Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Pathogen perspective: Lipoproteins are essential for the S. aureus -induced inflammatory response in bBMMs. The bBMMs were infected with S. aureus SA113 (WT), isogenic mutant lgt ::ermB (Δ lgt ), or its complemented strain, lgt ::ermB + pRB lgt (+pRB), at MOI 10:1 or not infected. The secretion of TNF-α, IL-1β and IL-10 were detected by ELISA (6, 9, and 12 h after infection) (A–C). The activation of the MAPK (P-ERK and P-p38) and NF-κB (P-p65) pathways was evaluated by western blotting at 15, 30, and 60 min post-infection (D). TLR2 , TLR4 and NLRP3 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at 4 h post-infection (E–G). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. * P < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

Article Snippet: The bBMMs (5 × 10 6 cells/well) were treated with the TLR2 inhibitor C29 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the TLR4 inhibitor TAK242 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the NLRP3 inhibitor MCC950 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 4 h before infection; the COX-2 inhibitor CAY10404 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was given for 40 min before infection, and the mPGES-1 inhibitor CAY10526 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was administered for 12 h before infection; the PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Infection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Host perspective: TLR2, TLR4, and NLRP3 play essential roles in the inflammatory response induced by S. aureus infection in bBMMs. bBMMs were pretreated with the TLR2 inhibitor (C29, 10 −5 M, before infection for 1 h), TLR4 inhibitor (TAK242, 10 −5 M, before infection for 1 h), and NLRP3 inhibitor (MCC950, 10 −5 M, before infection for 4 h). Then, bBMMs were infected SA113 (MOI 10:1), or uninfected. The activation of the MAPK (P-ERK and P-p38) and NF-κB (P-p65) pathways was evaluated by western blotting at 15, 30, and 60 min post-infection (A). The secretion of TNF-α, IL-1β and IL-10 were detected by ELISA (6, 9, and 12 h after infection) (B–D). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

Article Snippet: The bBMMs (5 × 10 6 cells/well) were treated with the TLR2 inhibitor C29 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the TLR4 inhibitor TAK242 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the NLRP3 inhibitor MCC950 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 4 h before infection; the COX-2 inhibitor CAY10404 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was given for 40 min before infection, and the mPGES-1 inhibitor CAY10526 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was administered for 12 h before infection; the PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Infection, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Host perspective: TLR2, TLR4, and NLRP3 play essential roles in PGE 2 production induced by S. aureus infection in bBMMs. bBMMs were pretreated with the TLR2 inhibitor (C29, 10 −5 M, before infection for 1 h), TLR4 inhibitor (TAK242, 10 −5 M, before infection for 1 h), and NLRP3 inhibitor (MCC950, 10 −5 M, before infection for 4 h). Then, bBMMs were infected SA113 (MOI 10:1), or uninfected. The experession of the COX-2 and mPGES-1 was evaluated by western blotting at 12 h and 24 h post-infection (A). COX-2 and mPGES-1 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at the 4 h and 8 h post-infection (B, C). The secretion of PGE 2 were detected by ELISA (9 h after infection) (D). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

Article Snippet: The bBMMs (5 × 10 6 cells/well) were treated with the TLR2 inhibitor C29 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the TLR4 inhibitor TAK242 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the NLRP3 inhibitor MCC950 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 4 h before infection; the COX-2 inhibitor CAY10404 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was given for 40 min before infection, and the mPGES-1 inhibitor CAY10526 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was administered for 12 h before infection; the PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Infection, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Cross-talk: PGE 2 regulates TLR2, TLR4, and NLRP3 expression and inflammatory responses in S. aureus -infected bBMMs. bBMMs were pretreated with the COX-2 inhibitor ( CAY10404 , 10 −5 M, before infection for 40 min), mPGES-1 inhibitor (CAY10526, 10 −5 M, before infection for 12 h). Then, bBMMs were infected SA113 (MOI 10:1), or uninfected. The secretion of PGE 2 were detected by ELISA (9 h after infection) (A). TLR2 , TLR4 and NLRP3 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at 4 h post-infection (B–D). The secretion of TNF-α, IL-1β and IL-10 were detected by ELISA (9 h and 12 h after infection) (E–J). Phagocytosis of Hoechst 33258 (blue)-labelled SA113 S. aureus within DiI-labelled macrophages (Orange) was analyzed by microscopy assay (×400, K). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

Article Snippet: The bBMMs (5 × 10 6 cells/well) were treated with the TLR2 inhibitor C29 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the TLR4 inhibitor TAK242 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the NLRP3 inhibitor MCC950 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 4 h before infection; the COX-2 inhibitor CAY10404 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was given for 40 min before infection, and the mPGES-1 inhibitor CAY10526 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was administered for 12 h before infection; the PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Microscopy

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Cross-talk: Excess PGE 2 exacerbates inflammation and impairs intracellular killing in S. aureus -infected bBMMs. bBMMs were pretreated with the PGE 2 (10 −6 M, before infection for 24 h). Then, bBMMs were infected SA113 (MOI 10:1), or uninfected. TLR2 , TLR4 and NLRP3 mRNA expression levels were detected by qRT-PCR and normalized to those of the housekeeping gene β-actin at 4 h post-infection (A–C). The activation of the MAPK (P-ERK and P-p38) and NF-κB (P-p65) pathways was evaluated by western blotting at 15, 30, and 60 min post-infection (D). The secretion of TNF-α, IL-1β and IL-10 were detected by ELISA (9 h and 12 h after infection) (E–G). Phagocytosis of Hoechst 33258 (blue)-labelled SA113 S. aureus within DiI-labelled macrophages (Orange) was analyzed by microscopy assay (×400, H). Results are expressed as mean ± SD of three independent experiments and were analyzed using two-way ANOVA with Bonferroni’s post-hoc test. *p < 0.05, ** p < 0.01, *** p < 0.001 and *** *p < 0.0001 indicated statistically significant differences between two experimental groups. These data are representative of three independent experiments.

Article Snippet: The bBMMs (5 × 10 6 cells/well) were treated with the TLR2 inhibitor C29 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the TLR4 inhibitor TAK242 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the NLRP3 inhibitor MCC950 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 4 h before infection; the COX-2 inhibitor CAY10404 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was given for 40 min before infection, and the mPGES-1 inhibitor CAY10526 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was administered for 12 h before infection; the PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Infection, Expressing, Quantitative RT-PCR, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Microscopy

Journal: The Veterinary Quarterly

Article Title: An underlying mechanism of bovine mastitis: PGE 2 regulates Staphylococcus aureus -induced inflammatory response through TLR2, TLR4, and NLRP3 in macrophages

doi: 10.1080/01652176.2026.2615759

Figure Lengend Snippet: Graphical abstract of the present study: the involvement of TLR2-, TLR4-, and NLRP3-dependent PGE 2 signaling in macrophage responses to S. aureus , which modulates inflammatory signaling and phagocytic activity and thereby contributes to the pathogenesis of bovine mastitis.

Article Snippet: The bBMMs (5 × 10 6 cells/well) were treated with the TLR2 inhibitor C29 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the TLR4 inhibitor TAK242 (10 −5 M, MCE, Monmouth Junction, NJ, USA) for 1 h before infection; the NLRP3 inhibitor MCC950 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 4 h before infection; the COX-2 inhibitor CAY10404 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was given for 40 min before infection, and the mPGES-1 inhibitor CAY10526 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI) was administered for 12 h before infection; the PGE 2 (10 −5 M, Cayman Chemical Company, Ann Arbor, MI, USA) for 24 h before infection.

Techniques: Activity Assay

Journal: International Journal of Molecular Medicine

Article Title: Targeting of CIRP attenuates osteoarthritis progression via suppressing TLR4/NF-κB/NLRP3 signaling axis

doi: 10.3892/ijmm.2025.5674

Figure Lengend Snippet: Dependence of CIRP-induced chondrocyte damage on TLR4/NF-κB signaling. (A) Cytotoxicity of BAY 11-7082 and TAK-242 on chondrocytes at various concentration for 48 h, assessed using a CCK-8 assay. (B and C) Western blotting showing the protein expression of IκBα in chondrocytes and p65 in the nucleus of chondrocytes following treatment with the two inhibitors. (D and E) Western blotting showing the protein expression NLRP3, cleaved-caspase-1, ASC and IL-1β following treatment with the two inhibitors. (F and G) Western blotting showing the expression of extracellular matrix proteins in chondrocytes following treatment with the two inhibitors. n=3, * P<0.05, ** P<0.01, *** P<0.001. CIRP, cold-inducible RNA-binding protein; TLR4, Toll-like receptor 4; NLRP3, NLR family pyrin domain containing 3; Cle, cleaved.

Article Snippet: TLR4 inhibitor TAK-242 was purchased from Shanghai Xianding Biotechnology Co., Ltd. Primary antibodies directed against inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), MMP1, MMP3, MMP13, Lamin B1, CD63, CD9, 130 kDa cis-Golgi matrix protein 1 (GM130) and GAPDH were purchased from Proteintech Group, Inc., ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) was purchased from ABclonal.

Techniques: Concentration Assay, CCK-8 Assay, Western Blot, Expressing, RNA Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Targeting of CIRP attenuates osteoarthritis progression via suppressing TLR4/NF-κB/NLRP3 signaling axis

doi: 10.3892/ijmm.2025.5674

Figure Lengend Snippet: Schematic illustration of targeting of CIRP attenuates osteoarthritis progression and the underlying mechanism. CIRP can be secreted in the form of exosomes and acts as a pro-inflammatory factor that activates the TLR4/NF-κB/NLRP3 signaling pathway, promoting the inflammatory response, ECM degradation and the progression of OA. Additionally, CIRP is identified as a target of miR-145, which inhibits its expression in OA. CIRP, cold-inducible RNA-binding protein; TLR4, Toll-like receptor 4; NLRP3, NLR family pyrin domain containing 3; OA, osteoarthritis; ECM, extracellular matrix; miR, microRNA; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; MMPs, matrix metalloproteinases.

Article Snippet: TLR4 inhibitor TAK-242 was purchased from Shanghai Xianding Biotechnology Co., Ltd. Primary antibodies directed against inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), MMP1, MMP3, MMP13, Lamin B1, CD63, CD9, 130 kDa cis-Golgi matrix protein 1 (GM130) and GAPDH were purchased from Proteintech Group, Inc., ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) was purchased from ABclonal.

Techniques: Expressing, RNA Binding Assay