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Neutrophils are involved in the development of endometritis, but it remains unknown how neutrophils induce inflammation and tissue damage. Neutrophil extracellular traps (NETs) clear invading pathogens during infection but induce pyroptosis, leading to inflammation and tissue damage. Thus, our objective was to investigate whether NETs participate in bovine endometrial epithelial cell (BEEC) pyroptosis during endometritis. To confirm this, NETs and caspase-1/4; apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC); nod-like receptor protein-3 (NLRP3); and gasdermin D N-terminal (GSDMD-N), TNF-a, IL-1β, IL-6, and IL-18 in endometrial tissue were detected. Pathological section and vaginal discharge smears were performed to visually determine endometritis in the uterus. BEECs were stimulated with NETs to induce pyroptosis, which was treated with DNase I against pyroptosis. Caspase-1/4, ASC, NLRP3, GSDMD-N, TNF-a, IL-1β, IL-6, and IL-18 in BEECs were analyzed in endometrial tissue. The results showed that NET formation, as well as pyroptosis-related proteins and proinflammatory, cytokines were elevated in the endometrial tissue of cows with endometritis. Pathological sections and vaginal discharge smears showed increased neutrophils and plasma cells in the uterus, as well as tissue congestion. In BEECs, NETs increased the level of pyroptosis-related proteins and proinflammatory cytokines and were diminished by DNase I. In summary NETs participate BEEC pyroptosis during endometritis in dairy cows.

Simple Summary: The progression of several inflammatory diseases, including endometritis, has been proven to be enhanced by the process of neutrophil extracellular traps (NETs) formation in pathogenesis. Therefore, averting the formation of NETs could be an effective method to combat endometritis. Cl-amidine has been reported to treat certain inflammatory diseases, but its therapeutic role in combating endometritis remains an enigma as it may likely attenuate NETs formation. Hence, we investigate the potential of NETs formation in the pathogenesis of endometritis using rats and the probable therapeutic indices of Cl-amidine against endometritis. It was revealed that LPS induced endometritis in rats, and the formation of NETs was detected in the rats' inflamed uterine tissues. The treatment with Cl-amidine showed the regression of LPS-induced endometritis and protection against uterine tissue damage. Cl-amidine effectively reduced the expression of specific proteins that could enhance the formation of NETs, thereby attenuating the inflammatory response to LPS-induced endometritis in rats. Our research showed that Cl-amidine possesses potentially therapeutic properties against endometritis. Endometritis is a common disease that affects the production in dairy cows and leads to severe losses in the dairy industry. Neutrophil extracellular traps (NETs) formation promotes pathogenic invasions of the lumen of the tissue, leading to inflammatory diseases such as mastitis, pancreatitis, and septic infection. However, research that could show the relationship between NETs and endometritis is scarce. Cl-amidine has been shown to ameliorate the disease squealing and clinical manifestation in various disease models. In this study, we investigated the role of NETs in LPS-triggered endometritis in rats and evaluated the therapeutic efficiency of Cl-amidine. An LPS-induced endometritis model in rats was established and found that the formation of NETs can be detected in the rat's uterine tissues in vivo. In addition, Cl-amidine treatment can inhibit NETs construction in LPS-induced endometritis in rats. Myeloperoxidase (MPO) activity assay indicated that Cl-amidine treatment remarkably alleviated the inflammatory cell infiltrations and attenuated the damage to the uterine tissue. The Western blot results indicated that Cl-amidine decreased the expression of citrullinated Histone H3 (Cit-H3) and high-mobility group box 1 protein (HMGB1) protein in LPS-induced rat endometritis. The ELISA test indicated that Cl-amidine treatment significantly inhibited the expression of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The NETs were determined by Quant-iTTMPicoGreen dsDNA kit®, which indicated that Cl-amidine significantly inhibited the NETs in rat serum. All results showed that Cl-amidine effectively reduced the expression of Cit-H3 and HMGB1 proteins by inhibiting the formation of NETs, thereby attenuating the inflammatory response to LPS-induced endometritis in rats. Hence, Cl-amidine could be a potential candidate for the treatment of endometritis. [ABSTRACT FROM AUTHOR]

Purpose: Endometritis is a female reproductive disease that affects the cattle industries development and microRNAs (miRNAs) play a pivotal role and critical regulators of the innate immune response in varieties of diseases. The present study intends to investigate the regulatory role of miR‐24-3p in the innate immune response involved in endometritis and evaluate its therapeutic potential. Methods: Whole mice uteri and bovine endometrial epithelial cells (BEECs) were separately stimulated with LPS. The BEECs were also transfected with miR-24-3p mimic and negative control; siTRAF6 and siNC; pcDNA3.1 empty and pcDNA3.1(+)TRAF6 separately with LPS stimulation. The expression levels of miR‐24-3p and TRAF6 were measured via quantitative real‐time polymerase chain reaction (qRT-PCR) and Western blot, respectively. LPS‐induced inflammatory response assessed by inflammatory cytokines secretion and expression via ELISA and qRT-PCR. Bioinformatics analysis and luciferase reporter assay validated the interaction between miR‐24-3p and TRAF6. The activation of the NF‐B/MAPK pathway and p65 phosphorylation was investigated by Western blot and immunofluorescence assay, respectively. Results: The expression of miR‐24-3p was decreased, and TRAF6 was elevated with an increased level of pro-inflammatory cytokines in LPS‐treated BEECs and mice uterus. The overexpression of miR‐24-3p suppressed LPS‐induced secretion of inflammatory cytokines (IL‐1β, IL‐6, IL-8 and TNF-α) and deactivation of NF‐B/MAPK pathways. The downregulation of TRAF6 inhibited LPS‐induced inflammatory response in BEECs. TRAF6 is validated as a target of miR‐24-3p, and miR‐24-3p reversed the overexpression of cloned TRAF6 on inflammation response in BEECs. Conclusion: Our findings demonstrate that the overexpression of miR‐24-3p attenuates endometrial inflammation and the expression of pro-inflammatory mediators via suppressing TRAF6. Therefore, modulating the pathogenesis of endometritis and possibly, a therapeutic potential against endometritis. [ABSTRACT FROM AUTHOR]

Endometritis is a major infectious disease affecting dairy development. MicroRNAs are recognized as critical regulators of the innate immune response. However, the role and mechanism of Bta-miR-24-3p in the development of endometritis are still unclear. This study aimed to investigate the effect of Bta-miR-24-3p on the inflammatory response triggered by lipopolysaccharide (LPS) and to clarify the possible mechanism. LPS-treated bovine endometrial epithelial cells (BEECs) were cultured to investigate the role of Bta-miR-24-3p. The expression levels of Bta-miR-24-3p were downregulated, and galectin-9 (LGALS9) were measured by quantitative real-time polymerase chain reaction. The LPS-induced inflammatory response was assessed by the elevated secretion of inflammatory cytokines measured by using enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction. Activation of nuclear factor-κB (NF-κB) and TLR4 pathway was assessed by Western blot. The interaction between Bta-miR-24-3p and LGALS9 was validated by bioinformatics analysis and a luciferase reporter assay. LPS-induction in BEECs with Bta-miR-24-3p was overexpressed leads inhibition of pro-inflammatory cytokines, LGALS9 expression, and TLR4/NF-ĸB pathway deactivation. Knockdown of LGALS9 inhibited the LPS-induced inflammatory response in BEECs. LGALS9 was validated as a target of Bta-miR-24-3p. Cloned overexpression of LGALS9 failed to alter the effect of Bta-miR-24-3p on the inflammatory response in BEECs. Overall, Bta-miR-24-3p attenuated the LPS-induced inflammatory response via targeting LGALS9. The immunotherapeutic stabilisation of Bta-miR-24-3p could give a therapeutic option for endometritis and other disorders commonly associated with endometritis, suggesting a novel avenue for endometritis treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: The provision of updated information on the molecular pathogenesis of bovine endometritis with host-pathogen interactions and the possibility of exploring the cellular sensors mechanism in a nanotechnology-based drug delivery system against persistent endometritis were reported in this review. The mechanism of Gram-negative bacteria and their ligands has been vividly explored, with the paucity of research detail on Gram-positive bacteria in bovine endometritis. The function of cell receptors, biomolecules proteins, and sensors were reportedly essential in transferring signals into cell signaling pathways to induce immuno-inflammatory responses by elevating pro-inflammatory cytokines. Therefore, understanding endometrial cellular components and signaling mechanisms across pathogenesis are essential for nanotherapeutic exploration against bovine endometritis. The nanotherapeutic discovery that could inhibit infectious signals at the various cell receptors and signal transduction levels, interfering with transcription factors activation and pro-inflammatory cytokines and gene expression, significantly halts endometritis. In order to control and prevent bovine endometritis, there is a need to understand the molecular pathogenesis of the infectious disease. Bovine endometrium is usually invaded by a massive mobilization of microorganisms, especially bacteria, during postpartum dairy cows. Several reports have implicated the Gram-negative bacteria in the pathogenesis of bovine endometritis, with information dearth on the potentials of Gram-positive bacteria and their endotoxins. The invasive bacteria and their ligands pass through cellular receptors such as TLRs, NLRs, and biomolecular proteins of cells activate the specific receptors, which spontaneously stimulates cellular signaling pathways like MAPK, NF-kB and sequentially triggers upregulation of pro-inflammatory cytokines. The cascade of inflammatory induction involves a dual signaling pathway; the transcription factor NF-κB is released from its inhibitory molecule and can bind to various inflammatory genes promoter. The MAPK pathways are concomitantly activated, leading to specific phosphorylation of the NF-κB. The provision of detailed information on the molecular pathomechanism of bovine endometritis with the interaction between host endometrial cells and invasive bacteria in this review would widen the gap of exploring the potential of receptors and signal transduction pathways in nanotechnology-based drug delivery system. The nanotherapeutic discovery of endometrial cell receptors, signal transduction pathway, and cell biomolecules inhibitors could be developed for strategic inhibition of infectious signals at the various cell receptors and signal transduction levels, interfering on transcription factors activation and pro-inflammatory cytokines and genes expression, which may significantly protect endometrium against postpartum microbial invasion. [ABSTRACT FROM AUTHOR]