Search Results (34)

Prostaglandin E₂ (PGE₂) plays a critical role in regulating uterine endometrial function and supporting embryonic development during early pregnancy in ruminants. However, its precise roles in shaping the uterine microenvironment remain unclear. Herein, 1 mg PGE₂ was infused daily into the uterus of dairy heifers from days 12 to 14 of the estrus cycle. ULF was subsequently collected for integrated proteomic, metabolomic, and targeted lipidomic analyses. In addition, bovine endometrial epithelial cells were used to evaluate the effects of PGE₂ on epithelial adhesion and responsiveness to interferon tau (IFNT). PGE₂ infusion resulted in 909 differentially abundant proteins (DAPs), which are primarily associated with early embryonic development, immune regulation, and cell adhesion. Untargeted metabolomics analysis identified 587 altered metabolites, which were enriched in sphingolipid, arachidonic acid, phenylalanine, and tryptophan metabolism. Proteomic–metabolomic analyses showed that these alterations were primarily associated with early embryonic development, immune regulation, and cell adhesion. Targeted lipidomic analysis showed a global reduction in lipid accumulation, with glycerophospholipid metabolism and choline metabolism most significantly affected. In vitro, PGE₂ reduced epithelial microvilli density, increased osteopontin (OPN) expression, and decreased the expression of junctional proteins (zona occludens-1 (ZO-1), E-cadherin (CDH1), and fibronectin 1 (FN1)). Moreover, PGE₂ enhanced the responsiveness of bEECs to IFNT by interferon alpha/beta receptor 1 (IFNAR1) and IFNAR2, and prostaglandin E receptor 4 (PTGER4) was identified as the primary receptor mediating this response. Collectively, these findings suggest that PGE₂ may modulate lipid metabolism and adhesion-related processes in the endometrium and influence endometrial responsiveness to IFNT, providing insights into molecular mechanisms associated with pregnancy establishment in dairy cows. Full article

Retained placenta (RP) is a significant postpartum complication in dairy cows. Although abnormal estradiol (E₂) levels are implicated, the underlying cellular mechanisms remain poorly defined. Through RNA-seq analysis of postpartum blood from cows with or without RP, we identified Serum and Glucocorticoid-regulated Kinase 1 (SGK1) as a differentially expressed gene candidate. Analysis of fetal cotyledonary tissues revealed that SGK1 expression was significantly elevated in these tissues, concomitant with markers of suppressed apoptosis, increased levels of tight junction proteins, and an inhibited epithelial–mesenchymal transition (EMT) phenotype. To explore a potential mechanistic link between E₂ and these cellular alterations, we investigated the E₂-SGK1 axis in bovine endometrial epithelial cells in vitro. E₂ treatment upregulated SGK1 expression, reduced apoptosis, increased tight junction protein levels, and suppressed EMT. Conversely, SGK1 knockdown induced apoptosis, disrupted tight junctions, and impaired EMT. Notably, E₂ could not rescue the apoptosis and EMT alterations in SGK1-knockdown cells, indicating that SGK1 is a critical mediator of these E₂ effects in this cellular model. Based on these initial correlative findings in tissues, combined with the subsequent mechanistic experiments in cells, we propose a novel model whereby dysregulation of the E₂- SGK1 axis could contribute to RP pathogenesis by stabilizing the placental interface. Our findings provide the first experimental evidence linking SGK1 to RP and establish a foundation for future in vivo validation. Full article

During the preimplantation period, the nutrition of the embryo is dependent on luminal secretions of the uterus, which can be modified by the health status of the animal. The aim of this study was to mimic the paracrine communication between healthy or LPS-stressed epithelial endometrial cells (EECs) and embryos using aa transwell plate. The rate of in vitro embryo production, size, and concentration of extracellular vesicles (EVs), and level of secretion of Galectin-9 (Gal-9) and leukaemia inhibitory factor (LIF) were detected. Embryos were produced with an established protocol of oocyte in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro embryo culture (IVC). On day 55 of IVC, one hour before the transfer of morulae in the basolateral compartment of the transwell, EECs were treated with 10 ng/mL of LPS, and IVC was continued until the eleventh day. Extracellular vesicles (EVs) were obtained from IVC medium by ultracentrifugation. Levels of Gal-9 and LIF were evaluated by ELISA. On day 7, the results did not show statistically different blastocyst rates between EECs+Embryo and EECs+LPS+Embryo (34.94 ± 1.95% and 33.06 ± 3.08%, respectively). On day 11, the rate of hatched blastocysts was 23.03 ± 3.18% in EECs+Embryo, while in EECs+LPS+Embryo, no hatching was observed. Nanosight revealed higher values in EV size and concentration in EECs+LPS+Embryo medium compared to EECs+Embryo (p < 0.05). In LPS-treated samples, there was a significant decrease in Gal-9 levels and a significant increase in LIF secretions compared with non-non-LPS-treated samples (p < 0.05). These results highlight how bidirectional secretions between EECs and embryos, crucial for embryo development, can be affected by endometritis. Full article

Endometritis, an inflammatory disease of the uterine endometrial tissue, is a major reproductive disorder in dairy cattle that causes extensive damage to endometrial epithelial cells. Excessive activation of the NLRP3 inflammasome is strongly associated with inflammatory pathology. Autophagy plays a critical role in clearing damaged proteins, organelles, and intracellular pathogens. Additionally, the zinc finger protein A20 exhibits potent anti-inflammatory effects across various inflammatory conditions. However, the roles of A20 and autophagy in regulating the NLRP3 inflammasome in BEECs remain poorly defined. This study shows that LPS significantly increased IL-1β expression, Caspase-1 activity, and lactate dehydrogenase (LDH) levels, while inducing numerous vesicular protrusions and membrane pores, resulting in severe inflammatory injury. A20 overexpression mitigated LPS-induced NLRP3 inflammasome activation and alleviated inflammatory injury. Conversely, autophagy inhibition or A20 silencing intensified LPS-induced NLRP3 inflammasome activation and inflammatory injury. Further analysis revealed that A20 promotes autophagy, and its inhibitory effect on the NLRP3 inflammasome was diminished when autophagy was suppressed. In conclusion, A20 reduces LPS-induced inflammatory injury in BEECs by enhancing autophagy and suppressing NLRP3 inflammasome activation. These results uncover a novel regulatory role for A20 in controlling excessive NLRP3 inflammasome activation in BEECs, suggesting its potential as a therapeutic target for bovine endometritis. Full article

Prostaglandin E₂ (PGE₂) has been implicated in multiple biological processes during pregnancy in ruminants. However, the regulatory effects of PGE₂ on endometrial function during the diestrus period and its underlying molecular mechanisms remain poorly understood. Herein, PGE₂ treatment promoted the accumulation of lipid droplets and induced cytoskeletal reorganization in bEECs. As a well-established inducer of lipid droplet formation, oleic acid (OA) treatment significantly increased the number of lipid droplets in bEECs, altered the distribution of F-actin and disrupted the expression patterns of key adhesion-related proteins. Transcriptomic analysis revealed that the PPAR signaling pathway was the key pathway that responded to PGE₂ treatment in bEECs, and its downstream target gene FABP3 was markedly up-regulated. Knockdown of FABP3 led to a reduced number of BTC spheroids and down-regulation of adhesion-related proteins in bEECs while increasing the density of microvilli and up-regulating the expression of epithelial markers. Prostaglandin E receptor 4 (PTGER4) was the primary receptor that responded to PGE₂ treatment, and PTGER4 knockdown or pharmacological inhibition with GW-627368 suppressed FABP3 expression in bEECs. Moreover, uterine samples from dairy cows at different stages of the estrous cycle showed that FABP3 expression was significantly elevated in the endometrium tissue during mid-diestrus compared to metestrus, with predominant localization in the luminal and superficial glandular epithelium. Collectively, these findings indicate that FABP3 regulates lipid droplet accumulation and adhesion ability in bEECs via the PGE₂/PTGER4/PPAR signaling axis, providing new insights into the metabolic regulation of endometrial receptivity in ruminants. Full article

Bovine endometritis can be caused by Escherichia coli (E. coli), from which the lipopolysaccharide (LPS) triggers TLR4/NF-κB-mediated inflammation and reactive oxygen species (ROS) overproduction, resulting in impaired reproductive performance. While NADPH oxidase (NOX) is a critical source of ROS generation, its role in bovine endometrial epithelial cells (BEEC) and modulation by selenium remains unexplored. In this study, primary BEEC was challenged by LPS to assess NOX2/4 expression kinetics. Inhibitors of NOX and NF-κB were applied to observe the role of NOX-derived ROS in BEEC inflammation and in selenomethionine (SeMet)-modulated anti-inflammation. ROS levels were measured by flow cytometry. The changes in inflammatory cytokines, and the proteins related to NOX4 and NF-κB, were analyzed via qPCR and Western blot. As a result, the inhibition of NOX decreased LPS-induced proinflammatory cytokine expression, ROS accumulation, NOX4 level, and the phosphorylation of NF-κB P65 and IκBα. Conversely, the suppression of NF-κB downregulated the levels of ROS and NOX4. Cotreatment with SeMet and a NOX inhibitor further suppressed the inflammatory response, ROS level, and NF-κB pathway activation compared to individual treatment, but had no additive effect on the NOX4 protein level. In conclusion, the NOX4/ROS/NF-κB axis forms a proinflammatory feedback loop in LPS-stimulated BEEC. SeMet mitigates oxidative stress and inflammation partially through NOX4 inhibition. Full article

Bovine endometritis is a common postpartum disease that significantly impairs reproductive performance and reduces economic sustainability in dairy and beef cattle. It is primarily caused by gram-negative and -positive bacteria, triggering strong inflammatory responses in the endometrium. Serum amyloid A (SAA) is an acute-phase protein and precursor of amyloid A (AA) in AA amyloidosis. In cattle, multiple SAA isoforms have been identified; however, the biological functions of SAA3 remain unclear. Hence, this study investigated the role of SAA3 in bovine endometrial epithelial cells (BEnEpCs) following stimulation with gram-negative or -positive bacterial antigens. BEnEpCs were treated with lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and, subsequently, the expression levels of SAA3 and SAA1 mRNA were compared by real-time PCR. To further investigate protein-level changes, immunocytochemistry (ICC) was performed to assess the expressions of SAA3 and SAA1. These analyses revealed that SAA3 mRNA expression was significantly enhanced by LPS and LTA, whereas SAA1 mRNA remained undetectable or showed only minimal responsiveness. Notably, only SAA3 protein expression increased in response to stimulation. These results indicate that SAA3 plays a crucial role in the innate immune response of BEnEpCs against gram-negative bacteria. Our in vitro findings may facilitate understanding of the innate immune activity in bovine uterus. Full article

Bovine endometritis is a common endocrine and reproductive disorder in postpartum dairy cows, closely associated with elevated systemic oxidative stress. This disease can lead to delayed uterine involution, repeated breeding failure, and significant economic losses in the dairy industry. Studies suggest that oxidative stress may contribute to the pathological progression of endometritis by regulating ECM remodeling, but the specific molecular mechanisms remain unclear. ECM homeostasis relies on the coordinated action of matrix metalloproteinases (e.g., MMP2, MMP9) and collagen (e.g., type IV collagen, COL-IV), while the TGFβ1/Smad3 signaling pathway is implicated in ECM metabolic regulation. Therefore, elucidating the regulatory mechanisms of oxidative-stress-mediated TGFβ1/Smad3 signaling on ECM remodeling is crucial for understanding the pathogenesis of endometritis. This study investigates postpartum bovine uterine tissues, comparing inflammatory cytokines (IL-1β, IL-6, TNF-α) and oxidative-stress-related factors (GPx, SOD, CAT) between healthy and endometritis groups. Additionally, the differences in ECM-remodeling-associated proteins (MMP2, MMP9, COL-IV) and TGFβ1/Smad3 pathway activity are analyzed. To further validate the mechanisms, an oxidative stress model is established in vitro by treating bovine endometrial epithelial cells (bEECs) with 200 μM H₂O₂ for 4 h, followed by the valuation of the same indicators. Furthermore, gene silencing to downregulate Smad3 expression or inhibitor-mediated suppression of TGFβ1/Smad3 pathway activity is performed to observe their regulatory effects on MMP2, MMP9, and COL-IV. The results demonstrate that oxidative-stress-mediated endometritis significantly upregulates MMP2, MMP9, and the TGFβ1/Smad3 pathway activity, while suppressing COL-IV expression. Functional genetic experiments further reveal the dual regulatory role of the TGFβ1/Smad3 pathway in ECM remodeling: (1) pathway activation promotes MMP2/MMP9 expression, accelerating COL-IV degradation; (2) Smad3 positively regulates COL-IV synthesis. These findings provide a theoretical basis for targeting the TGFβ1/Smad3 pathway to mitigate the pathological progression of endometritis. Full article

Meloxicam has been identified as an adjuvant therapeutic component in the management of bovine uterine diseases, exhibiting anti-inflammatory and antioxidant effects. However, the mechanisms underlying its antioxidant actions in the context of bovine uterine diseases remain incompletely understood. The objective of this research was to determine whether meloxicam exerts its antioxidant effects through the Nrf2/HO-1 signaling pathway. By employing N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), along with inhibitors directed against heme oxygenase-1 (HO-1) or nuclear factor erythroid 2-related factor 2 (Nrf2), we investigated the dynamic changes in oxidative stress markers (ROS and malondialdehyde) and antioxidant indices (comprising catalase, superoxide dismutase, and glutathione), as well as the expression profiles of Nrf2 and inflammation-associated genes and proteins in bovine endometrial epithelial cells (BEECs) subjected to lipopolysaccharide (LPS) stimulation. As a result, meloxicam alleviated the LPS-induced elevation of oxidative stress marker levels and the reduction in antioxidant enzyme activities and antioxidant substance contents in BEECs. Compared to NAC, meloxicam demonstrated superior efficacy in activating the Nrf2 pathway, with the promotion of NRF2 expression (~1.6-fold) and nuclear translocation. The pretreatment of cells with HO-1 or Nrf2 inhibitors markedly attenuated the antioxidant activity of meloxicam. In summary, meloxicam primarily alleviates LPS-induced oxidative stress through the activation of the Nrf2/HO-1 pathway in BEECs. Full article

Endometritis poses a significant challenge to the dairy industry, impairing bovine reproductive performance and causing substantial economic losses. Although Morinda officinalis oligosaccharides (MOO) exhibit anti-inflammatory properties, their therapeutic potential against endometritis remains unclear. This study investigated MOO’s protective effects against LPS-induced uterine injury in mice and inflammation in bovine endometrial epithelial cells (BENDs), and explored the underlying mechanisms. In mice, MOO attenuated uterine inflammation by improving histopathology, reducing pro-inflammatory cytokines and decreasing oxidative stress. In BEND cells, MOO alleviated LPS-induced inflammation, oxidative stress, and apoptosis via downregulating pro-inflammatory mediators (IL-1β, IL-6, TNF-α, IL-8, TLR4, RELA), restoring antioxidant enzymes (HMOX1, NQO1, Nrf2, NOX4), and modulating apoptosis markers (BAX, cleaved CASP3, CASP9, BCL2). MOO reduced ROS accumulation, preserved mitochondrial membrane potential, and inhibited calcium influx. Critically, the calcium channel agonist Bay K 8644 reversed MOO’s protective effects, confirming calcium signaling modulation as a key mechanism. This study provides the first evidence that MOO mitigates LPS-induced uterine damage and BENDs inflammation through calcium signaling regulation, suggesting its potential for treating inflammation-related reproductive disorders in livestock. Full article

Oxidative stress is a significant factor affecting reproductive efficiency in dairy cows, contributing to conditions such as endometritis that impair fertility and milk production. This study investigates the molecular mechanisms by which bta-let-7d modulates the oxidative stress responses induced by potassium permanganate (KMnO₄) in bovine endometrial epithelial cells (BEECs). Using KMnO₄ to induce oxidative stress, we observed significant increases in reactive oxygen species (ROS) and malondialdehyde (MDA) levels, accompanied by decreased activities of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD). Quantitative PCR and Western blot analyses indicated a negative correlation between IGF1R and bta-let-7d expression in oxidative-stress-affected tissues, suggesting opposing roles in managing stress responses. Following KMnO₄ treatment, there was marked downregulation of anti-apoptotic genes and an upregulation of pro-apoptotic markers, alongside diminished antioxidant capacity. Mechanistically, bta-let-7d targets IGF1R, leading to the suppression of the PI3K/AKT signaling pathway and exacerbating oxidative damage. In vivo experiments further confirmed the impact of KMnO₄ exposure on IGF1R expression. These findings provide novel insights into the mechanisms by which KMnO₄ induces oxidative stress and apoptosis in bovine uterus. They highlight the potential for therapeutic strategies targeting the bta-let-7d/IGF1R axis to enhance reproductive health management in dairy cows, offering a promising avenue for mitigating oxidative-stress-related reproductive disorders. Full article

Background: In dairy cows, the stress-related cortisol level increases the susceptibility to postpartum uterine diseases. Oxidative stress is an important component of the disease process and causes morphological and functional alterations in the bovine endometrium. Selenium (Se) has an antioxidant property, and an appropriate Se supplementation is recommended to enhance bovine disease resistance. Methods and results: Here, we aimed to answer two questions: (1) how does cortisol affect the oxidative status of bovine endometrial cells; and (2) does Se supplementation protect cells from oxidative injury with a high cortisol condition? The oxidative stress of primary bovine endometrial epithelial cells (BEECs) was established by Escherichia coli lipopolysaccharide (LPS) stimulation, as marked by the increased oxidative markers and the suppressions of antioxidant indicators and Nrf2 signaling. In the absence of LPS, cortisol levels of 15 ng/mL showed a more significant antioxidative effect than cortisol levels of 5 and 30 ng/mL. In the presence of LPS, cortisol levels of 15 and 30 ng/mL elicited antioxidation, whereas 5 ng/mL of cortisol did not. Regardless of LPS stimulation, Se pretreatment of 1, 2, and 4 μM protected BEEC from oxidative stress, as evidenced by the decreased oxidative markers, increased antioxidant indices, and the activated Nrf2 signaling. With the presence of 30 ng/mL of cortisol, there was an enhanced Se antioxidant effect, which can be abolished by the block of cortisol receptor. Conclusions: Both cortisol and Se elicited antioxidant properties in BEEC through the Nrf2 pathway. In addition, the Se antioxidation was enhanced by cortisol. Full article

The aim of this study is to investigate the therapeutic effect of Poria cocos polysaccharide (PCP) on bovine endometritis. Initially, an inflammation model was induced using LPS-treated bovine endometrial epithelial cells (BEND) to identify the differentially expressed genes (DEGs) between the control and LPS groups by transcriptome sequencing, and GO functional annotation and KEGG enrichment analysis were performed. Subsequently, the mechanism of PCP treatment for endometritis was further evaluated using protein immunoblotting and real-time fluorescence quantitative analysis. Finally, the efficacy of PCP in treating endometritis was evaluated using a rat model of endometritis established with a mixed bacterial infection. The results show that transcriptome sequencing identified 4367 DEGs, with enrichment analysis highlighting the primary influences on the cell cycle and apoptosis signaling pathways. Following treatment of BEND with LPS resulted in cell apoptosis and inflammatory response. However, the introduction of PCP intervention significantly inhibited the progression of apoptosis and inflammation. Animal test results indicate that PCP significantly decreases the levels of serum inflammatory in rats suffering from endometritis and enhances antioxidant capacity. Furthermore, it effectively improved uterine swelling and tissue vacuolization caused by bacterial infection. These findings suggest that PCP could alleviate endometritis by modulating the inflammatory response and suppressing cell apoptosis. Poria cocos polysaccharides demonstrate significant potential for applications in immune modulation, anti-inflammatory responses, and antioxidant activities. Their high safety profile makes them suitable candidates as alternative therapeutic agents for the treatment of endometritis in the veterinary field. Full article

High concentrations of non-esterified fatty acids (NEFA) in the blood contribute to various metabolic disorders and are linked to endometritis in dairy cows. Isorhamnetin (ISO), a flavonoid found in many plants, is known for its antioxidant, anti-inflammatory, and anti-obesity properties. This study systematically assessed NEFA-induced damage in bovine endometrial epithelial cells (bEECs) and investigated whether ISO alleviates NEFA-induced cell damage and its underlying molecular mechanisms. Our observations revealed that excessive NEFA inhibited proliferation and induced apoptosis in bEECs, accompanied by an increase in the expression of BAX and cleaved caspase-3. We further observed that NEFA could induce lipid accumulation, reactive oxygen species (ROS) generation, and the release of pro-inflammatory factors IL-1β, IL-6, and TNF-α in bEECs. RNA sequencing and Western blot analysis revealed that NEFA induced damage in bEECs by activating MAPK signaling pathway. Notably, ISO treatment ameliorated these effects induced by NEFA, as evidenced by decreased protein levels of BAX, cleaved caspase-3, and PPAR-γ, along with reductions in triglyceride content, ROS generation, and levels of IL-1β, IL-6, and TNF-α. Mechanistically, our experimental results demonstrated that ISO inhibited NEFA-induced activation of MAPK signaling. Overall, ISO shows promise for therapeutic development to address NEFA-related endometritis in dairy cows. Full article

Lipopolysaccharide (LPS) is one of the main factors inducing endometritis in dairy cows. However, the specific pathogenesis of LPS-induced endometritis in dairy cows is not fully understood. The objective of this study was to establish an in vitro endometritis model using LPS-induced bovine endometrial epithelial (BEND) cells. BEND cells were treated with LPS of different concentrations and times. The cell-counting kit-8 (CCK-8) was used to detect the cell survival rate after LPS treatment, and quantitative real-time PCR (RT-qPCR) was used to detect the expression of control group and LPS-treated group of inflammatory factors interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α). The results showed that the survival rate of endometrial epithelial cells stimulated by 5 μg/mL LPS for 6 h was 75.13%, and the expression of inflammatory factors was significantly increased. Therefore, 5 μg/mL LPS for 6 h could be selected as a suitable model for the study of inflammation. In addition, miRNA sequencing and target gene prediction was performed on normal and LPS-treated BEND cells. Among twenty-one differentially expressed miRNAs, six miRNAs were selected and their expression levels were detected by RT-qPCR, which were consistent with the sequencing results. Twenty-one differentially expressed miRNAs collectively predicted 17,050 target genes. This study provides a theoretical basis for further investigation of the pathogenesis of endometritis. Full article