Up-regulation of inflammatory, oxidative stress, and apoptotic mediators via inflammatory, oxidative stress, and apoptosis-associated pathways in bovine endometritis.

Endometritis is the inflammation of the endothelial lining of the uterine lumen and is multifactorial in etiology. Escherichia (E.) coli is a Gram-negative bacteria, generally considered as a primary causative agent for bovine endometritis. Bovine endometritis is characterized by the activation of Toll-like receptors (TLRs) by E. coli, which in turn triggers inflammation, oxidative stress, and apoptosis. The objective of this study was to investigate the gene expression of inflammatory, oxidative stress, and apoptotic markers related to endometritis in the uteri of cows. Twenty uterine tissues were collected from the abattoir. Histologically, congestion, edema, hyperemia, and hemorrhagic lesions with massive infiltration of neutrophil and cell necrosis were detected markedly (P < 0.05) in infected uterine samples. Additionally, we identify E. coli using the ybbW gene (177 base pairs; E. coli-specific gene) from infected uterine samples. Moreover, qPCR and western blot results indicated that TLR2, TLR4, proinflammatory mediators, and apoptosis-mediated genes upregulated except Bcl-2, which is antiapoptotic, and there were downregulations of oxidative stress-related genes in the infected uterine tissue. The results of our study suggested that different gene expression regimes related to the immune system reflex were activated in infected uteri. This research gives a novel understanding of active immunological response in bovine endometritis.

Transcription Factor Nrf2 Modulates Lipopolysaccharide-Induced Injury in Bovine Endometrial Epithelial Cells.

Endometritis in high-yield dairy cows adversely affects lactation length, milk quality, and the economics of dairy products. Endoplasmic reticulum stress (ERS) in bovine endometrial epithelial cells (BEECs) occurs as a consequence of diverse post-natal stressors, and plays a key role in a variety of inflammatory diseases. Nuclear-factor-erythroid-2-related factor 2 (Nrf2) is an important protective regulatory factor in numerous inflammatory responses. However, the mechanism by which Nrf2 modulates inflammation by participating in ERS remains unclear. The objective of the present study was to explore the role of Nrf2 in lipopolysaccharide (LPS)-induced injury to BEECs and to decipher the underlying molecular mechanisms of this injury. The expression of Nrf2- and ERS-related genes increased significantly in bovine uteri with endometritis. Isolated BEECs were treated with LPS to stimulate the inflammatory response. The expression of Nrf2 was significantly higher in cells exposed to LPS, which also induced ERS in BEECs. Activation of Nrf2 led to enhanced expression of the genes for the inflammation markers TNF-α, p65, IL-6, and IL-8 in BEECs. Moreover, stimulation of Nrf2 was accompanied by activation of ERS. In contrast, Nrf2 knockdown reduced the expression of TNF-α, p65, IL-6, and IL-8. Additionally, Nrf2 knockdown decreased expression of ERS-related genes for the GRP78, PERK, eIF2α, ATF4, and CHOP proteins. Collectively, our findings demonstrate that Nrf2 and ERS are activated during inflammation in BEECs. Furthermore, Nrf2 promotes the inflammatory response by activating the PERK pathway in ERS and inducing apoptosis in BEECs.

Prostaglandin E2 promotes Staphylococcus aureus infection via EP4 receptor in bovine endometrium.

Prostaglandin E2 (PGE2) enhances Staphylococcus aureus infection but its mechanism is not well understood. Here, we examined the effect of PGE2 on Staphylococcal Protein A (SPA) expression in bovine endometrium and determined the role of select PGE2 receptors (i.e., EP2 and EP4) in adhesion and internalization of S. aureus. S. aureus isolate SA113 was used for in vitro infection of bovine endometrial tissues and epithelial cells, with treatment conditions consisting of untreated control, SA113 treatment, SA113 + PGE2, SA113 + PGE2 + EP2 receptor antagonist (AH-6809), and SA113 + PGE2 + EP4 receptor antagonist (AH-23848). Immunofluorescence assay revealed that PGE2 could promote SPA expression in S. aureus-infected bovine endometrial tissues. PGE2 also enhanced the adhesion and internalization of S. aureus in bovine endometrial cells. The addition of EP4 antagonist, but not the EP2 antagonist, abrogated the ability of PGE2 to promote S. aureus SPA expression, adhesion, and internalization in endometrial cells. Our findings suggest that S. aureus infection in the endometrium is enhanced by PGE2 through the EP4 receptor. This result is essential for the development of new approach to treating S. aureus infection, such as the application of EP4 antagonist as an adjunct drug treatment.

Clinical endometritis with Trueperella pyogenes reduces reproductive performance and milk production in dairy cows.

The aim of this study was to identify the impact of Trueperella pyogenes in cows with clinical endometritis (CE) on reproductive performance and milk production in affected cows. In total, 230 lactating Holstein dairy cows from six commercial dairy herds were sampled once between 28 and 33 days post-partum. Cows included in the present study did not receive antibiotic or anti-inflammatory treatments prior to the experimental period. Clinical endometritis (CE) was characterized as cow with vaginal mucus score = 3 (>50% of purulent vaginal discharge) and >18% polymorphonuclear neutrophilic leukocyte (PMNL). The body condition scores (BCS) and milk production were evaluated at the time of enrolment. The identification of isolated bacteria was carried out through the analysis of MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry). According to uterine health, three groups of dairy cows were formed: healthy control cows without T. pyogenes (n = 147), CE cows with T. pyogenes (n = 22) and CE cows without T. pyogenes (n = 61). CE cows with T. pyogenes had lower BCS, milk production and conception at first AI (p < .01) than CE cows without T. pyogenes and control cows. Furthermore, CE cows with T. pyogenes had higher (p < .01) service per pregnancy and had greater (p < .01) days to get pregnant than CE cows without T. pyogenes and control cows. This study demonstrates that CE cows with T. pyogenes had impaired reproductive performance and milk production when compared to cows without CE and CE cows without T. pyogenes. This information can contribute to a strategic treatment in cows affected by clinical endometritis, favouring the rational use of antibiotics on dairy farms.

Differential proteomic profiling of endometrium and plasma indicate the importance of hydrolysis in bovine endometritis.

Endometritis is an important disease of dairy cows that leads to significant economic losses in the dairy cattle industry. To investigate the alteration of proteins associated with endometritis in the dairy cow, the isobaric tags for relative and absolute quantification (iTRAQ) technique was applied to quantitatively identify differentially expressed proteins (DEP) in the endometrium and peripheral plasma of Chinese Holstein cows with endometritis. Compared with the normal (control) group, 159 DEP in the endometrium and 137 DEP in the plasma were identified in cows with endometritis. Gene ontology analysis demonstrated that the predominant endometrial DEP were primarily involved in responses to stimulus and stress processes and mainly played a role in hydrolysis in the extracellular region. The predominant plasma DEP were mainly components of the cytosol and non-membrane-bound organelles, and they were involved in the response to stress and regulation of enzyme activity. Protein-protein interaction of tissue DEP revealed that some core seed proteins, such as RAC2, ITGB2, and CDH1 in the same network as CD14, MMP3, and MMP9, had important functions in the cross-talk of pathways related to extracellular proteolysis. In summary, significant enzymatic hydrolase activity in the extracellular region is proposed as a molecular mechanism by which altered proteins may promote inflammation and hence endometritis.

Enhanced expression of miR-204 attenuates LPS stimulated inflammatory injury through inhibiting the Wnt/ß-catenin pathway via targeting CCND2.

One of the most common bacterial diseases of the reproductive system in dairy cows is endometritis, which will cause huge economic loss. Here, we investigate the mechanisms of miR-204 on LPS-stimulated endometritis in vitro and in vivo. Experiments displayed that the expression of miR-204 was lower in bovine uterine tissue samples or bovine endometrial epithelial cell line (BEND) that stimulated by LPS. Compared with the negative group, miR-204 treatment significantly suppressed the production of proinflammatory factors and the Wnt/ß-catenin pathway activation. Additionally, the result of the dual luciferase assay showed that miR-204 targeted cyclin D2. More importantly, up-regulation of miR-204 alleviated LPS induced uterine injury was confirmed in vivo studies. Molecular experiments indicated that the expression level of tight junctional proteins Claudin3 and cadherin1 were both enchanced by miR-204 treatment. Accordingly, miR-204 may serve as a new measure to prevent and treat endometritis caused by LPS.

miR-92b ameliorates lipoteichoic acid induced endometritis by suppressing Wnt/ß-catenin pathway activation.

Endometritis is one of the important reasons for the low fecundity of dairy cows, which has brought huge economic losses to the dairy industry. Emerging evidence suggests that miR-92b is a novel therapeutic molecule that plays a crucial role in many inflammatory diseases. However, its mechanism in lipoteichoic acid (LTA) induced endometritis remains unclear. In the present study, we explored the mechanism of miR-92b on LTA-induced endometritis in vivo and in vitro. The result displayed that the expression of miR-92b was reduced in LTA induced mouse endometritis and bovine endometrial epithelial cell lines (BEND). Overexpression miR-92b significantly alleviated mouse uterine injury and reduced the protein levels of TNF-α, IL-1ß and the MPO activity. The reporter assay of luciferase showed that miR-92b directly targeted the transmembrane receptor Frizzled-10 (FZD10), a transmembrane-type Wnt receptor. Molecular experiments were further performed to explore the mechanism of miR-92b in protecting LTA induced endometritis. The results of in vitro suggested that miR-92b mimic decreased the protein levels of Wnt3a and ß-catenin in LTA stimulated BEND, which were abolished by overexpression of FZD10. As expected, miR-92b mimic decreased the expression levels of TNF-α and IL-1ß, while overexpression of FZD10 promoted the production of these pro-inflammatory cytokines. Collectively, the above findings indicated that miR-92b might be an effective strategy for treatment of LTA induced endometritis.

Melatonin Alleviates Lipopolysaccharide-Induced Endometritis by Inhibiting the Activation of NLRP3 Inflammasome through Autophagy.

Bovine endometritis is characterized by reduced milk production and high rates of infertility. Prior research has indicated that melatonin may possess anti-inflammatory and antioxidant properties that can counteract the progression of inflammatory diseases. In this research, we attempted to elucidate the protective effects of melatonin on LPS-induced endometritis. The results obtained from enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qRT-PCR) revealed that melatonin effectively reduced the production and release of pro-inflammatory cytokines in an LPS-induced bovine endometrial epithelial cell line (BEND cells). Furthermore, western blotting demonstrated that melatonin treatment reduced the expression levels of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome-related proteins, including NLRP3, activated caspase-1, and cleaved IL-1ß. Importantly, we further demonstrated that the anti-inflammatory effect of melatonin on BEND cells was related to autophagy by western blotting. Moreover, we used western blotting to detect autophagy-related proteins, MitoSOX to detect mitochondrial reactive oxygen species production (mtROS), and mitochondrial membrane potential (MMP) assay to detect mitochondrial membrane potential. The administration of melatonin demonstrated a significant enhancement in autophagy within BEND cells, leading to the effective elimination of impaired mitochondria. This process resulted in a reduction in the generation of reactive oxygen species within the mitochondria, restoration of mitochondrial membrane potential, and inhibition of the NLRP3 inflammasome activation. Moreover, in a mouse model of LPS-induced endometritis, melatonin treatment repressed the expression of pro-inflammatory cytokines by ELISA and qRT-PCR, alleviated pathological changes by hematoxylin-eosin staining (H&E), and inhibited myeloperoxidase (MPO) activity. In conclusion, our study showed that melatonin inhibited the activation of the NLRP3 inflammasome in BEND cells through autophagy, which may provide a novel therapeutic strategy for bovine endometritis.

The Endometrial Microbiota-16S rRNA Gene Sequence Signatures in Healthy, Pregnant and Endometritis Dairy Cows.

Endometritis is one of the most important causes of infertility in dairy cows, resulting in high economic losses in the dairy industry. Though the presence of a commensal uterine microbiota is now well established, the complex role of these bacteria in genital health, fertility, and susceptibility to uterine diseases remains unclear. In this study, we explore the endometrial microbiota through 16S rRNA gene profiling from cytobrush samples taken ex vivo from healthy, pregnant, and endometritis cows. There were no significant differences between healthy and pregnant cows, whose uterine microbiota were dominated by Streptococcus, Pseudomonas, Fusobacterium, Lactococcus and Bacteroides. Compared to pregnant and clinically healthy cows, the uterine bacterial community of endometritis cows was significantly decreased in species diversity (p < 0.05), reflecting uneven community composition in different patterns with either dominance of Escherichia-Shigella, Histophilus, Bacteroides and Porphyromonas or Actinobacteria.

Andrographolide attenuates inflammatory response induced by LPS via activating Nrf2 signaling pathway in bovine endometrial epithelial cells.

Endometritis is one of the main causes of bovine infertility, which causes serious economic losses to the industry. The endometrium is the first line of defense against invading microbial pathogens in the uterus. Andrographolide is the primary active component of A. paniculate, and has been shown to have anti-inflammatory and antioxidant effects. However, its effects on the LPS-induced signaling pathway in bovine endometrial epithelial cells (bEECs) have not been reported yet. The aim of this study was to investigate the anti-inflammatory effects and mechanism of andrographolide in the LPS-induced inflammatory response of bEECs. We found that andrographolide strongly reduced LPS-induced NO and iNOS expression. The production of cytokines that were upregulated by LPS was significantly suppressed. To investigate the anti-inflammatory mechanism of andrographolide, we examined the activation of Nrf2. The results shown that andrographolide inhibited the expression of Keap1 but increased the expression of Nrf2. The expression levels of target genes of Nrf2 including Ho-1 and Nqo-1 were increased by andrographolide. Taken together, these results suggest that andrographolide may serve as a candidate to protect against the LPS-induced inflammatory response by inducing Nrf2 activation.

Activation of alpha7 nicotinic acetylcholine receptor protects bovine endometrial tissue against LPS-induced inflammatory injury via JAK2/STAT3 pathway and COX-2 derived prostaglandin E2.

Bovine endometritis is one of the major postpartum diseases associated with infertility and subfertility, decreasing the benefit of dairy industry. It is important to develop alternate therapies for endometritis in the context of drug residues in the milk and hormone disorder in the estrous cycle. α7 nicotine acetylcholine receptor has been identified as the core of 'cholinergic anti-inflammatory pathway (CAP)', which is a potential drug target to inflammatory diseases. However, there has been still no study on its anti-inflammatory effects and mechanism on lipopolysaccharide (LPS)-induced bovine endometritis. This study aimed to demonstrate the underlying anti-inflammatory effects and mechanism of α7-nACh receptor on LPS-induced inflammation in bovine endometrial tissues cultured in vitro. The results suggested that activation of α7-nACh receptor significantly suppressed the mRNA expression levels of interleukin 1ß (IL-1ß), IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) in bovine endometrial tissues. Western blot and enzyme-linked immunosorbent assay (ELISA) detection results showed that activation of α7-nACh receptor inhibited LPS-induced phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Moreover, α7-nACh receptor agonist decreased the expression of cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase-1(mPGES-1), as well as prostaglandin E2 (PGE2) secretion. Interestingly, in COX-2 inhibition experiment, activation of α7-nACh receptor increased COX-2 expression and PGE2 production, compared with COX-2 inhibitor treatment. In conclusion, activation of the cholinergic system through α7-nACh receptor agonist has suppressed inflammation of bovine endometrial tissues via JAK2/STAT3 pathway and potential COX-2-derived PGE2.

Protective Effect of Hydroxytyrosol on LPS-Induced Inflammation and Oxidative Stress in Bovine Endometrial Epithelial Cell Line.

Bovine endometritis is a serious pathogen-induced infectious disease that affects the physiological processes of estrus, pregnancy and the postpartum condition. The inflamed endometrium responds by activating an inflammatory intracellular signaling cascade that leads to increased expression of proinflammatory cytokines and reactive oxygen species (ROS). Oxidative stress is closely related to several pathological conditions in perinatal dairy cows and play a key role in tissue damage. Hydroxytyrosol (HT), a natural phenolic alcohol with a strong antioxidant activity, displayed a wide range of biological effect. The aim of this study was to evaluate the protective effects of HT in an in vitro model of lipopolysaccharide (LPS)-induced inflammation in bovine uterine endometrial cells. Our results showed that HT had a significant protective effect in LPS-induced inflammation and oxidative stress. HT was also able to increase the capacity of endogenous antioxidant systems through the up-regulation of the NRF2 pathway. Furthermore, HT restored the tight junction protein expressions. In conclusion, our results showed the protective effects of HT in LPS-stimulated BEND cells. Therefore, the results of this study suggest an important protective role of HT in the treatment and prevention of uterine pathologies in dairy cows.

Chemical composition and antibacterial activity of essential oils against pathogens often related to cattle endometritis.

INTRODUCTION: Endometritis is a condition marked by inflammation of the endometrium that affects dairy cows from 21 days after parturition, causing damage to herd fertility and economic losses on farms. The use of active compounds obtained from plant sources has gained importance as disease treatment agents in farm animals due to the high resistance rates currently observed against traditional antibiotics commonly used. The study was carried out to examine the chemical composition and to investigate the antibacterial activity of rosemary, cinnamon, cloves, eucalyptus, lemon, oregano and thyme essential oils against the reference strain of Escherichia coli (ATCC 25922), Fusobacterium necrophorum (ATCC 25286), Trueperella pyogenes (ATCC 19411) and Staphylococcus aureus (ATCC 29213), considered as typical bacteria causing endometritis. METHODOLOGY: The chemical composition of the seven essential oils were analyzed by GC-MS and their antibacterial activity was evaluated by the disc diffusion method. RESULTS: Thirty-six components were identified in total using GC-MS analyzes. The main compounds were cinnamaldehyde (86.5% for cinnamon essential oil), eugenol (85.7% for clove essential oil), 1,8-cineol (80% for eucalyptus and 47.8% rosemary essential oils), limonene (65.5% for lemon essential oil), carvacrol (72.1% for oregano essential oil) and thymol (48.8% for thyme essential oil). The disc diffusion assay revealed that cinnamon, clove, oregano, and thyme essential oils showed the best results compared to the other three essential oils, showing the largest zone of inhibition against all bacteria evaluated. CONCLUSIONS: These findings indicated that essential oils are a potential agent to be used as an alternative for bovine endometritis treatment.

Dynamics of uterine infections with Escherichia coli, Streptococcus uberis and Trueperella pyogenes in post-partum dairy cows and their association with clinical endometritis.

The diversity and dynamics of the uterine microbiota of dairy cows are poorly understood although it is becoming increasingly evident that they play a crucial role in the development of metritis and endometritis. Fourier-transform infrared (FTIR) spectroscopy was used to monitor the bovine microbiota of 40 cows on the day of calving and days 3, 9, 15, and 21 after parturition, and to investigate the associations of selected species with clinical endometritis (CE). Trueperella pyogenes (43.5%), Escherichia coli (21.5%), Bacillus spp. (21.0%) and Streptococcus uberis (18.5%) were the most frequently isolated microbes. Analyses of different sampling time points revealed that the presence of S. uberis on day 3 increased the risk of subsequent T. pyogenes infection on day 9 (odds ratio [OR] = 5.1, 95% confidence interval [CI] = 1.2-22.6). T. pyogenes infection (OR = 36.0, 95% CI = 3.8-343.2) and retained fetal membranes (RFM) (OR = 12.4, 95%CI = 1.4-112.7) were significant risk factors for CE. Cows with S. uberis on day 3 tended to have greater odds of CE than S. uberis-negative cows (OR = 7.1, 95% CI = 0.9-55.6). Chemometric analysis revealed significant differences in the metabolic profile of S. uberis strains isolated from cows with different vaginal discharge scores. This is the first study showing the association of specific S. uberis subtypes with the uterine health status of post-partum dairy cows. The study demonstrates that uterine clearance is a highly dynamic process, during which time bacteria show distinct patterns of progression, and provides information about interactions between bacterial species involved in the occurrence of CE.

Diagnosis and therapeutic considerations of uterine infections in dairy cattle: [review] / Consideraciones diagnósticas y terapéuticas para infecciones uterinas en vacas lecheras: [revisión]

From an animal health-well being, welfare, and performance perspective, the postpartum period is comprised of an early window where acute health and cow survival is an issue with the development of septic metritis. Subsequently, sustained presence of endometritis is likely to be contributing to sub-fertility in lactating dairy cows with an overall herd pregnancy rate of approximately 16%. The challenge is to integrate on commercial dairies preventive medicine programs with reproductive management to improve herd fertility. Cows affected by periparturient disorders such as hypocalcemia, dystocia, and retained fetal membranes are more likely to contract uterine infections than cows that calve normally are. Thus, appropriate management of the transition period is critical in the prevention of uterine infections at the herd level. In addition, a postpartum program to monitor health to provide treatment to cows in the early stages of disease should be implemented.

Desde el punto de vista del bienestar animal y de la perspectiva de productividad, el periodo postparto puede estar comprometido por una pequeña ventana durante el postparto temprano, en la cual la salud y supervivencia de las vacas es un problemática, debido a la presentación de metritis séptica. Esta se perpetuán subsecuentemente con la presentación de endometritis, la cual muy posiblemente contribuye con los patrones de sub-fertilidad en vacas lactantes, generando en general bajas tasas de preñez del 16 %. El reto es entonces integrar en las explotaciones comerciales un programa de medicina veterinaria preventiva con el manejo reproductivo, con el fin de mejorar la fertilidad del hato. Se ha descrito que las vacas que sufren problemas durante el periparto tales como hipocalcemia y retención de las membranas fetales son más susceptibles de contraer infecciones uterinas, comparadas con aquellas vacas que tienen un parto normal. Por ello un manejo apropiado del periodo de transición es crítico en la prevención de infecciones uterinas a nivel del hato. Así mismo, es necesario implementar un programa adecuado de monitoreo postparto con el fin de proveer tratamiento oportuno a las vacas durante los estados iniciales de enfermedad.