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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.

Bovine uterine infection is commonly caused by Escherichia coli (E. coli). Elevated concentrations of plasma cortisol have been reported in postpartum cows. However, the direct role of cortisol in the inflammatory response of bovine endometrial stromal cells (BESCs) remains unclear. Therefore, the aim of the study was to explore the regulatory effect of cortisol on lipopolysaccharide (LPS)-induced inflammatory response in BESCs. Both the primary and immortalized BESCs were used in this study. BESCs were treated with cortisol (5, 15, and 30 ng/mL) in the presence of LPS. The mRNA expression of inflammatory cytokines and chemokines was detected using RT-qPCR. Western blot and immunofluorescence were used to analyze the activation of the NF-κB and MAPK signaling pathways. The results revealed that cortisol downregulated the LPS-induced overexpression of interleukin(IL)-1β, IL-6, IL-8, TNF-α, COX-2, iNOS in BESCs. Moreover, cortisol inhibited LPS-induced phosphorylation levels of IκB, p65, ERK1/2, JNK and p38, and p65 nuclear translocation in BESCs. These results indicated that cortisol inhibited LPS-induced inflammatory response in BESCs, which may be mediated by suppressing the NF-κB and MAPK signaling pathways.

Bacterial keratitis is a common eye disease in dogs and can seriously affect vision. This study investigated the anti-inflammatory effect of MCC950 in the cornea of canines infected with Staphylococcus pseudintermedius (S. pseudintermedius).In vitro, canine cornea epithelial cells were pretreated with MCC950 and PDTC and then infected with S. pseudintermedius. The key proteins of the NF-κB pathway and NLRP3 inflammasome were detected by Western blotting, the levels of inflammatory factors were detected by qPCR, and the levels of MDA and LDH were detected by assay kit. In vivo, the canine keratitis model was established by injecting S. pseudintermedius into the corneal stroma layer. After treatment with MCC950, slit-lamp examinations were performed. Cornea tissue protein and RNA were extracted, and Western blotting was used to detect key proteins of the NF-κB pathway and NLRP3 inflammasome. qPCR was used to detect the inflammatory factors. Paraffin sections of corneal tissue were prepared for HE staining and immunohistochemical staining.After MCC950 treatment, the expression levels of key proteins in the NF-κB pathway and NLRP3 inflammasome in canine cornea epithelial cells and corneal tissues were decreased, and the expression levels of IL-1β, IL-6, IL-8, IL-18 and TNF-α were reduced. Cellular MDA and LDH levels were decreased. In vivo, the degree of corneal opacity, edema, neovascularization and corneal injury area decreased after MCC950 treatment. Canine corneal sections showed that MCC950 attenuated neutrophil infiltration.MCC950 alleviates the inflammatory response to canine keratitis caused by S. pseudintermedius by inhibiting the activation of the NLRP3 inflammasome.

The bovine uterus is subject to infection after calving, which may lead to endometritis. Elevated cortisol levels have been observed in postpartum cattle. However, the role of cortisol in the inflammatory response of the uterus has not been reported. The aim of this study was to investigate the anti-inflammatory effects of cortisol on lipopolysaccharide (LPS)-induced primary bovine endometrial epithelial cells (BEECs). BEECs were treated with various concentrations of cortisol (5, 15 and 30 ng/mL) in the presence of LPS. The mRNA expression of TLR4 and proinflammatory cytokines was measured with qPCR. The activation of NF-κB and MAPK signalling pathways was detected with Western blotting and immunofluorescence. Cortisol induced the down-regulation of the mRNA expression of toll-like receptor 4 (TLR4) and proinflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor–α (TNF-α), cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS). Cortisol inhibited the activity of nuclear factor-κB (NF-κB) via blocking the phosphorylation and degradation of IκB. Cortisol suppressed the phosphorylation of mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK1/2), p38MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). These results demonstrated that cortisol may exert its anti-inflammatory actions by regulating NF-κB activation and MAPK phosphorylation.

Postpartum uterine infections are common reproductive diseases in postpartum cows. Evidence has shown that plasma β-endorphins increase during bovine uterine inflammation. However, the effect of β-endorphins on the inflammatory response in bovine endometrium has not been clarified. The aim of this study was to investigate the effect of β-endorphins on the inflammatory response of bovine endometrial epithelial and stromal cells, and to explore the possible mechanism. The cells were treated with E. coli lipopolysaccharide (LPS) to simulate inflammation, which was characterized by the significant activation of NF-κB signaling pathway and the increased gene expression of the downstream proinflammatory cytokines (approximately 1.2- to 15-fold increase, P 0.05). By using Western blot and qPCR techniques, we found that β-endorphins inhibited the key protein expression of NF-κB pathway, and the gene expressions of TNF, IL1B, IL6, CXCL8, nitric oxide synthase 2, and prostaglandin-endoperoxide synthase 2 (P 0.05). The co-treatment of β-endorphins and opioid antagonists showed that the anti-inflammatory effect of β-endorphins could be blocked (P 0.05) by non-selective opioid antagonist naloxone or δ opioid receptor antagonist ICI 154129, but not the μ opioid receptor antagonist CTAP (P 0.05). In conclusion, β-endorphins may inhibit the inflammatory response of bovine endometrial epithelial and stromal cells through δ opioid receptor.

Selenium can alleviate the inflammatory reaction infected by Staphylococcus aureus (S. aureus). However, the role of selenium on the autophagy in RAW264.7 macrophages infected by S. aureus has not been reported. The goal of this study was to clarify the effect of selenium on the autophagy and related inflammatory pathways (MAPK and NF-κB) in RAW264.7 macrophages infected by S. aureus. RAW264.7 macrophages were co-treated with Na2SeO3 and S. aureus. The expression of related inflammatory pathways (MAPK and NF-κB) and autophagy-related proteins were detected by Western blotting. The microtubule-binding protein light chain 3 (LC3) puncta were measured with immunofluorescence staining. The ultrastructure of RAW264.7 macrophages infected by S. aureus was detected by transmission electron microscope (TEM). And plate counting method was used to detect the proliferation of S. aureus in RAW264.7 macrophages. The results showed that the expression levels of LC3 II increased and the expression levels of p62 decreased after adding selenium, compared with S. aureus infection group. Compared with S. aureus infection group, the intracellular LC3 puncta and autophagic vesicles, autophagosomes, and autolysosomes increased with selenium supplementation. The number of S. aureus proliferation decreased with addition of selenium, compared with S. aureus infection group. Selenium could significantly inhibit the phosphorylation of MAPK and NF-κB signaling pathway key proteins, compared with S. aureus infection group. In summary, selenium could promote the autophagy in macrophages infected by S. aureus, alleviate the blockade of autophagic flow, depress the transcription of MAPK and NF-κB signaling pathways, and inhibit the proliferation of S. aureus in RAW264.7 macrophages.

Glucocorticoids have been widely used as anti-inflammatory therapies. The mechanisms of cortisol action in goat does with endometritis, however, have not been reported. The aim of this study was to investigate the mechanism of cortisol in modulation of effects of E. coli-induced endometritis in the does. Does (n = 24) were assigned to four groups (n = 6): control, E. coli, cortisol, and E. coli + cortisol groups. Does in the cortisol and E. coli + cortisol group were treated with cortisol from 3 days before E. coli inoculations occurred to 36 days post-partum. Does in the E. coli and inoculation groups were administered via intrauterine infusion E. coli O55 (109 CFU/mL) at 0 h. Physical indicators, macroscopic and microscopic changes in the endometrium, uterine secretion cytology and bacteriology were evaluated before (0 h) and at 6, 12, 24, 48, and 72 h after E. coli inoculation. The TLR4 and pro-inflammatory cytokine mRNA transcripts were detected using qPCR. The activations of NF-κB and MAPK signaling pathways were detected using Western blot procedures. As a result, cortisol inhibited the inflammatory response of does by reducing the clinical symptoms, morphological endometrial damage, % PMN in uterine secretions, relative abundance of inflammatory gene mRNA transcripts in the endometrium of does. Cortisol inhibited NF-κB activity by reducing MyD88 and IκB phosphorylation. Treatment with cortisol suppressed the phosphorylation of ERK1/2, p38MAPK, and JNK. These results indicate the anti-inflammatory effect of cortisol in the endometrium of does may be regulated by NF-κB and MAPK pathways.

Progesterone suppresses the innate immune function of bovine endometrium, making the uterus susceptible to bacterial infection. The bovine endometrial epithelial cells (BEEC) are the first line of defense against bacteria, such as Escherichia coli (E.coli) that causes inflammation of endometrium through the recognition of lipopolysaccharide (LPS). The aim of this study was to investigate the effect of progesterone on the inflammatory response and its potential mechanism using E.coli- or LPS-induced BEEC. Concentrations of 1, 3 and 5 ng/mL progesterone were selected. The mRNA expressions of proinflammatory cytokines were determined using qPCR. The activations of NF-κB and MAPK pathways were detected by Western blot and immunofluorescence. An increase in the mRNA expression of IL-1β, IL-6, IL-8 and TNF-α was observed in BEEC treated with progesterone, LPS or E.coli alone. Progesterone inhibited the E.coli- or LPS-induced gene expression of these cytokines. Progesterone treatment alone showed little influence on NF-κB or MAPK pathway. In BEEC stimulated with E.coli or LPS, progesterone inhibited the phosphorylations of IκB, p65, ERK1/2, p38MAPK and JNK, and the translocation of p65 into the nucleus. These results suggested that progesterone has anti-inflammatory effect, which may be mediated by inhibiting NF-κB activation and MAPK phosphorylation in BEEC.