Journal: Frontiers in Immunology

Article Title: Laminaria japonica polysaccharide mitigates acute neuroinflammation in cerebral ischemia-reperfusion injury through Csf3-modulated pathways

doi: 10.3389/fimmu.2026.1801746

Figure Lengend Snippet: LJP attenuates OGD/R-induced inflammation in BV2 microglia via Csf3. (A) CCK-8 assay of cell viability. (B, C) . Intracellular ROS levels were measured by DCFH-DA fluorescence. (D) qPCR shows the expression of Csf3, IL-1b, Tnfα and IL-6 in OGD/R-stimulated BV2 cells. (E) Representative immunofluorescence images of Csf3 (red) and DAPI (blue) in BV2 cells. (F) ELISA present the levels of IL-1b, Tnfα and IL-6 were detected in the supernatant of OGD/R-stimulated BV2 cells (n = 4).

Article Snippet: Cell Culture and OGD/R Model: BV2 microglia (Procell Life Science) were cultured in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin under standard conditions (37 °C, 5% CO2).

Techniques: CCK-8 Assay, Fluorescence, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Forsythoside A Alleviates Neuroinflammatory Damage via Inhibiting TLR4 / NF ‐ κB / NLRP3 Activation‐Induced Astrocyte Pyroptosis in Cerebral Ischemia–Reperfusion

doi: 10.1002/cns.70867

Figure Lengend Snippet: FA inhibits activation of NLRP3 inflammatory vesicles in OGD/R cells. (A) CCK‐8 assay detected the viability of U251 cells after 24 h of FA (5, 10, 20, 40, 80, 100, 120 μM) treatment. (B) CCK8 assay showed that FA (20–100 μM) treatment attenuated U251 cell viability impairment caused by OGD/R. (C–D) Immunofluorescence co‐localization measured that OGD/R resulted in increased fluorescence intensity of NLRP3 in U251 cells, and FA treatment decreased NLRP3 level (20×, 100 μm). (E–F) Western blot measured that OGD/R resulted in elevated NLRP3, Cl‐Caspase‐1/Caspase‐1, and ASC levels in U251 cells, whereas FA treatment decreased these proteins. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Control; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. OGD/R.

Article Snippet: For the FA + NLRP3 agonist nigericin (Nig) group, U251 cells were first exposed to FA (80 μM) for 3 h, and the OGD/R cell model was constructed in the culture medium supplemented with Nig (10 μM, HY‐127019, MedChemExpress) [ ].

Techniques: Activation Assay, CCK-8 Assay, Immunofluorescence, Fluorescence, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Forsythoside A Alleviates Neuroinflammatory Damage via Inhibiting TLR4 / NF ‐ κB / NLRP3 Activation‐Induced Astrocyte Pyroptosis in Cerebral Ischemia–Reperfusion

doi: 10.1002/cns.70867

Figure Lengend Snippet: FA suppresses OGD/R astrocyte pyroptosis and inflammation through hindering NLRP3 inflammasome activation. (A–B) Hoechst 33,342 and PI staining showed that 80 μM FA reduced pyroptosis in U251 cells after OGD/R, whereas NLRP3 agonist Nig resulted in increased pyroptosis (20×, 100 μm). (C) LDH assay showed that LDH release from U251 cells was increased following OGD/R FA inhibited LDH release, while Nig led to a rebound in LDH release. (D–E) Immunofluorescence co‐localization measured that OGD/R resulted in increased GSDMD‐N fluorescence intensity in U251 cells, FA treatment decreased GSDMD‐N level, and Nig attenuated the effect of FA (20×, 100 μm). (F–I) ELISA indicated that OGD/R resulted in increased IL‐1β, TNF‐α, IL‐18, and IL‐6 levels; FA treatment declined these inflammatory factors, but addition of Nig resulted in increased levels. (J–M) Western blot measured that OGD/R caused elevated iNOS, IL‐1β, and GSDMD‐N levels in U251 cells, and FA treatment decreased these protein levels, whereas the addition of Nig caused a rebound in these protein levels. (N) SEM to observe the morphological changes associated with pyroptosis in U251 cells. *** p < 0.001 vs. Control; ** p < 0.01 vs. OGD/R; && p < 0.01 vs. 80 μM FA.

Article Snippet: For the FA + NLRP3 agonist nigericin (Nig) group, U251 cells were first exposed to FA (80 μM) for 3 h, and the OGD/R cell model was constructed in the culture medium supplemented with Nig (10 μM, HY‐127019, MedChemExpress) [ ].

Techniques: Activation Assay, Staining, Lactate Dehydrogenase Assay, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: Forsythoside A Alleviates Neuroinflammatory Damage via Inhibiting TLR4 / NF ‐ κB / NLRP3 Activation‐Induced Astrocyte Pyroptosis in Cerebral Ischemia–Reperfusion

doi: 10.1002/cns.70867

Figure Lengend Snippet: FA inhibits NLRP3 inflammasome activation through regulating TLR4/NF‐κB. (A–B) Immunofluorescence co‐localization measured that OGD/R resulted in increased p‐NF‐κB‐p65 fluorescence intensity in U251 cells, and FA treatment decreased p‐NF‐κB‐p65 levels, whereas the addition of LPS, the TLR4 agonist, resulted in a rebound of p‐NF‐κB‐p65 levels (20×, 100 μm). (C–D) OGD/R caused increased TLR4, p‐NF‐κB‐p65/NF‐κB‐p65 levels measured by Western blot, and FA treatment decreased the expression levels, while LPS weakened the inhibitory effect of FA. (E–F) Immunofluorescence co‐localization measured OGD/R led to increased NLRP3 fluorescence intensity in U251 cells, FA treatment decreased NLRP3 levels, and LPS led to a rebound in NLRP3 levels (20×, 100 μm). (G–H) OGD/R led to increased NLRP3, Cl‐Caspase 1/Caspase 1, and ASC levels revealed through Western blot, and FA decreased the levels of all three, whereas LPS weakened the inhibitory effect of FA. *** p < 0.001 vs. Control; * p < 0.05, ** p < 0.01 vs. OGD/R; && p < 0.01 vs. 80 μM FA.

Article Snippet: For the FA + NLRP3 agonist nigericin (Nig) group, U251 cells were first exposed to FA (80 μM) for 3 h, and the OGD/R cell model was constructed in the culture medium supplemented with Nig (10 μM, HY‐127019, MedChemExpress) [ ].

Techniques: Activation Assay, Immunofluorescence, Fluorescence, Western Blot, Expressing, Control