lipopolysaccharide lps  (InvivoGen)

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