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: A total of 30 μ g protein/lane was loaded and separated by 10% SDS-PAGE, transferred onto PVDF membranes and blocked with 5% BSA at room temperature for 2 h. Following rinsing with TBST containing 0.1% Tween-20, the membrane underwent overnight incubation with primary antibodies against synapsin-1 (SYP-1, cat. no. 51-5200), inducible nitric oxide synthase (iNOS; cat. no. PA5-17106l; both 1:1,000), arginase 1 (Arg 1, cat. no. PA5-29645, 1:5,000), postsynaptic density protein-95 (PSD-95; cat. no. 51-6900), brain-derived neurotrophic factor (BDNF; cat. no. PA5-85730; both 1:500), growth-associated protein 43 (GAP-43; cat. no. PA5-34943, 1:5,000), GSDMD (cat. no. PA5-116815; all Invitrogen; Thermo Fisher Scientific, Inc.), cleaved-caspase-1 (cat. no. HY- P80622 ; both 1:500, MedChemExpress), pro-caspase-1 (cat. no. PA5-87536, 1:2,000, Invitrogen; Thermo Fisher Scientific, Inc.), ASC (cat. no. ab283684), GSDMD-N (cat. no. ab215203; both 1:1,000; both Abcam), NLRP3 (cat. no. MA5-32255, 1:500), IL-1β (cat. no. P420B, 1:1,000), IL-18 (cat. no. PA5-79479, 1:2,000), phosphorylated (p-)NF-κB-p65 (cat. no. 44-711G, 1:1,000), MFG-E8 (cat. no. PA5-109955; all Invitrogen; Thermo Fisher Scientific, Inc.) and NF-κB-p65 (cat. no. ab76311; both 1:2,000, Abcam) at 4°C.

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: 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: A total of 30 μ g protein/lane was loaded and separated by 10% SDS-PAGE, transferred onto PVDF membranes and blocked with 5% BSA at room temperature for 2 h. Following rinsing with TBST containing 0.1% Tween-20, the membrane underwent overnight incubation with primary antibodies against synapsin-1 (SYP-1, cat. no. 51-5200), inducible nitric oxide synthase (iNOS; cat. no. PA5-17106l; both 1:1,000), arginase 1 (Arg 1, cat. no. PA5-29645, 1:5,000), postsynaptic density protein-95 (PSD-95; cat. no. 51-6900), brain-derived neurotrophic factor (BDNF; cat. no. PA5-85730; both 1:500), growth-associated protein 43 (GAP-43; cat. no. PA5-34943, 1:5,000), GSDMD (cat. no. PA5-116815; all Invitrogen; Thermo Fisher Scientific, Inc.), cleaved-caspase-1 (cat. no. HY- P80622 ; both 1:500, MedChemExpress), pro-caspase-1 (cat. no. PA5-87536, 1:2,000, Invitrogen; Thermo Fisher Scientific, Inc.), ASC (cat. no. ab283684), GSDMD-N (cat. no. ab215203; both 1:1,000; both Abcam), NLRP3 (cat. no. MA5-32255, 1:500), IL-1β (cat. no. P420B, 1:1,000), IL-18 (cat. no. PA5-79479, 1:2,000), phosphorylated (p-)NF-κB-p65 (cat. no. 44-711G, 1:1,000), MFG-E8 (cat. no. PA5-109955; all Invitrogen; Thermo Fisher Scientific, Inc.) and NF-κB-p65 (cat. no. ab76311; both 1:2,000, Abcam) at 4°C.

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: A total of 30 μ g protein/lane was loaded and separated by 10% SDS-PAGE, transferred onto PVDF membranes and blocked with 5% BSA at room temperature for 2 h. Following rinsing with TBST containing 0.1% Tween-20, the membrane underwent overnight incubation with primary antibodies against synapsin-1 (SYP-1, cat. no. 51-5200), inducible nitric oxide synthase (iNOS; cat. no. PA5-17106l; both 1:1,000), arginase 1 (Arg 1, cat. no. PA5-29645, 1:5,000), postsynaptic density protein-95 (PSD-95; cat. no. 51-6900), brain-derived neurotrophic factor (BDNF; cat. no. PA5-85730; both 1:500), growth-associated protein 43 (GAP-43; cat. no. PA5-34943, 1:5,000), GSDMD (cat. no. PA5-116815; all Invitrogen; Thermo Fisher Scientific, Inc.), cleaved-caspase-1 (cat. no. HY- P80622 ; both 1:500, MedChemExpress), pro-caspase-1 (cat. no. PA5-87536, 1:2,000, Invitrogen; Thermo Fisher Scientific, Inc.), ASC (cat. no. ab283684), GSDMD-N (cat. no. ab215203; both 1:1,000; both Abcam), NLRP3 (cat. no. MA5-32255, 1:500), IL-1β (cat. no. P420B, 1:1,000), IL-18 (cat. no. PA5-79479, 1:2,000), phosphorylated (p-)NF-κB-p65 (cat. no. 44-711G, 1:1,000), MFG-E8 (cat. no. PA5-109955; all Invitrogen; Thermo Fisher Scientific, Inc.) and NF-κB-p65 (cat. no. ab76311; both 1:2,000, Abcam) at 4°C.

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

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 upregulates MFG-E8 to inhibit activation of NF-κB/NLRP3 pathway. (A) Western blotting of MFG-E8, p-NF-κB, NF-κB, NLRP3, ASC, cleaved-caspase-1, pro-caspase-1, GSDMD-N, GSDMD, IL-18, IL-1β, and GAPDH (internal reference). (B-F) Western blot analysis revealed decreased MFG-E8 (B) levels in tMCAO mice, along with elevated p-NF-κB/NF-κB (C), NLRP3, ASC, IL-1β, IL-18 (D), cleaved-caspase-1/pro-caspase-1 (E), GSDMD-N/GSDMD (F) levels. PPF reduced these protein levels. (G) Western blotting protein bands for MFG-E8 and GAPDH (internal reference). (H) Western blotting detected the expression of MFG-E8 in the brain tissue of mice after injection of si-MFG-E8/si-NC. (I) Western blotting protein bands for MFG-E8, p-NF-κB, NF-κB, NLRP3, ASC, cleaved-caspase-1, pro-caspase-1, GSDMD-N, GSDMD, IL-18, IL-1β, and GAPDH (internal reference). (J-N) Western blot analysis revealed that si-MFG-E8 diminished the therapeutic efficacy of PPF, leading to deceased MFG-E8 level (J) and elevated p-NF-κB/NF-κB (K), NLRP3, ASC, IL-1β, and IL-18 levels (L), and cleaved-caspase-1/pro-caspase-1 (M) and GSDMD-N/GSDMD (N) levels in tMCAO mice. * P<0.05, ** P<0.01. PPF, Propofol; tMCAO, transient middle cerebral artery occlusion; MFG-E8, milk fat globule-EGF factor 8; p-, phosphorylated; ASC, apoptosis-associated speck-like protein containing a CARD; ns, not significant; GSDMD, gasdermin D; si, small interfering; NC, negative control.

Article Snippet: A total of 30 μ g protein/lane was loaded and separated by 10% SDS-PAGE, transferred onto PVDF membranes and blocked with 5% BSA at room temperature for 2 h. Following rinsing with TBST containing 0.1% Tween-20, the membrane underwent overnight incubation with primary antibodies against synapsin-1 (SYP-1, cat. no. 51-5200), inducible nitric oxide synthase (iNOS; cat. no. PA5-17106l; both 1:1,000), arginase 1 (Arg 1, cat. no. PA5-29645, 1:5,000), postsynaptic density protein-95 (PSD-95; cat. no. 51-6900), brain-derived neurotrophic factor (BDNF; cat. no. PA5-85730; both 1:500), growth-associated protein 43 (GAP-43; cat. no. PA5-34943, 1:5,000), GSDMD (cat. no. PA5-116815; all Invitrogen; Thermo Fisher Scientific, Inc.), cleaved-caspase-1 (cat. no. HY- P80622 ; both 1:500, MedChemExpress), pro-caspase-1 (cat. no. PA5-87536, 1:2,000, Invitrogen; Thermo Fisher Scientific, Inc.), ASC (cat. no. ab283684), GSDMD-N (cat. no. ab215203; both 1:1,000; both Abcam), NLRP3 (cat. no. MA5-32255, 1:500), IL-1β (cat. no. P420B, 1:1,000), IL-18 (cat. no. PA5-79479, 1:2,000), phosphorylated (p-)NF-κB-p65 (cat. no. 44-711G, 1:1,000), MFG-E8 (cat. no. PA5-109955; all Invitrogen; Thermo Fisher Scientific, Inc.) and NF-κB-p65 (cat. no. ab76311; both 1:2,000, Abcam) at 4°C.

Techniques: Activation Assay, Western Blot, Expressing, Injection, Drug discovery, Negative Control

Journal: International Journal of Oncology

Article Title: Hypoxia-induced exosomal CAMTA1 promotes radio-resistance in MDA-MB-231 cells by regulating NRG1 to mediate M2 macrophage polarization

doi: 10.3892/ijo.2026.5875

Figure Lengend Snippet: Exosomal CAMTA1 promotes tumor growth in vivo . (A) The transfection efficacy of Ov-CAMTA1 was detected using RT-qPCR and immunoblotting analysis. (B) The appearance of tumor. The tumor (C) volume and (D) weight. (E) The mRNA expression of CAMTA1 was detected using RT-qPCR. (F) The level of IL-10 was detected using ELISA-related IL-10 assay kits. (G) The level of CD163 was detected using immunohistochemistry analysis. (H) H&E staining. (I) The expression of Caspase 3 was detected using immunohistochemistry analysis. (J) The Spearman correlation analysis of CAMTA1 and NRG1. (K) The expression of NRG1 was detected using immunohistochemistry analysis. * P<0.05, ** P<0.01 and *** P<0.001. CAMTA1, Calmodulin-binding Transcription Activator 1; Ov, overexpression; RT-qPCR, reverse transcription-quantitative PCR; H&E, hematoxylin and eosin; NRG1, neuregulin 1.

Article Snippet: The slices were then incubated with primary antibody against NRG1 (cat. no. 83323-6-RR; 1:500; Proteintech Group, Inc.), CD163 (cat. no. 83285-4-RR; 1:2,000; Proteintech Group, Inc.) and Caspase-3 (cat. no. 19677-1-AP; 1:500; Proteintech Group, Inc.) overnight at 4°C.

Techniques: In Vivo, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Binding Assay, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction