation of NLRP3 inflammasome and inhibited pyroptosis in BEECs.The results of the study suggest that NETs enter cells by releasing HMGB1 and interacting with RAGE receptors.Then,they enter lysosomes under the action of dynamin,leading to lysosome rupture and cat B activation.This,in turn,activates the NLRP3 inflammasome in the cytoplasm,leading to caspase 1 activation and subsequent pyroptosis.The pyroptosis process also triggers enhanced release of inflammatory cytokines and LDH.In conclusion,the study provides evidence that NETs are associated with tissue inflammation and damage in cow endometrium.The study shows that NETs induce BEEC pyroptosis,inflammation,and injury,and that HMGB1 plays a key role in this process.The study also demonstrates that inhibition of NETs formation effectively alleviates uterine inflammation and tissue damage in rats.The mechanism of NETs-mediated BEEC pyroptosis in the context of cow endometritis appears to be associated with the HMGB1/RAGE/dynamin/cat B/NLRP3/caspase 1 signaling pathway.Overall,this study clarifies the mechanisms of endometrial tissue inflammation and damage induced by NETs in cow endometritis,and may provide a basis for the clinical management of this condition.