Journal: Frontiers in Pharmacology

Article Title: Licochalcone D mitigates intracerebral hemorrhage-induced ferroptosis of neurons through COX2 inhibition

doi: 10.3389/fphar.2025.1566724

Figure Lengend Snippet: LCD Improves Ferroptosis in ICH Neuronal Cells In Vitro by Targeting the COX2/PGE2/EP1 Pathway. (A–B) PC12 or SH-SY5Y cells were transfected with a COX2-overexpressing (COX2-OE) plasmid or a control vector for 24 h, followed by exposure to LCD (5 or 10 µM) or FE-1 (1 µM) for 2 h. This was followed by a 24 h incubation with CoCl 2 (400 μg/mL). After the incubation period, protein levels of COX2, EP1-4, and GPX4 were quantified by Western blot, using β-Actin as the loading control. Data are presented as mean ± SD (n = 3). (C–D) PC12 or SH-SY5Y cells were subjected to the same treatment as in (A). After 24 h incubation with CoCl 2 , the ratio of GSH/GSSG was measured using a dedicated GSH/GSSG quantification assay. Data are presented as mean ± SD (n = 3). (E–F) PC12 or SH-SY5Y cells underwent the same treatment regimen as described in (A). Following the 24 h incubation with CoCl 2 , mitochondrial lipid ROS levels were quantified using BODIPY RED fluorescence. The oxidized dye was measured using FITC fluorescence ( green ), the reduced dye was quantified using BODIPY RED fluorescence ( red ). The ratio of the fluorescence intensities at FITC and BODIPY RED channels was calculated to assess lipid peroxidation in cells. Quantification of fluorescence density were normalized before plotting. Scale bars are provided 20 μm. Bars represent the mean ± SD (n = 3). (G–H) PC12 or SH-SY5Y cells were subjected to the identical treatment regimen as in (A) . After the 24 h incubation period, the protein level of PGE2 was measured using an ELISA assay. Data are presented as mean ± SD (n = 3). Statistical significance is indicated as follows: ** p < 0.01, * p < 0.05, and n. s.; not significant.

Article Snippet: The production of PGE2 was measured using a PGE2 ELISA kit (EK8103/2, Lianke Biotech, China), following the kit’s instructions.

Techniques: In Vitro, Transfection, Plasmid Preparation, Control, Incubation, Western Blot, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Licochalcone D mitigates intracerebral hemorrhage-induced ferroptosis of neurons through COX2 inhibition

doi: 10.3389/fphar.2025.1566724

Figure Lengend Snippet: LCD Improves Ferroptosis in Rat ICH by Inhibiting the COX2/PGE2/EP1 Pathway. (A) The CAT activity in brain tissues was were quantified using a CAT assay and normalized prior to plotting. Data are presented as mean ± SD (n = 3). (B) The levels of MDA in brain tissue were measured using an MDA assay and normalized before plotting. Data are expressed as mean ± SD (n = 3). (C) The ratio of GSH/GSSG in brain tissue was quantified using a GSH/GSSG assay and normalized prior to plotting. Data are expressed as mean ± SD (n = 3). (D) The levels of PGE2 in brain tissue were assessed using a PGE2 ELISA assay and normalized before plotting. Data are expressed as mean ± SD (n = 3). (E) Immunohistochemical staining of COX2 was performed on coronal sections of brain tissue, and the results were normalized before plotting. Data are expressed as mean ± SD (n = 3). Scale bars of 1 mm or 2 mm are provided. (F) Western blot analysis was conducted to quantify the relative protein expression of COX2, EP1-EP4, and GPX4 proteins in brain tissue, β-Actin was used as an internal loading control. Error bars represent standard deviation and data are expressed as the mean ± SD, n = 3. Statistical significance is denoted as: ** p < 0.01, * p < 0.05 and n. s., no significant difference.

Article Snippet: The production of PGE2 was measured using a PGE2 ELISA kit (EK8103/2, Lianke Biotech, China), following the kit’s instructions.

Techniques: Activity Assay, Multiple Displacement Amplification, GSSG Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Western Blot, Expressing, Control, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: Licochalcone D mitigates intracerebral hemorrhage-induced ferroptosis of neurons through COX2 inhibition

doi: 10.3389/fphar.2025.1566724

Figure Lengend Snippet: Proposed model. Propose a model demonstrating LCD’s pharmacological effects in ameliorating ferroptosis in neuronal cells following spontaneous ICH, achieved through targeted inhibition of the COX2-driven PGE2/EP1 pathway.

Article Snippet: The production of PGE2 was measured using a PGE2 ELISA kit (EK8103/2, Lianke Biotech, China), following the kit’s instructions.

Techniques: Inhibition

Journal: Frontiers in Pharmacology

Article Title: Licochalcone D mitigates intracerebral hemorrhage-induced ferroptosis of neurons through COX2 inhibition

doi: 10.3389/fphar.2025.1566724

Figure Lengend Snippet: LCD Improves Ferroptosis in ICH Neuronal Cells In Vitro by Targeting the COX2/PGE2/EP1 Pathway. (A–B) PC12 or SH-SY5Y cells were transfected with a COX2-overexpressing (COX2-OE) plasmid or a control vector for 24 h, followed by exposure to LCD (5 or 10 µM) or FE-1 (1 µM) for 2 h. This was followed by a 24 h incubation with CoCl 2 (400 μg/mL). After the incubation period, protein levels of COX2, EP1-4, and GPX4 were quantified by Western blot, using β-Actin as the loading control. Data are presented as mean ± SD (n = 3). (C–D) PC12 or SH-SY5Y cells were subjected to the same treatment as in (A). After 24 h incubation with CoCl 2 , the ratio of GSH/GSSG was measured using a dedicated GSH/GSSG quantification assay. Data are presented as mean ± SD (n = 3). (E–F) PC12 or SH-SY5Y cells underwent the same treatment regimen as described in (A). Following the 24 h incubation with CoCl 2 , mitochondrial lipid ROS levels were quantified using BODIPY RED fluorescence. The oxidized dye was measured using FITC fluorescence ( green ), the reduced dye was quantified using BODIPY RED fluorescence ( red ). The ratio of the fluorescence intensities at FITC and BODIPY RED channels was calculated to assess lipid peroxidation in cells. Quantification of fluorescence density were normalized before plotting. Scale bars are provided 20 μm. Bars represent the mean ± SD (n = 3). (G–H) PC12 or SH-SY5Y cells were subjected to the identical treatment regimen as in (A) . After the 24 h incubation period, the protein level of PGE2 was measured using an ELISA assay. Data are presented as mean ± SD (n = 3). Statistical significance is indicated as follows: ** p < 0.01, * p < 0.05, and n. s.; not significant.

Article Snippet: The production of PGE2 was measured using a PGE2 ELISA kit (EK8103/2, Lianke Biotech, China), following the kit’s instructions.

Techniques: In Vitro, Transfection, Plasmid Preparation, Control, Incubation, Western Blot, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Licochalcone D mitigates intracerebral hemorrhage-induced ferroptosis of neurons through COX2 inhibition

doi: 10.3389/fphar.2025.1566724

Figure Lengend Snippet: LCD Improves Ferroptosis in Rat ICH by Inhibiting the COX2/PGE2/EP1 Pathway. (A) The CAT activity in brain tissues was were quantified using a CAT assay and normalized prior to plotting. Data are presented as mean ± SD (n = 3). (B) The levels of MDA in brain tissue were measured using an MDA assay and normalized before plotting. Data are expressed as mean ± SD (n = 3). (C) The ratio of GSH/GSSG in brain tissue was quantified using a GSH/GSSG assay and normalized prior to plotting. Data are expressed as mean ± SD (n = 3). (D) The levels of PGE2 in brain tissue were assessed using a PGE2 ELISA assay and normalized before plotting. Data are expressed as mean ± SD (n = 3). (E) Immunohistochemical staining of COX2 was performed on coronal sections of brain tissue, and the results were normalized before plotting. Data are expressed as mean ± SD (n = 3). Scale bars of 1 mm or 2 mm are provided. (F) Western blot analysis was conducted to quantify the relative protein expression of COX2, EP1-EP4, and GPX4 proteins in brain tissue, β-Actin was used as an internal loading control. Error bars represent standard deviation and data are expressed as the mean ± SD, n = 3. Statistical significance is denoted as: ** p < 0.01, * p < 0.05 and n. s., no significant difference.

Article Snippet: The production of PGE2 was measured using a PGE2 ELISA kit (EK8103/2, Lianke Biotech, China), following the kit’s instructions.

Techniques: Activity Assay, Multiple Displacement Amplification, GSSG Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Western Blot, Expressing, Control, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: Licochalcone D mitigates intracerebral hemorrhage-induced ferroptosis of neurons through COX2 inhibition

doi: 10.3389/fphar.2025.1566724

Figure Lengend Snippet: Proposed model. Propose a model demonstrating LCD’s pharmacological effects in ameliorating ferroptosis in neuronal cells following spontaneous ICH, achieved through targeted inhibition of the COX2-driven PGE2/EP1 pathway.

Article Snippet: The production of PGE2 was measured using a PGE2 ELISA kit (EK8103/2, Lianke Biotech, China), following the kit’s instructions.

Techniques: Inhibition