yo pro 1 pi  (Beyotime)

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 NLRP3-mediated pyroptosis in BV2 cells following OGD/R. (A) SEM observation of BV2 cell morphology (magnification, ×20,000; scale bar, 2 μ m). (B) Yo-Pro-1 and Hoechst 33342 staining for assessing the pyroptosis levels in BV-2 cells. (C) Quantitative analysis of Yo-Pro-1 and Hoechst 33342 staining revealed that PPF deceased pyroptosis levels in BV-2 cells. (D) Immunofluorescence staining of NLRP3 in BV2 cells (magnification, ×40; scale bar, 50 μ m). (E) Immunofluorescence staining of ASC in BV2 cells (magnification, ×40; scale bar, 50 μ m). (F) Quantitative analysis of immunofluorescence staining revealed that NLRP3 and ASC fluorescence intensity increased in OGD/R-treated BV2 cells, while PPF decreased the fluorescence intensity of both proteins. (G) Caspase-1 activity was detected in BV2 cells following OGD/R using Caspase-1 Activity Assay kit. (H) Western blotting of NLRP3, ASC, cleaved-caspase-1, pro-caspase-1, GSDMD-N, GSDMD, IL-18, IL-1β, and GAPDH (internal reference). (I) Western blot analysis revealed elevated NLRP3, ASC, IL-1β and IL-18, cleaved-caspase-1/pro-caspase-1 levels (J), and GSDMD-N/GSDMD levels (K) in OGD/R-treated BV2 cells, PPF treatment reduced these proteins levels. (L) ELISA showed that PPF decreased IL-1β and IL-18 levels. ** P<0.01. PPF, Propofol; OGD/R, Oxygen-glucose deprivation/reoxygenation; ASC, apoptosis-associated speck-like protein containing a CARD; GSDMD, gasdermin D.

Article Snippet: Hoechst 33342 (cat. no. C1025) and Yo-Pro-1 staining solution (500 μ l; cat. no. C1356S; both Beyotime Institute of Biotechnology) were added at 37°C for 30 min.

Techniques: Staining, Immunofluorescence, Fluorescence, Activity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

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: Hoechst 33342 (cat. no. C1025) and Yo-Pro-1 staining solution (500 μ l; cat. no. C1356S; both Beyotime Institute of Biotechnology) were added at 37°C for 30 min.

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