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.

Avian pathogenic Escherichia coli (APEC) causes bloodstream infections mainly by resisting the bactericidal action of host serum. Although various protein and polysaccharide factors involved in serum resistance have been identified, the role of small non-coding RNA (sRNA) in serum resistance has rarely been studied. The sRNA RyhB contributes to serum resistance in APEC, but the regulation mechanism of RyhB to serum resistance-related targets remains unknown. Here, we studied the regulatory mechanism of RyhB on capsule synthesis and how RyhB regulates serum resistance, macrophage phagocytosis resistance, and pathogenicity to natural hosts by regulating capsule synthesis. The results showed that RyhB upregulates capsular synthesis by interacting with the promoter regions of the capsule gene cluster and activating the translation of the capsule. The deletion of ryhB and/or neu reduced the ability of resistance to serum, macrophage phagocytosis, and pathogenicity of APEC in ducks. It can be concluded that RyhB directly upregulates the expression of capsular gene cluster and capsular synthesis and then indirectly promotes resistance to serum and macrophage phagocytosis and pathogenicity to ducks.

The global emergence of hypervirulent Klebsiella pneumoniae (hvKP) poses significant threats to public health. The iucABCD (iuc) operon, a plasmid-borne genetic determinant encoding the siderophore aerobactin, serves as a reliable biomarker for hvKP identification. This study aimed to characterize the iuc3 (type 3 iuc)-positive K.pneumoniae isolates from diseased cows as hvKP strains and to elucidate the contribution of iuc3-positive plasmids to the hypervirulence phenotype, the evolutionary pathways and the spreading risk of the iuc3-positive plasmids. Eight of 108 K. pneumoniae isolates from 763 cow milk samples contained the iuc3 operon, and all iuc3 operons were located on T4SS-bearing plasmids. Notably, these isolates showed hypervirulent phenotype compared to the iuc3 knockout and plasmid-cured strains, including severe tissue damage, high bacterial loads in typical organs and high mortality in animal models. Analysis of NCBI database showed iuc3-positive K. pneumoniae strains have been widely distributed across animal, food, and humans, and exhibit an independent evolutionary pathways compared to the human-associated iuc1-type hvKP. The IS elements likely drive the acquisition of various resistance genes and the emergence of iuc3-positive resistance plasmids. The iuc3-bearing plasmids in this study demonstrated highly conjugative ability, low fitness costs, and the ability to replace the iuc1-bearing classical virulence plasmid in human hvKP strain. Besides, they could completely fuse with broad-host-range resistance plasmid (IncX3-blaNDM-5), becoming carbapenem-resistant hvKP strains. The iuc3- and blaNDM-5- fusion plasmids showed strong in vivo retention and dissemination capabilities in mouse models. Our findings emphasize the necessity for long-term monitoring of iuc3-positive plasmids in K. pneumoniae isolates from both animals and humans.

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.

Brucellosis, a zoonotic disease caused by Brucella species, presents significant public health challenges due to its complex diagnosis and the limited availability of rapid detection methods. To address these challenges, we developed a novel detection method that integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, enabling dual readout through fluorescence (FL) and lateral flow strip (LFS) detection. The RPA-CRISPR/Cas12a-FL assay demonstrated an impressive detection limit of 1 copy/μL, which is 10 times more sensitive than quantitative polymerase chain reaction, while the RPA-CRISPR/Cas12a-LFS method achieved a detection limit of 10 copies/μL, comparable to nested PCR. Specificity testing confirmed the robustness of the assay, as it produced strong signals exclusively for Brucella without cross-reactivity with other bacterial species. Clinical validation using serum samples from 24 confirmed brucellosis patients and six healthy controls demonstrated a 100% concordance with serological results, underscoring the reliability of this method for clinical applications. This assay provides a rapid, sensitive, and specific tool for Brucella detection, suitable for both laboratory and field settings, and holds significant potential for enhancing the diagnosis and control of brucellosis.IMPORTANCEBrucellosis is a significant zoonotic disease, and rapid and accurate diagnosis is crucial for its treatment and control. To address this need, we developed a novel detection method that combines recombinant enzyme polymerase amplification with a CRISPR/Cas12a system, achieving dual readout through fluorescence and lateral flow strips. The test demonstrates excellent sensitivity and specificity, with clinical validation confirming complete concordance with serological results. This approach offers a fast, reliable, and field-deployable solution for brucellosis diagnosis, significantly enhancing disease management and public health outcomes.

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.

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.

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.

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.

By analyzing the fecal microbiome data of patients with non-severe aplastic anemia (NSAA), this study aims to explore the potential role of dysbiosis in the immune-mediated pathogenesis of NSAA. This study included 21 newly diagnosed NSAA patients from the Affiliated Hospital of Nanjing University of Chinese Medicine between July 2018 and June 2021, along with 24 healthy controls who underwent routine health checkups. Fecal samples were collected for DNA extraction. At the species level of phylum, class, order, family, genus and species, the intestinal microbial community of NSAA patients was significantly different from that of healthy controls. Alpha diversity analysis showed that the Chao and Observed species indices were significantly lower in the NSAA group compared to the control group ( P < 0.05 ), indicating that the gut microbiota diversity in NSAA patients was lower than that in normal individuals. Further multivariate statistical analysis revealed that the relative abundance of the following gut microbiota was higher in the NSAA group: Actinobacteriota, Coriobacteriia, Bifidobacteriales, Coriobacteriales, Pasteurellales, Prevotellaceae, Muribaculaceae, Bifidobacteriaceae, Coriobacteriaceae, Pasteurellaceae, Prevotella spp., Muribaculum spp., Barnesiella spp., Bifidobacterium spp., Mitsuokella spp., Collinsella spp., Alloprevotella spp. Meanwhile, the abundance of Lachnospirales spp. in the gut microbiota of NSAA patients was found to be lower than that of healthy controls. Correlation and model prediction analyses of the dominant microbial species in both the NSAA and control groups revealed a strong competitive relationship between Bacteroides spp. and Prevotella spp. NSAA patients exhibit a decrease in the abundance of Lachnospirales spp. and an increase in the abundance of Prevotella spp., which may be closely related to immune dysfunction mediated by NSAA Treg/Th17 imbalance, which, in turn, may contribute to the development of hematopoietic failure.

The bovine mastitis caused by Klebsiella pneumoniae (K. pneumoniae) infection, lead to high death rate and severe economic loss. Currently, the regional epidemiological characteristics of K. pneumoniae in China are still unclear, especially the surveillance data on prevalence of extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae in the cow are still limited. In this study, the epidemiology of K. pneumoniae from mastitis-infected cows in large-scale dairy farms in China were investigated. A total of 108 isolates of K. pneumoniae were isolated from 763 mastitis-associated milk samples collected from dairy farms in Jiangsu, Anhui, Hebei, Fujian, Guangdong, Shanxi and Zhejiang provinces in China. The whole genomes of 108 strains of K. pneumoniae were sequenced to identify their phylogenetic relationships, antimicrobial resistance and virulence genes. The antimicrobial susceptibility was determined by micro-broth dilution method according to the Clinical Laboratory and Standards Institute (CLSI) guidelines M100-S31. The K. pneumoniae isolates were divided into 42 different sequence types (STs) and 43 K Locus (KL) types. No dominant ST and KL were identified in this study, reflecting the great genetic divergence in the K. pneumoniae population. While ST1049 and KL5 were relatively prevalent, potentially indicating a robust transmission capability. Among the 78 virulence genes identified, eight isolates carried the aerobactin-encoding gene cluster iucABCD/iutA (iuc), which might be hypervirulent strains. The K. pneumoniae isolates showed variable resistance levels to cefotaxime, ceftiofur, cephalexin, ceftazidime, meropenem, florfenicol, sulfamethoxazole, trimethoprim/sulfamethoxazole, amikacin, gentamicin, streptomycin and tetracycline, while sensitive to ciprofloxacin, polymyxin B or tigecycline. A total of 73 kinds of antimicrobial resistance (AMR) genes were identified, and 28 strains were determined to be ESBL-producing bacteria. The pKP83 plasmid 2, which carried blaTEM and blaCTX-M-3 genes, was highly homological to three ESBL plasmids from human. The horizontal transfer potential of pKP83 plasmid 2 was further verified by conjugation to Escherichia coli J53 and K. pneumoniae K12016. It is necessary to conduct the regular monitoring of the antibiotic resistance of clinical K. pneumoniae from bovine mastitis, in order to prevent and control the transmission of K. pneumoniae along the food chain.