Journal: Journal of Animal Science and Biotechnology

Article Title: Extracellular vesicles containing microbial DNA contribute to ruminal dysbiosis-induced mastitis by activating cGAS-STING-NF-κB/NLRP3 pathway

doi: 10.1186/s40104-025-01316-4

Figure Lengend Snippet: HCD goats and HC‑RMT mice induces mastitis through activating cGAS-STING-NF-κB/NLRP3 axis. A and B Representative western blot images of cGAS-STING signaling in the mammary glands from the goats and relative intensity analysis. C and D Representative western blot images of NF-κB signaling in the mammary glands from the goats and relative intensity analysis. E–G Representative western blot images of NLRP3 signaling in the mammary glands from the goats and relative intensity analysis. H and I Representative western blot images of cGAS-STING signaling and relative intensity analysis in the mammary glands of RMT mice. J and K Representative western blot images of NF-κB signaling and relative intensity analysis in the mammary glands of RMT mice. L and M Representative western blot images of NLRP3 signaling and relative intensity analysis in the mammary glands of RMT mice. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate significance ( n = 6)

Article Snippet: After a 10-min block with 1 × protein-free rapid blocking solution at room temperature, specific antibodies including cGAS, STING, TBK1, p-TBK, IRF3, p-IRF3, p-p65, p65, p-IκB, IκB, ZO-1, Occludin, Claudin-3 and β-actin (Affinity Biosciences, Jiangsu, China) were used at the appropriate final concentration following the manufacturer's instructions.

Techniques: Western Blot

Journal: Journal of Animal Science and Biotechnology

Article Title: Extracellular vesicles containing microbial DNA contribute to ruminal dysbiosis-induced mastitis by activating cGAS-STING-NF-κB/NLRP3 pathway

doi: 10.1186/s40104-025-01316-4

Figure Lengend Snippet: The leakage of mEVs can cause mastitis and enhances the activation of the cGAS-STING-NF-κB/NLRP3 pathways in the mammary gland. Mice were tail vein injected with mEVs. After 4 weeks of adoptive transfer of mEVs, relevant indicators were measured in recipient mice. A Representative H&E-stained images of mammary tissues from mice in mEVs treatment groups (scale bar, 50 μm). B The mammary gland histological scores derived from H&E-stained sections. C The pro-inflammatory cytokine of mice mammary from various groups, such as the levels of IL-1β, TNF-α and IL-6. D MPO activity in mammary gland. E Tight junction proteins including Claudin-3 and ZO-1 in mammary tissue are detected by immunohistochemistry. F and G Western blotting and intensity analysis were used to measure the tight junction levels of mice mammary Claudin-3, Occludin, and ZO-1. H– J Representative western blot images of cGAS-STING signaling in the mammary glands and relative intensity analysis in the control, CEV and SEV groups. K and L Representative western blot images of NF-κB signaling in the mammary glands and relative intensity analysis in the control, CEV and SEV groups. M and N Representative western blot images of NLRP3 signaling in the mammary glands and relative intensity analysis. β-actin was used as a control. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate significance ( n = 6)

Article Snippet: After a 10-min block with 1 × protein-free rapid blocking solution at room temperature, specific antibodies including cGAS, STING, TBK1, p-TBK, IRF3, p-IRF3, p-p65, p65, p-IκB, IκB, ZO-1, Occludin, Claudin-3 and β-actin (Affinity Biosciences, Jiangsu, China) were used at the appropriate final concentration following the manufacturer's instructions.

Techniques: Activation Assay, Injection, Adoptive Transfer Assay, Staining, Derivative Assay, Activity Assay, Immunohistochemistry, Western Blot, Control

Journal: Journal of Animal Science and Biotechnology

Article Title: Extracellular vesicles containing microbial DNA contribute to ruminal dysbiosis-induced mastitis by activating cGAS-STING-NF-κB/NLRP3 pathway

doi: 10.1186/s40104-025-01316-4

Figure Lengend Snippet: mEVs can cause cellular inflammation and microbial DNA are the key pathogenic cargoes within mEVs that induce mastitis. To make DNA-free SEV, we depleted microbial DNA carriers SEV by electroporating SEV and then treating these SEV with DNase I. RAW 264.7 cells were preincubated in 6-well plates for 24 h and then treated with SEV and DNA-free SEV for the next 24 h to collect the supernatants. A– C The relative levels of pro-inflammatory IL-1β, IL-6, and TNF-α in control and SEV groups. D– F The relative levels of pro-inflammatory IL-1β, IL-6, and TNF-α in control, SEV and DNA-free SEV groups. For animal experiments, mice were tail vein injected with mEVs (including the SEV and DNA-free SEV). After 4 weeks of adoptive transfer of mEVs, relevant indicators were measured in recipient mice. G and H Representative H&E-stained images of mammary tissues from mice (scale bar, 50 μm) and mammary gland histological scores derived from H&E-stained sections. I and J The levels of IL-1β, IL-6, TNF-α, and MPO activity in mice mammary tissue. K – O Representative western blot images of cGAS-STING-NF-κB signaling in the mammary glands and relative intensity analysis. β-actin was used as a control. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate significance ( n = 6)

Article Snippet: After a 10-min block with 1 × protein-free rapid blocking solution at room temperature, specific antibodies including cGAS, STING, TBK1, p-TBK, IRF3, p-IRF3, p-p65, p65, p-IκB, IκB, ZO-1, Occludin, Claudin-3 and β-actin (Affinity Biosciences, Jiangsu, China) were used at the appropriate final concentration following the manufacturer's instructions.

Techniques: Control, Injection, Adoptive Transfer Assay, Staining, Derivative Assay, Activity Assay, Western Blot

Journal: Journal of Animal Science and Biotechnology

Article Title: Extracellular vesicles containing microbial DNA contribute to ruminal dysbiosis-induced mastitis by activating cGAS-STING-NF-κB/NLRP3 pathway

doi: 10.1186/s40104-025-01316-4

Figure Lengend Snippet: Microbial DNA within mEVs trigger the activation of cGAS-STING pathway that enhances mammary gland inflammatory responses. For cGAS inhibition, the cells were pretreated with RU.521 for 2 h, followed by SEV treatment. Twenty-four hours after SEV treatment, the supernatants and cells were collected. The protein levels of the cGAS-STING-NF-κB/NLRP3 pathways from the indicated groups were determined by western blotting. The levels of pro-inflammatory cytokine from the indicated group were detected by ELISA. A– C The relative levels of pro-inflammatory IL-1β, IL-6, and TNF-α. D – O Representative western blot images of cGAS-STING-NF-κB/NLRP3 signaling in the mammary glands and relative intensity analysis. β-actin was used as a control. For STING inhibition, we used STING −/− and WT mice. Mice were injected with SEV in the tail vein, and 4 weeks after SEV was transferred by adoption, the correlation index was measured in recipient mice. P Representative H&E-stained images of mammary tissues from mice (scale bar, 50 μm) and mammary gland histological scores derived from H&E-stained sections. Q– S The pro-inflammatory cytokine of mice mammary from various groups, such as the levels of IL-1β, TNF-α and IL-6. T MPO activity. Data are expressed as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001 indicate significance ( n = 6)

Article Snippet: After a 10-min block with 1 × protein-free rapid blocking solution at room temperature, specific antibodies including cGAS, STING, TBK1, p-TBK, IRF3, p-IRF3, p-p65, p65, p-IκB, IκB, ZO-1, Occludin, Claudin-3 and β-actin (Affinity Biosciences, Jiangsu, China) were used at the appropriate final concentration following the manufacturer's instructions.

Techniques: Activation Assay, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Injection, Staining, Derivative Assay, Activity Assay