Journal: International Journal of Molecular Medicine

Article Title: Propofol upregulates MFG-E8 in BV2 cells to inhibit pyroptosis mediated by the NF-κB/NLRP3 pathway, thereby ameliorating ischemic-reperfusion neuronal injury

doi: 10.3892/ijmm.2026.5786

Figure Lengend Snippet: Overexpression of MFG-E8 suppresses NF-κB and NLRP3 activation in OGD/R-induced BV2 cells. (A) Western blotting protein bands for MFG-E8, p-NF-κB, NF-κB, and GAPDH (internal reference). (B and C) Following OGD/R, western blotting revealed decreased MFG-E8 expression (B) and elevated p-NF-κB/NF-κB levels (C) in BV2 cells. (D) Western blotting protein bands for MFG-E8 and GAPDH (internal reference). (E) Following transfection of OE-MFG-E8 into BV2 cells, MFG-E8 expression was elevated markedly. (F) Western blotting protein bands for NLRP3, ASC, p-NF-κB, NF-κB, and GAPDH (internal reference). (G and H) Following transfection with OE-MFG-E8, p-NF-κB/NF-κB levels (G), and NLRP3 and ASC levels (H) were detected using western blotting. (I) Western blotting protein bands for NLRP3, ASC, p-NF-κB, NF-κB, and GAPDH (internal reference). Following treatment with the NF-κB activator LPS, p-NF-κB/NF-κB (J), and NLRP3 and ASC levels (K) were detected by western blotting. (L) Western blotting protein bands for p-NF-κB, NF-κB, and GAPDH (internal reference). (M) Following transfection with OE-MFG-E8, p-NF-κB/NF-κB expression was decreased, whereas treatment with LPS increased p-NF-κB/NF-κB levels. (N) Immunofluorescence staining of NLRP3 in BV2 cells (magnification, ×40; scale bar, 50 μ m). (O) Immunofluorescence staining of ASC in BV2 cells (magnification, ×40; scale bar, 50 μ m). (P) Immunofluorescence revealed OE-MFG-E8 reduced the fluorescence intensity of NLRP3 and ASC, while LPS diminished the effect of MFG-E8 overexpression. (Q) Western blotting protein bands for NLRP3, ASC, cleaved-caspase-1, pro-caspase-1, GSDMD-N, GSDMD, IL-1β, IL-18, and GAPDH (internal reference). (R-T) Western blotting revealed that OE-MFG-E8 reduced cleaved-caspase-1/pro-caspase-1 levels (R), GSDMD-N/GSDMD levels (S), NLRP3, ASC, IL-1β, and IL-18 levels (T) in OGD/R-induced BV2 cells, LPS attenuated the impact of OE-MFG-E8. ** P<0.01. PPF, Propofol; OGD/R, Oxygen-glucose deprivation/reoxygenation; ASC, apoptosis-associated speck-like protein containing a CARD; MFG-E8, milk fat globule-EGF factor 8; GSDM, gasdermin; OE, overexpression; p-, phosphorylated; LPS, lipopolysaccharide; NC, negative control; ns, not significant.

Article Snippet: For the OGD/R + OE-MFG-E8 + LPS (NF-κB agonist) group, cells were transfected with OE-MFG-E8, treated with OGD and exposed to LPS (1 μ g/ml; cat. no. HY-D1056, MedChemExpress) during reoxygenation ( ).

Techniques: Over Expression, Activation Assay, Western Blot, Expressing, Transfection, Immunofluorescence, Staining, Fluorescence, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Propofol upregulates MFG-E8 in BV2 cells to inhibit pyroptosis mediated by the NF-κB/NLRP3 pathway, thereby ameliorating ischemic-reperfusion neuronal injury

doi: 10.3892/ijmm.2026.5786

Figure Lengend Snippet: PPF suppresses pyroptosis caused by NF-κB/NLRP3 signaling by upregulating MFG-E8. (A) Western blotting protein bands for MFG-E8, p-NF-κB, NF-κB, and GAPDH (internal reference) in BV2 cells. (B) Western blotting revealed that PPF increased MFG-E8 and decreased p-NF-κB/NF-κB levels (C) in BV2 cells. (D) Western blotting for MFG-E8 and GAPDH in BV2 cells. (E) Following transfection of si-MFG-E8 into BV2 cells, MFG-E8 protein levels were significantly decreased. (F) Dual luciferase reporter gene assay confirmed that NF-κB is the target of MFG-E8. (G) Western blotting protein bands for p-NF-κB, NF-κB, and GAPDH (internal reference) in BV2 cells. (H) si-MFG-E8 attenuated the effects of PPF, resulting in elevated p-NF-κB/NF-κB levels. (I) Yo-Pro-1 and Hoechst 33342 staining for assessing the pyroptosis levels in BV-2 cells (magnification, ×40; scale bar, 50 μ m). (J) Yo-Pro-1 and Hoechst 33342 staining showed that PPF decreased Yo-Pro-1 positivity, while silencing MFG-E8 increased Yo-Pro-1 positivity. ELISA showed that PPF decreased TNF-α (K) and IL-1β (L) levels, silencing MFG-E8 reversed this effect. (M and N) ELISA showed that PPF raised IL-10 levels (M) and decreased IL-6 levels (N), silencing MFG-E8 reversed this effect. (O) Western blotting protein bands for NLRP3, ASC, cleaved-caspase-1, pro-caspase-1, GSDMD-N, GSDMD, IL-1β, IL-18, and GAPDH (internal reference) in BV2 cells. (P-R) Western blot analysis indicated that PPF decreased NLRP3, ASC, IL-1β, and IL-18 levels (P), GSDMD-N/GSDMD levels (Q), cleaved-caspase-1/pro-caspase-1 levels (R), silencing MFG-E8 increased these protein levels. ** P<0.01. PPF, propofol; MFG-E8, milk fat globule-EGF factor 8; OGD/R, Oxygen-glucose deprivation/reoxygenation; ASC, apoptosis-associated speck-like protein containing a CARD; GSDM, gasdermin; OE, overexpression; p-, phosphorylated; LPS, lipopolysaccharide; NC, negative control; ns, not significant; si, small interfering; WT, wild-type; MUT, mutant.

Article Snippet: For the OGD/R + OE-MFG-E8 + LPS (NF-κB agonist) group, cells were transfected with OE-MFG-E8, treated with OGD and exposed to LPS (1 μ g/ml; cat. no. HY-D1056, MedChemExpress) during reoxygenation ( ).

Techniques: Western Blot, Transfection, Luciferase, Reporter Gene Assay, Staining, Enzyme-linked Immunosorbent Assay, Over Expression, Negative Control, Mutagenesis

Journal: Frontiers in Cellular Neuroscience

Article Title: Structure matters: commensal Phocaeicola vulgatus lipopolysaccharide induces attenuated microglial activation and preserves neuronal integrity

doi: 10.3389/fncel.2026.1796397

Figure Lengend Snippet: Differential effects of E. coli and P. vulgatus LPS on BV2 microglial cells. (A) BV2 cell viability after stimulation with increasing concentrations of E. coli or P. vulgatus LPS (0.1–100 ng/mL) using the MTT assay. Unstimulated cells (NS) served as negative control. (B) Immunofluorescence analysis of microglial marker Iba-1 (red) to evaluate cell morphology and number following exposure to 100 ng/mL of LPS (scale bar = 100 μm). (C) The relative number of Iba 1 + cells from the experiments in (B) , normalized to NS (set at 100%). (D) Iba-1 immunofluorescence intensity was calculated as the mean area of the positive signal per cell. (E) Nitrite quantification and (F) western blot analysis of iNOS expression. Data are presented as mean ± SEM. Statistical significance was assessed by ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. # p < 0.05; #### p < 0.0001 vs. NS; * p < 0.05; *** p < 0.001, **** p < 0.0001 vs. LPS.

Article Snippet: Cells were seeded in 96-well plates at 1 × 10 4 cells/well and treated for 24 h with E. coli LPS (LPS-EB ultrapure; InvivoGen) or P. vulgatus LPS at concentrations of 0.1, 1, 10, or 100 ng/mL.

Techniques: MTT Assay, Negative Control, Immunofluorescence, Marker, Western Blot, Expressing, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Structure matters: commensal Phocaeicola vulgatus lipopolysaccharide induces attenuated microglial activation and preserves neuronal integrity

doi: 10.3389/fncel.2026.1796397

Figure Lengend Snippet: Differential modulation of cytokine release and intracellular signaling in BV2 microglia. BV2 cells were treated with increasing concentrations (0.1, 1, 10, and 100 ng/mL) of LPS derived from E. coli or P. vulgatus . Unstimulated cells (NS) served as negative controls. (A) Cytokine levels (IL-1β, IL-6, and TNF-α) were quantified in the culture supernatants by using ELLA assay. (B) Activation of intracellular signaling pathways (STAT3, Nf-kb, ERK1/2) was assessed by western blotting. Data are presented as mean ± SEM. Statistical significance was assessed by ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. # p < 0.05; ## p < 0.01; ### p < 0.001, #### p < 0.0001 vs. NS; * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001 vs. LPS.

Article Snippet: Cells were seeded in 96-well plates at 1 × 10 4 cells/well and treated for 24 h with E. coli LPS (LPS-EB ultrapure; InvivoGen) or P. vulgatus LPS at concentrations of 0.1, 1, 10, or 100 ng/mL.

Techniques: Derivative Assay, Activation Assay, Protein-Protein interactions, Western Blot, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Structure matters: commensal Phocaeicola vulgatus lipopolysaccharide induces attenuated microglial activation and preserves neuronal integrity

doi: 10.3389/fncel.2026.1796397

Figure Lengend Snippet: Differential responses of human HMC3 microglia to LPS from E. coli and P. vulgatus . (A) HMC3 cell viability was assessed after stimulation with increasing concentrations of LPS (0.1, 1, 10, and 100 ng/mL) from E. coli or P. vulgatus , using the MTT assay. Unstimulated cells (NS) were used as negative control. (B) Representative immunofluorescence Iba-1 + cells (green) and corresponding brightfield phase-contrast microscopy images of HMC3 cells (scale bar = 100 μm). (C) Quantification of Iba-1 + cell number from the experiments in B, expressed as percentage relative to NS condition (set at 100%). (D) Iba-1 immunofluorescence intensity calculated as the mean area of the positive signal per cell. (E) Western blot analysis of TLR4-associated signaling pathways. (F) Cytokine levels (IL-8, IL-6, TNF-α) measured in culture supernatants by ELLA assay. Data are presented as mean ± SEM. Statistical significance was assessed by ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. # p < 0.05; ## p < 0.01; ### p < 0.001, #### p < 0.0001 vs. NS; ** p < 0.01; *** p < 0.001, **** p < 0.0001 vs. LPS.

Article Snippet: Cells were seeded in 96-well plates at 1 × 10 4 cells/well and treated for 24 h with E. coli LPS (LPS-EB ultrapure; InvivoGen) or P. vulgatus LPS at concentrations of 0.1, 1, 10, or 100 ng/mL.

Techniques: MTT Assay, Negative Control, Immunofluorescence, Microscopy, Western Blot, Protein-Protein interactions, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Structure matters: commensal Phocaeicola vulgatus lipopolysaccharide induces attenuated microglial activation and preserves neuronal integrity

doi: 10.3389/fncel.2026.1796397

Figure Lengend Snippet: Effects of LPS-treated HMC3 conditioned media on neuronal integrity (A) Bright-field images of differentiated PC12 cells (10 × and 40 × magnification) and (B) MAP2 immunofluorescence staining. (C) Immunofluorescence analysis was assessed after exposure to conditioned media (CM) from HMC3 cells treated with 1 μg/mL or 100 ng/mL. LPS from E. coli or P. vulgatus . Unstimulated PC12 cells (NS) from the experiments in (B) served as control and (D) the accompanying quantification confirms the differences observed. (E) Western blot analysis of MAP2 protein levels in PC12 cells exposed to CM from HMC3 cells treated with 1 μg/mL LPS. Data are presented as mean ± SEM. Statistical significance was assessed by ordinary one-way ANOVA followed by Tukey’s multiple-comparison test. # p < 0.05; ### p < 0.001, #### p < 0.0001 vs. NS; ** p < 0.01; vs. LPS.

Article Snippet: Cells were seeded in 96-well plates at 1 × 10 4 cells/well and treated for 24 h with E. coli LPS (LPS-EB ultrapure; InvivoGen) or P. vulgatus LPS at concentrations of 0.1, 1, 10, or 100 ng/mL.

Techniques: Immunofluorescence, Staining, Control, Western Blot, Comparison