ABSTRACT
Stress and infection seriously threaten the reproductive performance and health of dairy cows. Various perinatal stresses increase plasma cortisol concentrations in cows, and chronically high cortisol levels may increase the incidence and severity of the uterine diseases. Selenium (Se) enhances antioxidant capacity of cows. The aim of this study was to explore how Se affects the oxidative stress of primary bovine endometrial stromal cells (BESC) with high cortisol background. The levels of reactive oxygen species (ROS) and other biomarkers of oxidative stress were measured using flow cytometry and assay kits. The changes in nuclear NF-E2-related factor 2 (Nrf2) pathway were detected by Western blot, qPCR, and immunofluorescence. The result showed that lipopolysaccharide (LPS) increased (P < 0.01) ROS and malondialdehyde (MDA) content and reduced (P <0.01) superoxide dismutase (SOD) concentration, provoking BESC oxidative stress. The elevated levels of cortisol resulted in the accumulation (P < 0.05) of ROS and MDA and inhibition (P < 0.05) of SOD in unstimulated BESC, but demonstrated an antioxidative effect in LPS-stimulated cells. Pretreatment with Se reduced (P < 0.01) the levels of ROS and MDA, while increasing (P < 0.05) the antioxidant capacities and the relative abundance of gene transcripts and proteins related to the Nrf2 pathway in BESC. This antioxidant effect was more pronounced in the presence of high cortisol level. Se alleviated the LPS-induced cellular oxidative stress, which is probably achieved through activating Nrf2 pathway. At high cortisol levels, Se supplement has a more significant protective effect on BESC oxidative stress. This study provided evidence for the protective role of Se in bovine endometrial oxidative damage of stressed animals and suggested the potential regulatory mechanism in vitro.
ABSTRACT
BACKGROUND: An elevated endogenous cortisol level due to the peripartum stress is one of the risk factors of postpartum bovine uterine infections. Selenium is a trace element that elicits anti-inflammation and antioxidation properties. This study aimed to reveal the modulatory effect of selenium on the inflammatory response of primary bovine endometrial stromal cells in the presence of high-level cortisol. The cells were subjected to lipopolysaccharide to establish cellular inflammation. The mRNA expression of toll-like receptor 4 (TLR4), proinflammatory factors, and selenoproteins was measured with qPCR. The activation of NF-κB and MAPK signalling pathways was detected with Western blot and immunofluorescence. RESULTS: The pretreatment with sodium selenite (2 and 4 µΜ) resulted in a down-regulation of TLR4 and genes encoding proinflammatory factors, including interleukin (IL)-1ß, IL-6, IL-8, tumour necrosis factor α, cyclooxygenase 2, and inducible nitric oxide synthase. Selenium inhibited the activation of NF-κB and the phosphorylation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38MAPK and c-Jun N-terminal kinase/stress-activated protein kinase. The suppression of those genes and pathways by selenium was more significant in the presence of high cortisol level (30 ng/mL). Meanwhile the gene expression of glutathione peroxidase 1 and 4 was promoted by selenium, and was even higher in the presence of cortisol and selenium. CONCLUSIONS: The anti-inflammatory action of selenium is probably mediated through NF-κB and MAPK, and is augmented by cortisol in primary bovine endometrial stromal cells.
Subject(s)
Anti-Inflammatory Agents , Endometrium , Hydrocortisone , Selenium , Stromal Cells , Animals , Cattle , Female , Endometrium/drug effects , Endometrium/metabolism , Endometrium/cytology , Hydrocortisone/pharmacology , Stromal Cells/drug effects , Stromal Cells/metabolism , Selenium/pharmacology , Anti-Inflammatory Agents/pharmacology , NF-kappa B/metabolism , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 4/genetics , Cells, Cultured , Lipopolysaccharides/pharmacologyABSTRACT
Endometritis is a common postpartum disease in cows. It delays uterine involution and impairs normal physiological function. This can result in long-term or even lifelong infertility and cause significant losses to the dairy farming industry. Traditional treatments like antibiotics possess certain shortcomings, such as antibiotic residues, the abuse of antibiotics, and increased antimicrobial resistance of pathogens. Alternative treatment strategies are needed to minimize the utilization of antibiotics in dairy production. As an essential trace element in animals, selenium (Se) plays a vital role in regulating immune function, the inflammatory response, and oxidative stress, affecting the speed and completeness of tissue repair. This paper reviewed previous studies to analyse the potential of Se in the prevention and treatment of bovine endometritis, aiming to provide a new direction to increase production capacity in the future.
Subject(s)
Cattle Diseases , Endometritis , Selenium , Animals , Cattle , Endometritis/veterinary , Endometritis/prevention & control , Endometritis/drug therapy , Female , Selenium/therapeutic use , Selenium/administration & dosage , Selenium/pharmacology , Cattle Diseases/prevention & control , Cattle Diseases/drug therapy , Oxidative Stress/drug effectsABSTRACT
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/ß-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 ß-catenin translocation from the cytoplasm to the nucleus and activated the Wnt/ß-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/ß-catenin signaling pathways.
Subject(s)
Proto-Oncogene Proteins c-akt , Selenium , Female , Cattle , Animals , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/pharmacology , Lipopolysaccharides/toxicity , Lipopolysaccharides/metabolism , Selenium/pharmacology , Selenium/metabolism , beta Catenin/metabolism , Phosphatidylinositol 3-Kinases/metabolism , bcl-2-Associated X Protein/pharmacology , Wnt Signaling Pathway , Epithelial Cells , Cell Proliferation , ApoptosisABSTRACT
Staphylococcus pseudintermedius (S. pseudintermedius) is a common pathogen that causes canine corneal ulcers. However, the pathogenesis remained unclear. In this study, it has been demonstrated that S. pseudintermedius invaded canine corneal epithelial cells (CCECs) intracellularly, mediating oxidative damage and pyroptosis by promoting the accumulation of intracellular reactive oxygen species (ROS) and activating the NLRP3 inflammasome. The canine corneal stroma was infected with S. pseudintermedius to establish the canine corneal ulcer model in vivo. The intracellular infectious model in CCECs was established in vitro to explore the mechanism of the ROS - NLRP3 signalling pathway during the S. pseudintermedius infection by adding NAC or MCC950. Results showed that the expression of NLRP3 and gasdermin D (GSDMD) proteins increased significantly in the infected corneas (p < 0.01). The intracellular infection of S. pseudintermedius was confirmed by transmission electron microscopy and immunofluorescent 3D imaging. Flow cytometry analysis revealed that ROS and pyroptosis rates increased in the experimental group in contrast to the control group (p < 0.01). Furthermore, NAC or MCC950 inhibited activation of the ROS - NLRP3 signalling pathway and pyroptosis rate significantly, by suppressing pro-IL-1ß, cleaved-IL-1ß, pro-caspase-1, cleaved-caspase-1, NLRP3, GSDMD, GSDMD-N, and HMGB1 proteins. Thus, the research confirmed that oxidative damage and pyroptosis were involved in the process of CCECs infected with S. pseudintermedius intracellularly by the ROS - NLRP3 signalling pathway. The results enrich the understanding of the mechanisms of canine corneal ulcers and facilitate the development of new medicines and prevention measures.
Subject(s)
NLR Family, Pyrin Domain-Containing 3 Protein , Pyroptosis , Staphylococcus , Animals , Dogs , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Reactive Oxygen Species/metabolism , Ulcer , Cell Line , Inflammasomes/metabolism , Epithelial Cells/metabolism , SulfonamidesABSTRACT
PROBLEM: Endometritis is a common disease that affects dairy cow reproduction. Autophagy plays a vital role in cellular homeostasis and modulates inflammation by regulating interactions with innate immune signaling pathways. However, little is known about the regulatory relationship between autophagy and inflammation in bovine endometrial epithelial cells (BEECs). Thus, we aimed to determine the role of autophagy in the inflammatory response in BEECs. METHODS OF STUDY: In the present study, the expression levels of proinflammatory cytokines were measured by quantitative real-time polymerase chain reaction. Changes in the nuclear factor-κB (NF-κB) pathway and autophagy were determined using immunoblotting and immunocytochemistry. The induction of autophagosome formation was visualized by transmission electron microscopy. RESULTS: Our results demonstrated that autophagy activation was inhibited in LPS-treated BEECs, while activation of the NF-κB pathway and the mRNA expression of IL-6, IL-8, and TNF-α were increased. Furthermore, blocking autophagy with the inhibitor chloroquine increased NF-κB signaling pathway activation and proinflammatory factor expression in LPS-treated BEECs. Conversely, activation of autophagy with the agonist rapamycin inhibited the NF-κB signaling pathway and downregulated proinflammatory factors. CONCLUSIONS: These data indicated that LPS-induced inflammation was related to the inhibition of autophagy in BEECs. Thus, the activation of autophagy may represent a novel therapeutic strategy for eliminating inflammation in BEECs.
Subject(s)
Lipopolysaccharides , NF-kappa B , Female , Cattle , Animals , NF-kappa B/metabolism , Inflammation/metabolism , Epithelial Cells , AutophagyABSTRACT
Bovine endometritis severely inhibits uterine repair and causes considerable economic loss. Besides, parturition-induced high cortisol levels inhibit immune function, reduce cell proliferation, and further inhibit tissue repair. Selenium (Se) is an essential trace element for animals to maintain normal physiological function and has powerful antioxidant functions. This study investigated whether Se supplementation reduces endometrial damage and promotes tissue repair in cows with endometritis under stress and explored the underlying mechanism. Primary bovine endometrial epithelial cells were isolated and purified from healthy cows. The cells were treated with different combinations of lipopolysaccharide (LPS), cortisol, and various concentrations of Se. Data showed that LPS stimulation inhibited cell proliferation and increased cell apoptosis. High levels of cortisol further exacerbated these effects. Flow cytometry, scratch wound healing tests, and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assays showed that Se supplementation promoted cell cycle progression, cell migration, and cell proliferation in the presence of LPS and cortisol. The quantitative PCR results showed that the expression of related growth factors was increased after Se supplementation. After administering various inhibitors, we further demonstrated that Se supplementation decreased the activity of glycogen synthetase kinase 3ß (GSK-3ß) through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway to reduce the degradation of ß-catenin except the Wnt signal to promote cell proliferation. In conclusion, Se supplementation attenuated the cell damage induced by LPS at high cortisol levels and increased cell proliferation to promote uterine repair by elevating the mRNA expression of TGFB3 and VEGFA and activating the PI3K/AKT/GSK-3ß/ß-catenin signaling pathway.
After parturition, endometritis is a common bovine disease, which hinders endometrial repair and reduces bovine economic value. Besides, parturition-induced high cortisol levels cause immunosuppression, aggravate infection, and further inhibit cell proliferation and tissue repair. As an essential trace element, adding selenium to feed helps to maintain the normal physiological function of animals. This study developed a cellular model using lipopolysaccharide (LPS) and cortisol to simulate cows with endometritis in stress conditions. The results showed that Se supplementation attenuated bovine endometrial epithelial cell damage and promoted their proliferation in the presence of LPS and high cortisol levels, which are positively correlated with the concentration of Se. Besides, this study proved another molecular mechanism for Se to regulate ß-catenin except for the Wnt signal by affecting the ß-catenin degradation pathway.
Subject(s)
Cattle Diseases , Endometritis , Selenium , Female , Cattle , Animals , Proto-Oncogene Proteins c-akt/metabolism , Endometritis/chemically induced , Endometritis/genetics , Endometritis/veterinary , Lipopolysaccharides/toxicity , Hydrocortisone/metabolism , Selenium/pharmacology , Selenium/metabolism , beta Catenin/metabolism , Glycogen Synthase Kinase 3 beta/metabolism , Glycogen Synthase Kinase 3 beta/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Cell Proliferation , Epithelial Cells/metabolism , Dietary Supplements , Cattle Diseases/geneticsABSTRACT
Klebsiella pneumoniae (K. pneumoniae) is one of the major pathogens causing bovine clinical mastitis. Autophagy maintains cellular homeostasis and resists excessive inflammation in eukaryotic organisms. Selenomethionine (Se-Met) is commonly used as a source of selenium supplementation for dairy cows. This study aimed to investigate the effects of Se-Met on inflammatory responses mediated by nuclear factor-kappa B (NF-κB) through autophagy. We infected bovine mammary epithelial cell line (MAC-T) with K. pneumoniae and examined the expression of autophagy-related proteins and changes in autophagic vesicles, LC3 puncta, and autophagic flux at various intervals. The results showed that K. pneumoniae activated the early-stage autophagy of MAC-T cells. The levels of LC3-II, Beclin1, and ATG5, as well as the number of LC3 puncta and autophagic vesicles, increased after 2 h post-treatment. However, the late-stage autophagic flux was blocked. Furthermore, the effect of autophagy on NF-κB-mediated inflammation was investigated with different autophagy levels. The findings showed that enhanced autophagy inhibited the K. pneumoniae-induced inflammatory responses of MAC-T cells. The opposite results were found with the inhibition of autophagy. Finally, we examined the effect of Se-Met on NF-κB-mediated inflammation based on autophagy. The results indicated that Se-Met alleviated K. pneumoniae-induced autophagic flux blockage, inhibited NF-κB-mediated inflammation, and decreased the adhesion of K. pneumoniae to MAC-T cells. The inhibitory effect of Se-Met on NF-κB-mediated inflammation could be partially blocked by the autophagy inhibitor chloroquine (CQ). Overall, Se-Met attenuated K. pneumoniae-induced NF-κB-mediated inflammatory responses by enhancing autophagic flux.
Subject(s)
NF-kappa B , Selenomethionine , Female , Cattle , Animals , NF-kappa B/metabolism , Selenomethionine/pharmacology , Selenomethionine/metabolism , Klebsiella pneumoniae , Autophagy , Inflammation/metabolism , Epithelial Cells/metabolismABSTRACT
BACKGROUND: Targeted next-generation sequencing (NGS) is a powerful and suitable approach to comprehensively identify multiple types of variants in tumors. RNA-based NGS is increasingly playing an important role in precision oncology. Both parallel and sequential DNA- and RNA-based approaches are expensive, burdensome, and have long turnaround times, which can be impractical in clinical practice. A streamlined, unified DNA- and RNA-based NGS approach is urgently needed in clinical practice. METHODS: A DNA/RNA co-hybrid capture sequencing (DRCC-Seq) approach was designed to capture pre-capture DNA and RNA libraries in a single tube and convert them into one NGS library. The performance of the DRCC-Seq approach was evaluated by a panel of reference standards and clinical samples. RESULTS: The average depth, DNA data ratio, capture ratio, and target coverage 250 (×) of the DNA panel data had a negative correlation with an increase in the proportion of RNA probes. The SNVs, indels, fusions, and MSI status were not affected by the proportion of RNA probes, but the copy numbers of the target genes were higher than expected in the standard materials, and many unexpected gene amplifications were found using D:R (1:2) and D:R (1:4) probe panels. The optimal ratio of DNA and RNA probes in the combined probe panel was 1:1 using the DRCC-Seq approach. The DRCC-Seq approach was feasible and reliable for detecting multiple types of variants in reference standards and real-world clinical samples. CONCLUSIONS: The DRCC-Seq approach is more cost-effective, with a shorter turnaround time and lower labor requirements than either parallel or sequential targeted DNA NGS and RNA NGS. It is feasible to identify multiple genetic variations at the DNA and RNA levels simultaneously in clinical practice.
Subject(s)
Neoplasms , Nucleic Acids , Humans , Neoplasms/genetics , RNA/genetics , RNA Probes , Precision Medicine , DNA , High-Throughput Nucleotide SequencingABSTRACT
Animal infection models are crucial for studying various aspects of Ehrlichia canis infections. To understand the pathogenesis of the first Chinese isolate of E. canis and simulate the natural progression of canine ehrlichiosis, we developed a model with 18 Beagle dogs that consisted of E. canis initial infection (days 0-17), treatment with doxycycline or rifampicin (days 18-32), recovery (days 33-66), E. canis reinfection (days 67-91), and Babesia vogeli superinfection (days 92-116). We measured body weight and rectal temperature every other day, drew blood every 4 days for routine hematology and biochemistry tests, and for quantification of E. canis and B. vogeli by quantitative PCRs. In this study, the first isolate of E. canis from China was used to experimentally infect dogs, and the infected dogs exhibited clinical signs of acute severe ehrlichiosis, including high fever, loss of appetite, dehydration, and body weight loss, confirming the similar pathogenicity of E. canis in China as compared to isolates from other regions. Infection with E. canis and B. vogeli led to reduced body weight and fever in dogs. Doxycycline treatment led to absence of E. canis DNA in infected dogs, while rifampicin treatment lowered the blood E. canis copy number up to 1.5 folds. E. canis-free infected dogs after doxycycline treatment were successfully re-infected with E. canis, indicating dogs with antibodies are still at risk of re-infection. Super-infection with B. vogeli resulted in higher fever, more severe anemia, and a reduced number of platelets. Splenectomized dogs showed significantly higher E. canis numbers during recovery and re-infection than intact dogs. The histological changes were observed in brain, lung, kidney, liver and spleen of the infected dogs. The findings in this study provide insights into clinical and hematologic responses, as well as effective treatment options, for dogs infected with the first Chinese isolate of E. canis, and may contribute to our understanding of the diagnosis and prevention of tick-borne diseases in dogs, including canine monocytic ehrlichiosis.
ABSTRACT
Endometritis is a common postpartum disease of female animals that causes significant losses to the goat industry. High levels of cortisol induced by various stresses after delivery severely inhibit innate immunity and tissue repair. The repair ability of the endometrium is closely related to the reproductive performance of goats. Selenium (Se) is an essential trace element in animals that has powerful antioxidant and immunity-enhancing functions. In this study, we established a goat model of endometritis at high cortisol (Hydrocortisone) levels to investigate the effect of Se (supplement additive) on endometrial repair. The results showed that the clinical symptoms, %PMN in uterine secretions, morphological endometrial damage, and the gene expression of BAX were reduced in the goats with Se supplementation compared with those in the model group. Se increased the gene expression of BCL2, VEGFA, TGFB1, and PCNA and activated the PI3K/AKT and Wnt/ß-catenin signaling pathways in goats with Se supplementation. In conclusion, Se reduced the inflammatory response, increased the proliferation, and decreased the apoptosis of endometrial cells to promote endometrial tissue repair in goats with endometritis at high cortisol levels. It probably achieved this effect of promoting repair by activating the Wnt/ß-catenin and PI3K/AKT pathways and affecting the gene expression of VEGFA, TGFB1, PCNA, BCL2, and BAX.
ABSTRACT
KEY MESSAGE: VAMP726/VAMP725 and SYP131 can form a part of a SNARE complex to mediate vesicle secretion at the pollen tube apex. Secretory vesicle fusion with the plasma membrane of the pollen tube tip is a key step in pollen tube growth. Membrane fusion was mediated by SNAREs. However, little is known about the composition and function of the SNARE complex during pollen tube tip growth. In this study, we constructed a double mutant vamp725 vamp726 via CRISPRâCas9. Fluorescence labeling combined with microscopic observation, luciferase complementation imaging, co-immunoprecipitation and GST pull-down were applied in the study. We show that double mutation of the R-SNAREs VAMP726 and VAMP725 significantly inhibits pollen tube growth in Arabidopsis and slows vesicle exocytosis at the apex of the pollen tube. GFP-VAMP726 and VAMP725-GFP localize mainly to secretory vesicles and the plasma membrane at the apex of the pollen tube. In addition, fluorescence recovery after photobleaching (FRAP) experiments showed that mCherry-VAMP726 colocalizes with Qa-SNARE SYP131 in the central region of the pollen tube apical plasma membrane. Furthermore, we found that VAMP726 and VAMP725 can interact with the SYP131. Based on these results, we suggest that VAMP726/VAMP725 and SYP131 can form a part of a SNARE complex to mediate vesicle secretion at the pollen tube apex, and vesicle secretion may mainly occur at the central region of the pollen tube apical plasma membrane.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Pollen Tube/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Membrane/metabolism , SNARE Proteins/genetics , SNARE Proteins/metabolismABSTRACT
The bovine uterus is susceptible to infection, and the elevated cortisol level due to stress are common in cows after delivery. The essential trace element selenium plays a pivotal role in the antioxidant and anti-inflammatory defence system of body. This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high-level cortisol. The primary bovine endometrial epithelial cells were subjected to Escherichia coli lipopolysaccharide to establish cellular inflammation model. The gene expression of inflammatory mediators and proinflammatory cytokines was measured by quantitative PCR. The key proteins of NF-κB and MAPK signalling pathways were detected by Western blot and immunofluorescence. The result showed that pre-treatment of Na2 SeO3 (1, 2 and 4 µΜ) decreased the mRNA expression of proinflammatory genes, inhibited the activation of NF-κB and suppressed the phosphorylation of extracellular signal-regulated kinase, P38MAPK and c-Jun N-terminal kinase. This inhibition of inflammation was more apparent in the presence of high-level cortisol (30 ng/mL). These results indicated that selenium has an anti-inflammatory effect, which is mediated via NF-κB and MAPK signalling pathways and is augmented by cortisol in bovine endometrial epithelial cells.
Subject(s)
NF-kappa B , Selenium , Female , Cattle , Animals , NF-kappa B/metabolism , Lipopolysaccharides/pharmacology , Hydrocortisone/pharmacology , Selenium/pharmacology , Inflammation/drug therapy , Anti-Inflammatory Agents/pharmacology , Cytokines/metabolism , Epithelial Cells/metabolism , MAP Kinase Signaling SystemABSTRACT
Meloxicam is a selective cyclooxygenase-2 inhibitor and has been widely used in combination with antibiotics to alleviate uterine inflammation and provide analgesia in postpartum cows. Studies have shown that meloxicam has antioxidant and anti-inflammatory effects. However, the link between meloxicam and uterine inflammation and oxidative stress in dairy cows has not been studied. The purpose of this study was to research the effects of meloxicam (0.5 or 5 µM) on oxidative stress and inflammatory response of primary bovine endometrial epithelial cells (BEEC) stimulated by Escherichia coli lipopolysaccharide (1 µg/mL LPS). As a result, LPS stimulated the production of oxidative stress markers and the expression of inflammatory factors, accompanied by a decrease in the activity and the gene transcription of antioxidant enzymes. Co-treatment of meloxicam and LPS reduced the content of oxidative stress markers and the mRNA levels of the pro-inflammatory genes, and improved antioxidant enzyme activities and the corresponding gene expression as compared with the cells treated with LPS alone. Meloxicam attenuated the inhibitory effect of the Nrf2 pathway and the phosphorylation levels of p65 and IκBα caused by LPS. In conclusion, meloxicam alone had no effect on BEEC, but prevented oxidative stress and inflammatory response in LPS-stimulated BEEC.
Subject(s)
Lipopolysaccharides , NF-kappa B , Female , Cattle , Animals , NF-kappa B/metabolism , Meloxicam/therapeutic use , Meloxicam/metabolism , Meloxicam/pharmacology , Lipopolysaccharides/pharmacology , NF-E2-Related Factor 2/metabolism , Antioxidants/pharmacology , Antioxidants/metabolism , Oxidative Stress , Inflammation/drug therapy , Inflammation/metabolism , Epithelial CellsABSTRACT
Mitochondria are cellular organelles that are involved in various metabolic processes, and damage to mitochondria can affect cell health and even lead to disease. Mitophagy is a mechanism by which cells selectively wrap and degrade damaged mitochondria to maintain cell homeostasis. However, studies have not focused on whether mitophagy is involved in the occurrence of Staphylococcus aureus (S. aureus)-induced mastitis in dairy cows. Here, we found that S. aureus infection of bovine macrophages leads to oxidative damage and mitochondria damage. The expression of LC3, PINK1 and Parkin was significantly increased after intracellular infection. We observed changes in the morphology of mitochondria and the emergence of mitochondrial autolysosomes in bovine macrophages by transmission electron microscopy and found that enhanced mitophagy promoted bacterial proliferation in the cell. In conclusion, this study demonstrates that S. aureus infection of bovine macrophages induces mitophagy through the PINK1/Parkin pathway, and this mechanism is used by the bacteria to avoid macrophage-induced death. These findings provide new ideas and references for the prevention and treatment of S. aureus infection.
Subject(s)
Macrophages , Mitophagy , Staphylococcus aureus , Animals , Cattle , Mitochondria/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Macrophages/metabolismABSTRACT
BACKGROUND: The stromal layer is the thickest layer of the cornea, and corneal stromal cells play an important role in the inflammatory response and wound repair. This study investigated the effect of MCC950, an inhibitor of NLRP3 inflammasome, on the inflammatory response and proliferation of canine corneal stromal cells (CCSCs) induced by Staphylococcus pseudintermedius (S. pseudintermedius). METHODS: CCSCs were pretreated with MCC950 and infected with S. pseudintermedius. The phosphorylation of p65, IκBα, PI3K, and AKT and the expression of NLRP3, caspase-1 p20, cleaved IL-1ß, ASC, ß-catenin, c-Myc, and CyclinD1 were detected by western blotting. The expression of inflammatory factors (IL-1ß, IL-6, IL-8, IL-18, and TNF-α) and growth factors (EGF, FGF, TGF-ß1, VEGF, and CTGF) were measured by RT-PCR. The levels of MDA content and LDH activity were detected by an assay kit. The cell cycle was detected by flow cytometry. RESULTS: MCC950 down-regulated the phosphorylation of p65, IκBα, PI3K, and AKT and decreased the expression of NLRP3, caspase-1 p20, cleaved IL-1ß, ASC, ß-catenin, c-Myc, and CyclinD1 compared to those in the S. pseudintermedius infection group (p < 0.05). MCC950 significantly inhibited the expression of inflammatory factors (IL-1ß, IL-6, IL-8, IL-18, and TNF-α) and growth factors (EGF, FGF, TGF-ß1, VEGF, and CTGF) induced by S. pseudintermedius (p < 0.01). Compared to the S. pseudintermedius infection group, the MDA content and LDH activity of CCSCs were significantly decreased after treatment with MCC950 (p < 0.01). CONCLUSION: MCC950 attenuates S. pseudintermedius-induced inflammatory responses in CCSCs. At the same time, MCC950 can inhibit excessive proliferation of cells, which is beneficial for alleviating corneal fibrosis healing.
Subject(s)
Interleukin-18 , NLR Family, Pyrin Domain-Containing 3 Protein , Animals , Dogs , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NF-KappaB Inhibitor alpha , Transforming Growth Factor beta1 , beta Catenin , Tumor Necrosis Factor-alpha , Interleukin-6 , Epidermal Growth Factor , Interleukin-8 , Proto-Oncogene Proteins c-akt , Vascular Endothelial Growth Factor A , Heterocyclic Compounds, 4 or More Rings/pharmacology , Sulfones/pharmacology , Caspase 1 , Stromal Cells/metabolism , Cornea/metabolism , Cell Proliferation , Phosphatidylinositol 3-KinasesABSTRACT
Staphylococcus aureus (S. aureus) is known to induce chronic and persistent bovine mammary infection, which affects milk quality and leads to premature culling. The ability of S. aureus to invade mammalian cells protects it from clearance by the immune system. Mitophagy is important in cell homeostasis, and can be utilized by pathogens for immune escape. However, mitophagy's role in S. aureus-associated bovine mastitis remains unclear. Here, S. aureus infection induced mitophagy and enhanced mitochondrial translocation of parkin in MAC-T cells. After mitophagy inhibition by Mdivi-1 treatment or PTEN-induced putative kinase 1 (PINK1) silencing in MAC-T cells infected with S. aureus, NOD-like receptor protein 3 (NLRP3) inflammasome activation and p65 and IκBα phosphorylation were increased. Meanwhile, PINK1 overexpression had the opposite effects. In addition, NLRP3 inflammasome overactivation and enhanced p65 and IκBα phosphorylation caused by PINK1 silencing were reversed by MitoTEMPO. Furthermore, PINK1/parkin-mediated mitophagy promoted S. aureus survival and contributed to persistent S. aureus infection. These findings provide new insights into S. aureus invasion in bovine mastitis.
Subject(s)
Mastitis, Bovine , Staphylococcal Infections , Animals , Cattle , Female , Epithelial Cells/metabolism , Inflammasomes/metabolism , Mitophagy , NF-kappa B/metabolism , NF-KappaB Inhibitor alpha , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Proteins , Protein Kinases/genetics , Protein Kinases/metabolism , Staphylococcal Infections/veterinary , Staphylococcus aureus/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Canine bacterial keratitis is a common infection that can potentially threaten vision. Staphylococcus pseudintermedius (S. pseudintermedius) is an opportunistic pathogen that has been isolated from the canine conjunctival sac but there are only a few reports on the role of this bacterium in canine keratitis. This study focused on the distribution rate of S. pseudintermedius in the canine conjunctival sac, and the antibiotic resistance, biofilm-producing ability, and dissemination of virulence factors in strains of S. pseudintermedius isolated from healthy dogs and dogs with keratitis. The study included 35 healthy dogs and 40 dogs with keratitis. Bacterial species were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Strains of S. pseudintermedius were screened for resistance against nine different antibiotics by the Kirby-Bauer assay. The ability to produce biofilm was investigated by microtiter plate assay (MtP) and amplification of icaA and icaD genes. Virulence factors in the strains were also evaluated. A total of 132 aerobic bacteria were isolated from the 119 samples in the study. Among them, 67 bacterial strains were isolated from 70 eyes of healthy dogs, and 65 bacterial strains were isolated from 49 eyes of dogs with keratitis. The prevalence of S. pseudintermedius, which was the most frequent bacterial isolate in both the groups, was 20.9% in the healthy group and 23.08% in the keratitis group. Most of the isolates of S. pseudintermedius were sensitive to rifampin (96.6%), oxacillin (100%), and neomycin (96.6%), and resistant to tetracycline (96.6%). Virulence factors such as lip (96.6%), hlgB (96.6%), and hlgA (96.6%) were found in most of the isolates, and 89.66% of isolates were classed as biofilm producers. In conclusion, S. pseudintermedius was the common bacterium in the conjunctivital sac of the healthy dogs and dogs with keratitis in Yangzhou, China, and the presence of virulence factors and biofilm-formation ability were high in the strains isolated from the dogs with keratitis.
ABSTRACT
A photocatalyst- and additive-free visible-light-induced α-C(sp3)-H phosphinylation of unactivated ethers involving a C-O bond cleavage with molecular oxygen as the sole oxidant at room temperature has been achieved. This method provides a sustainable access to α-hydroxyphosphine oxides in up to 88% yield with good functional group compatibility under mild and neutral conditions (34 examples). Moreover, the subsequent two-step conversion of the resulting dihydroxy diarylphosphine oxides afforded α-phosphinylated cyclic ethers in good overall yields (10 examples).
ABSTRACT
Autophagy is crucial for the maintenance of homeostasis under stimuli related to infection. Selenium (Se) plays variable roles in defence against infection and Selenomethionine (Se-Met) is a common Se supplementation. This study aimed to understand whether Se-Met could regulate the nuclear factor-kappa B (NF-κB) signaling pathway through autophagy. Mammary alveolar cell-T (MAC-T) was challenged with Escherichia coli (E. coli). Western blotting and real-time quantitative PCR (RT-qPCR) were used to detect the protein expression and mRNA expression of cytokines. Immunofluorescence assays were performed to observe the expression of intracellular LC3. The results showed that E. coli inhibited autophagy by decreasing the LC3-â ¡ protein levels, and the Atg5 and Beclin1 protein levels were increased after 4 h. Infection also decreased the number of LC3 puncta. E. coli increased the phosphorylation of p65 and IκBα protein. Concomitantly, the levels of interleukin (IL)-1ß, IL-6, IL-8 and tumour necrosis factor (TNF)-α mRNA increased at 3 and 4 h post-infection. We further explored the regulatory role of autophagy on NF-κB-mediated inflammation with autophagy modulators and shAtg5. The results indicated that the autophagy activator reduced the phosphorylation of p65 and IκBα and the mRNA expression of IL-1ß, IL-6, IL-8 and TNF-α. Additionally, activating autophagy weakened the adhesion to MAC-T of E. coli. Autophagy inhibitors exacerbated NF-κB-mediated inflammation and strengthened the adhesion of E. coli to cells. We then examined the effects of Se-Met on NF-κB-mediated inflammation through autophagy. The data suggested that Se-Met enhanced LC3-II expression, inhibited the E. coli-induced phosphorylation of p65 and IκBα, and suppressed the adhesion ability of E. coli to MAC-T and that the effects of Se-Met in attenuating NF-κB-mediated inflammation were partially blocked by an autophagy inhibitor. In summary, Se-Met alleviated NF-κB-mediated inflammation induced by E. coli by enhancing autophagy in bovine mammary epithelial cells.