Endometritis in dairy cows is characterized by excessive nuclear factor-κB (NF-κB)-mediated inflammation. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key negative regulator of inflammatory signaling. However, whether it mediates the anti-inflammatory effects of selenomethionine (SeMet) in bovine endometrial epithelial cells (BEEC) remains unclear. Here, we investigated the role of PPARγ in SeMet-mediated regulation of lipopolysaccharide (LPS)-induced inflammatory responses. LPS stimulation suppressed PPARγ expression and activated NF-κB signaling, whereas SeMet pretreatment restored PPARγ expression and attenuated NF-κB activation. Functional analyses using lentiviral-mediated knockdown and overexpression demonstrated that PPARγ plays a critical regulatory role in the anti-inflammatory effects mediated by SeMet. PPARγ knockdown exacerbated inflammation and blunted SeMet efficacy, while PPARγ overexpression produced the opposite effects. Furthermore, SeMet-mediated inhibition of p65 phosphorylation and nuclear translocation was dependent on PPARγ expression, with a significant interaction between SeMet treatment and PPARγ status confirmed by two-way ANOVA. Collectively, these findings identify PPARγ as a critical mediator linking selenium to NF-κB signaling and establish a SeMet-PPARγ-NF-κB regulatory axis in BEEC, providing mechanistic support for selenium-based strategies to control endometritis in ruminants.

Bovine mastitis caused by Klebsiella pneumoniae (K. pneumoniae) severely threatens the dairy industry. The mitochondrial dysfunction plays a pivotal role in the pathogenesis of mastitis caused by K. pneumoniae. Forkhead box O 3 (FOXO3), a key transcription factor known to regulate mitochondrial function, has not been explored in the mastitis induced by K. pneumoniae. This study aims to elucidate the regulatory mechanism of FOXO3 in K. pneumoniae infection-induced mastitis. In vivo and in vitro mastitis models were established using K. pneumoniae infection. K. pneumoniae infection significantly increased FOXO3 mRNA expression but reduced its protein levels in bovine mammary epithelial cells (BMECs) and mammary tissues. Mechanistically, K. pneumoniae activated folliculin-interacting protein 1 (FNIP1) to inhibit AMP-activated protein kinase (AMPK)-mediated FOXO3 phosphorylation, thereby reducing nuclear FOXO3 localization. Concurrently, K. pneumoniae upregulated mouse double minute 2 (MDM2), promoting FOXO3 ubiquitination and degradation via the ubiquitin-proteasome system. Inhibition of FNIP1 attenuated FOXO3 ubiquitination by suppressing MDM2 expression. FOXO3 overexpression ameliorated mitochondrial dysfunction, reduced milk synthesis defects, and alleviated inflammation induced by K. pneumoniae. In summary, these findings demonstrate that K. pneumoniae infection activates FNIP1, which subsequently reduces FOXO3 activity by inhibiting AMPK-mediated phosphorylation and promoting FOXO3 ubiquitination via the E3 ubiquitin ligase MDM2. These dual effects of FNIP1 on FOXO3 contribute to mitochondrial dysfunction, inhibit milk synthesis, and exacerbate inflammation, suggesting that activation of FOXO3 may represent a promising strategy for the prevention and control of K. pneumoniae-induced mastitis.

Endometritis is a common disease of the reproductive system that leads to decreased fertility and potential miscarriage, resulting in substantial economic losses. Ferroptosis is an iron-dependent mode of cell death driven by lipid peroxidation. Basic helix-loop-helix family member E40 (BHLHE40) has been developed as a molecular marker and a therapeutic target for bacterial infectious diseases. However, the underlying role of BHLHE40 needs to be further studied. This study investigated whether BHLHE40 drives Escherichia coli (E. coli) -induced endometrial ferroptosis by transcriptionally repressing carnitine palmitoyltransferase 1B (CPT1B) and attenuating the NRF2 signaling pathway, with the aim of elucidating the regulatory mechanism of the BHLHE40-CPT1B-NRF2 axis. In endometrial tissues from E. coli-infected cows (n = 8 per group, in vivo), the expression of BHLHE40 was increased by 3.6 times (p < 0.05), and its protein level positively correlated with the severity of tissue damage (Pearson r = 0.6722, p = 0.0034). Functionally, knockdown of BHLHE40 conferred significant protection against E. coli-induced cell death (decreased from 36% to 18%, p < 0.01), concurrently reversing the features of ferroptosis (p < 0.05), including reduced lipid peroxidation, decreased intracellular Fe2+ concentration, and restored glutathione levels and the expression of anti-ferroptosis proteins. Integrated analysis of CUT&Tag and RNA-sequencing data revealed that BHLHE40 binds to the promoter region of CPT1B (enrichment increased by 5 times, p < 0.01) and transcriptionally represses its expression. Further investigation demonstrated that BHLHE40 mediates CPT1B repression by recruiting HDAC1 to the CPT1B promoter, a process reversible upon HDAC1 inhibition. Crucially, BHLHE40-mediated suppression of CPT1B led to functional impairment of the NRF2 signaling pathway. Overexpression of CPT1B inhibited ferroptosis by reactivating NRF2 and its downstream targets (p < 0.05), whereas inhibition of NRF2 abolished the protective effect (cell death increased from 16% to 27%, p < 0.01), confirming NRF2 as the critical downstream effector. This study elucidates the BHLHE40-CPT1B-NRF2-ferroptosis axis as a novel pathway and potential target for E. coli to cause endometrial epithelial injury.

Klebsiella pneumoniae (K. pneumoniae)-induced mastitis poses a severe threat to livestock health with limited therapeutic options. Selenium nanoparticles (SeNP) offer promising biocompatibility and antioxidant potential, but their efficacy and mechanism against bacterial mastitis remain unexplored. This study demonstrated that the oral administration of SeNP (20 μg/kg BW) attenuated K. pneumoniae-induced mastitis in goats. The treatment reduced histopathological damage and neutrophil infiltration. Furthermore, it restored anti-inflammatory IL-10 mRNA expression and suppressed proinflammatory IL-1β and IL-6 mRNA levels. Ultrasonography and Doppler analysis confirmed that mammary hemodynamics and echotextural abnormalities were improved. The SeNP supplementation contributed to increased concentrations of selenium in the serum, enhanced the activity of mammary glutathione, activated the peroxiredoxin 6 (PRDX6)/selenophosphate synthetase 2 (SEPHS2) axis, and upregulated glutathione peroxidase 4 (GPX4) expression. Immunofluorescence revealed that SeNP strengthened the PRDX6-SEPHS2 interaction to improve the activity of GPX4. Crucially, SeNP suppressed ferroptosis by inhibiting nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, reducing iron overload and lipid peroxidation, and attenuating NCOA4-ferritin heavy chain 1 colocalization. The findings established that SeNP concurrently target inflammatory, antioxidant, hemodynamic, and ferroptotic pathways to prevent bacterial mastitis, suggesting a novel intervention for the prevention and control of mastitis.

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.

Klebsiella pneumoniae (K. pneumoniae) is one of the pathogens causing clinical mastitis of bovine. Previous studies have demonstrated that mitochondrial damage and dysfunction are important mechanisms of mastitis in dairy cattle. Folliculin interacting protein 1 (FNIP1) is a major metabolic regulator of mitochondrial function with proinflammatory capabilities, but its role in K. pneumoniae-induced mastitis is yet to be elucidated. Thus, the studies were conducted to clarify the role of FNIP1-mediated mitochondrial function in mastitis caused by K. pneumoniae in vivo and in vitro. The experiments verified that K. pneumoniae caused decrease of milk fat and protein synthesis evidently in the mammary glands and bovine mammary epithelial cells (BMECs), accompanied by an imbalance in mitochondrial fission and fusion, increased mitochondrial permeability transition pore opening, decreased membrane potential and ATP content. While the enhancement of mitochondrial function alleviated K. pneumoniae-induced BMECs injury via relieving milk fat and protein dyssynthesis. Notably, transcriptomic analysis revealed that FNIP1 expression was upregulated in BMECs induced by K. pneumoniae. Further investigations revealed FNIP1 silencing improved milk synthesis by alleviating mitochondrial dysfunction caused by K. pneumoniae infection, and further inhibiting the activation of inflammatory factors, which in turn prompted the mammary recovery. In conclusion, K. pneumoniae inhibited mitochondrial function by activating FNIP1, which reducing the synthesis of milk fat and protein, thereby in turn lowers milk quality and induced mastitis. This study showed that FNIP1 has the potential as a novel target for the prevention and control of bovine mastitis.

During the postpartum period, domestic ruminants suffer elevated endogenous cortisol levels, which are associated with an increased risk of uterine infections. Selenium is a trace mineral nutrient with beneficial impacts on animals. The study aimed to investigate whether selenium yeast (SeY) could attenuate Escherichia coli (E. coli)-induced endometrial injury in goats with high cortisol background. Goats were examined after oral SeY administration for 21 days and were treated with glacial acetic acid, E. coli, and hydrocortisone to establish an endometritis model with high cortisol background. The results showed that endometrial injury caused by E. coli was aggravated under high cortisol background. Supplementation with SeY alleviated endometrial inflammation and serum LDH content. The mRNA expression of pro-inflammatory cytokines and defensin beta 2 and the phosphorylation level of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-b (NF-κB) signaling pathways were decreased by SeY supplementation. Total antioxidant capacity and antioxidant enzymes activities were increased by SeY supplementation, but malondialdehyde and 4-hydroxynonenal content were decreased. Moreover, nuclear factor erythroid-2 related factor 2 (NRF2) in the nucleus, heme oxygenase-1, and NAD(P)H quinone dehydrogenase 1 were increased by SeY supplementation. So, supplementation with SeY alleviated E. coli-induced endometritis in goats by activating the NRF2 pathway and inhibiting the activation of the MAPK and NF-κB pathways under postpartum stress.

Klebsiella pneumoniae (K. pneumoniae), a prominent causative agent of mastitis in dairy cattle, remains enigmatic in its pathogenic mechanisms. This study aimed to reveal the effects of K. pneumoniae on mammary glands via the induction of ferroptosis, as well as the protective role of Ferrostatin-1 (Fer-1) against this pathogen-mediated damage in bovine mammary epithelial cells (BMECs). Holstein cows were used to establish an intramammary infection model of K. pneumoniae. In vitro, primary BMECs were treated with 10 μM Fer-1 and K. pneumoniae alone or in combination. The results showed that mammary glands infected with K. pneumoniae exhibited increased transcriptional levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). Concurrently, significant elevations in iron, 4-hydroxynonenal, and reactive oxygen species (ROS) levels were observed. Conversely, K. pneumoniae infection downregulated nuclear factor erythroid 2-related factor 2 (Nrf2), cystine/glutamate antiporter (xCT), glutathione peroxidase 4 (GPX4), and glutathione levels. Following K. pneumoniae invasion, intracellular Fe2+ and lipid ROS accumulated in the BMECs, impeding activation of Nrf2/xCT/GPX4 signal transduction. Additionally, Fer-1 facilitated the nuclear translocation of Nrf2 protein, upregulating the protein levels of Nrf2/xCT/GPX4 while downregulation transcriptional levels of IL-1β, IL-6, IL-8, and TNF-α. In conclusion, Fer-1 alleviates K. pneumoniae-induced inflammatory factor activation and ferroptosis in BMECs via upregulation of the Nrf2/xCT/GPX4 pathway, supporting ferroptosis inhibition holds promise as a feasible therapeutic agent for the control of mastitis.

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.

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.

The incidence of bovine mastitis caused by K. pneumoniae increases during summer. This experiment aimed to clarify the connection between heat stress and K. pneumoniae-induced mastitis in terms of inflammatory responses and barrier function. From day 9-16 of the postpartum period, lactating rats were exposed to 35 °C in the daytime and 29 °C at night to establish a heat stress model. On day 8 of the heat stress treatment, the rat mastitis model was established by intramammary infection with K. pneumoniae through the milk ducts. At 12 h post-infection, the rectal temperature, serum biochemistry, K. pneumoniae burden, as well as histopathology, epithelium integrity, inflammatory response, and HSP70 expression of the mammary glands were detected. The results showed that heat-stressed rats with K. pneumoniae infection displayed higher rectal temperatures, more neutrophil infiltration, and more significant pathological damage to the acinar lumen. Heat stress promoted the disruption of the structural integrity of tight junction and the downregulation of relative protein expressions (ZO-1, Occludin, and Claudin-3) as a consequence of the increased production of inflammatory parameters (endotoxin, MPO, IL-1β, IL-6, and TNF-α) by accelerating NF-κB pathway activation and HSP70 expression after K. pneumoniae infection. Thus, heat stress disrupts mammary epithelium integrity, contributing to the pathogen invasion, and aggravates intramammary damage during K. pneumoniae mastitis by facilitating barrier disruption and inflammatory response, which could trigger more severe mastitis.

Klebsiella pneumoniae, a major bovine mastitis pathogen, disrupts mammary gland function through poorly understood mechanisms. This study demonstrates that K. pneumoniae infection triggers ferroptosis in bovine mammary epithelial cells (BMECs) via nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy, leading to lactation impairment. RNA sequencing revealed enrichment in ferroptosis, autophagy, and iron metabolism pathways. Infection time-dependently suppressed glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression, causing mitochondrial membrane potential collapse and lipid peroxidation. Crucially, K. pneumoniae activated NCOA4-dependent ferritin degradation, elevating intracellular Fe2+. NCOA4 knockdown alleviated ferroptosis, restored GPX4/SLC7A11 expression, reduced the bacterial load, and partially rescued lactation markers (fatty acid synthase, acetyl-CoA carboxylase 1, and β-casein). In vivo, infected mammary glands showed iron overload, 4-hydroxynonenal accumulation, lactation protein suppression, and pathological damage, correlating with NCOA4/ferritin heavy chain 1 upregulation. These findings establish NCOA4-mediated ferritinophagy as a key driver of K. pneumoniae-induced ferroptosis and lactation dysfunction, suggesting novel therapeutic targets for mastitis.

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.

Enterohemorrhagic Escherichia coli (EHEC) and dyspeptic diarrhea are significant health concerns in calves, leading to substantial economic losses in the livestock industry. This study investigated the impact of EHEC infection and dyspeptic diarrhea on calf health, focusing on blood parameters, fecal microbiota, and metabolite profiles. Thirty-two holstein calves were divided into three groups: healthy group (C Group), EHEC-infected group (E Group), and indigestion-induced diarrhea group (I Group). Significant alterations in diarrheic calves were noted in peripheral blood parameters, including hematological, biochemical, and blood gas indices. And then fecal microbiota analysis revealed decreased diversity, with reduced Actinobacteria and increased Proteobacteria and Fusobacteriota in E and I group. Metabolomic profiling showed significant reductions in organic acids and lipids in diarrheic calves. The study concludes that microbial and metabolic alterations play critical roles in the pathogenesis of EHEC- and indigestion-induced diarrhea, with Scorzoside identified as a potential biomarker for differentiating healthy calves from those with diarrhea. These findings provide insights for designing targeted interventions to enhance gut health and reduce disease burden in the livestock.

The infection of the bovine endometrium by Escherichia coli often results in endometrial injury with lower fertility in the bovine industry. However, what functions does ferroptosis play during endometritis? This is not completely explained. Therefore, this study aimed to elucidate the potential mechanism of ferroptosis during endometrial injury induced by E. coli. The endometrial tissues from sixteen Holstein cows with E. coli infection were harvested for pathological examination and RNA-seq. Primary bovine endometrial epithelial cells (BEECs) were used to establish an E. coli infection model in vitro. The mechanism by which ferroptosis is involved in endometritis was explored by western blot, flow cytometry, and immunofluorescence methods. The results revealed that the injury of endometrial tissues and BEECs induced by E. coli was associated with ferroptosis via Fe2+ accumulation, increased lipid peroxidation, decreased glutathione concentration, and inhibited expression of solute carrier family 7-member 11, glutathione peroxidase 4, and ferritin heavy chain 1. Ferroptosis inhibition alleviated BEEC damage induced by E. coli. Further analysis revealed that the E. coli-induced ferroptosis was associated with impaired nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense, which is characterized by mitigated NRF2 degradation and blunted induction of target antioxidant enzymes, such as NAD(P)H quinone oxidoreductase 1. In contrast, promoting the expression of NRF2 mitigated the cellular damage and ferroptosis induced by E. coli. In particular, glycogen synthase kinase (GSK)-3β exhibited sustained expression and hyperactivity in endometrial tissue and BEECs infected with E. coli, which confirmed that the suppression of GSK-3β could significantly inhibit NRF2 degradation and attenuate ferroptosis. This study demonstrated that E. coli can trigger ferroptosis in endometrial tissues and BEECs through GSK-3β-mediated NRF2 degradation. Modulating GSK-3β/NRF2 holds promise as a potential therapeutic strategy for alleviating endometritis induced by E. coli.

Endometritis represents a prevalent condition in perinatal dairy cows. Bovine endometrial epithelial cells (BEECs), as the primary interface between cavity and the external environment, are particularly vulnerable to infection by pathogenic bacteria following parturition. A20 is essential for regulating inflammation and modulating immune responses. Nevertheless, the exact role of A20 in the BEECs in response to inflammatory response is not fully understood. An endometritis model infected by Escherichia coli (E. coli) in vivo and a BEECs inflammation model induced with lipopolysaccharide (LPS) in vitro were built to investigate the function and governing mechanisms of A20 in endometritis. The results showed that infection with E. coli resulted in endometrial damage, inflammatory cell infiltration, and upregulation of inflammatory factors in dairy cows. Furthermore, A20 expression was upregulated in the endometrium of cows with endometritis and in BEECs following LPS stimulation. A20 overexpression attenuated the level of proinflammatory cytokines in LPS-stimulated BEECs; conversely, A20 knockdown lead to an exacerbated response to LPS stimulation. The overexpression of A20 was shown to activate autophagy and suppress the NF-κB signaling pathway in LPS-stimulated BEECs. However, blocking autophagy with chloroquine notably attenuated the anti-inflammatory effect of A20, leading to the activation of the NF-κB signaling pathway. In summary, the study demonstrated that A20’s suppression of inflammation in LPS-stimulated BEECs is associated with the activation of autophagy. Therefore, the A20 protein showed potential as a novel treatment focus for managing endometritis in dairy cows.

Background Endometritis is a common bovine postpartum disease. Rapid endometrial repair is beneficial for forming natural defense barriers and lets cows enter the next breeding cycle as soon as possible. Selenium (Se) is an essential trace element closely related to growth and development in animals. This study aims to observe the effect of Se on the proliferation of bovine endometrial epithelial cells (BEECs) induced by lipopolysaccharide (LPS) and to elucidate the possible underlying mechanism. Results In this study, we developed a BEECs damage model using LPS. Flow cytometry, cell scratch test and EdU proliferation assay were used to evaluate the cell cycle, migration and proliferation. The mRNA transcriptions of growth factors were detected by quantitative reverse transcription-polymerase chain reaction. The activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin pathways were detected by Western blotting and immunofluorescence. The results showed that the cell viability and BCL-2/BAX protein ratio were significantly decreased, and the cell apoptosis rate was significantly increased in the LPS group. Compared with the LPS group, Se promoted cell cycle progression, increased cell migration and proliferation, and significantly increased the gene expressions of TGFB1 , TGFB3 and VEGFA . Se decreased the BCL-2/BAX protein ratio, promoted β-catenin translocation from the cytoplasm to the nucleus and activated the Wnt/β-catenin and PI3K/AKT signaling pathways inhibited by LPS. Conclusions In conclusion, Se can attenuate LPS-induced damage to BEECs and promote cell proliferation and migration in vitro by enhancing growth factors gene expression and activating the PI3K/AKT and Wnt/β-catenin signaling pathways.

The bovine uterus is susceptible to bacterial infections after calving, particularly from Escherichia coli (E. coli), which often results in endometritis. Additionally, postpartum stress in cows can elevate cortisol levels in the body, inhibiting endometrial regeneration and reducing immune function, thereby further increasing the risk of infection. Selenium (Se) is a common feed additive in dairy farming, known for its anti-inflammatory and antioxidant effects. The aim of this study was to investigate the regulatory role of Se in the growth of bovine endometrial stromal cells (BESCs) under the conditions of LPS-induced inflammatory damage at high cortisol levels. BESCs were treated with 1, 2, 4 μM Se in combination with co-treatment of LPS and cortisol. The results indicated that LPS inhibited the cell viability and reduced the mRNA expression of CTGF, TGF-β1, and TGF-β3. Additionally, LPS increased apoptosis, hindered the cell cycle progression by blocking it in the G0/G1 phase, and suppressed the PI3K/AKT/GSK-3β and Wnt/β-catenin signaling pathways. Furthermore, increased concentrations of cortisol can exacerbate the impacts of LPS on cell proliferation and apoptosis. Conversely, the supplementation of Se promoted cell viability, increased the mRNA expression of TGF-β1 and TGF-β3, and enhanced cell cycle progression, while simultaneously repressing cell apoptosis as well as activating the PI3K/AKT/GSK-3β and Wnt/β-catenin signaling pathways. The above findings demonstrated that Se can promote cell proliferation, reduce cell apoptosis, and aid in the growth of BESCs damaged by LPS under high levels of cortisol. The potential mechanisms may be associated with the regulation of the PI3K/AKT/GSK-3β and Wnt/β-catenin signaling pathways.

Klebsiella pneumoniae (K. pneumoniae) is a significant pathogen associated with clinical mastitis in cattle. Anti-inflammatory drugs are necessary to alleviate pain and inflammation during clinical mastitis. Among many drugs, meloxicam (MEL) has been widely used in clinical mastitis because of its excellent inhibitory effect on the cyclooxygenase-2 (COX-2) enzyme. However, the effectiveness of MEL on the inflammatory response and oxidative stress induced by K. pneumoniae are unclear. In the present study, primary BMECs were infected with K. pneumoniae in the presence or absence of plasma maintenance concentration of MEL (0.5 and 5 μM). Following 1 or 3 h of combined treatment with K. pneumoniae and MEL, BMECs were gathered to assess the related indicators. The results showed that MEL at plasma maintenance concentrations exerted no influence on the viability of uninfected BMECs and also had no impact on bacterial load in BMECs. At these concentrations, MEL was able to inhibit the mRNA expression of COX-2, Interleukin (IL)-1β, Tumor necrosis factor α (TNF-α), and IL-6 while simultaneously elevating the mRNA levels of IL-8 in K. pneumoniae-infected BMECs. MEL had clear effects on relieving oxidative stress by increasing the activity of superoxide dismutase (SOD) and catalase (CAT) and the level of total antioxidant capacity (T-AOC). The mechanisms by which MEL mitigated the inflammatory response and oxidative stress were partially attributed to inhibition of the nuclear transcription factor-kappa B (NF-κB) signaling pathway and improvement of the activation of the nuclear factor erythroid 2-related factors (Nrf2) signaling pathway. To conclude, the results manifested that MEL at plasma maintenance concentrations protected BMECs from inflammatory and oxidative damage induced by K. pneumoniae.

The resistance of pathogenic bacteria to various clinical antibiotics is the major problem in treating bacterial keratitis. Dimethyl fumarate (DMF) has good anti-fungal and anti-inflammatory effects in fungal keratitis, but its effect on bacterial keratitis is unclear. This study aims to investigate DMF's anti-inflammatory and antibacterial effects. The pyroptosis model was constructed by intracellular infection of canine corneal epithelial cells (CCECs) with Staphylococcus pseudintermedius (S. pseudintermedius), and 200 μM DMF was added to explore its function. Western blot, ELISA, immunostaining, flow cytometry, qRT-PCR, and bacterial counts were used to examine the expression of the NLRP3-GSDMD signaling pathway, virulence genes, and oxidant mediators. 111 clinical keratitis isolates or S. pseudintermedius were treated with different concentrations of DMF to detect bacterial growth and biofilm formation. Adding DMF resulted in the inhibition of the NLRP3-GSDMD pathway while activating the NRF2 pathway. This led to a decrease in pyroptosis rate, intracellular bacteria count, and ROS content. Additionally, DMF blocked the mRNA expression of virulence genes ebpS, hlgB, siet, lukS-I, PVL, icaA, icaD, spsD, and spsL associated with S. pseudintermedius infection. Furthermore, DMF demonstrated concentration-dependent inhibition of the growth of clinical isolates and the formation of S. pseudintermedius biofilm. In conclusion, our results indicate that DMF can inhibit pyroptosis and the growth of various clinical isolates, making it a novel ophthalmic drug with anti-inflammatory and antibacterial properties.