Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: Caffeic acid (CA) activate kelch-like ECH-associated protein 1/nuclear factor erythroid 2 related factor 2 (Keap1/Nrf2) signaling pathway. (A–E) Western blot and gray value analysis of Keap1 protein expression. (F) Western blot and gray value analysis of Nrf2 protein expression in the nucleus and cytoplasm. (G, H) Representative images of immunofluorescence staining of Keap1 (Green) and Nrf2 (Red). (I) Western blot and gray value analysis of heme oxygenase-1 (HO-1) and nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase quinone 1 (NQO1) protein expression. The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4′,6-diamidino-2′-phenylindole.

Article Snippet: Interaction analysis of caffeic acid (CA) analogs with kelch-like ECH-associated protein 1 (Keap1) Kelch . (A–D) The affinity of Keap1 Kelch binding with different components was detected by Biacore. (A) CGA with Keap1 Kelch , (B) CA-derivative with Keap1 Kelch , (C) Protocatechuic acid with Keap1 Kelch , D) Gallic acid with Keap1 Kelch . (E-H) The CA analogs and Keap1 Kelch interaction diagram. (E) Molecular docking of CGA with Keap1 Kelch , (F) Molecular docking of CA-derivative with Keap1 Kelch , (G) Molecular docking of protocatechuic acid with Keap1 Kelch , (H) Molecular docking of gallic acid with Keap1 Kelch , This figure is exported from the Ligplot software.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Standard Deviation, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: Caffeic acid (CA) induced kelch-like ECH-associated protein 1 (Keap1) degradation via p62-dependent autophagy. (A) Western blot and gray value analysis of Keap1 and p62 protein expression. (B) Immunofluorescence staining of p62 (Red) was analyzed by treating cells with different doses of CA. (C–E) Western blot and gray value analysis of Keap1 and LC3B-II protein expression were conducted both with and without the addition of MG132. (F–H) Western blot and gray value analysis of Keap1 and LC3B-II protein expression were conducted both with and without the addition of CQ (I) The LC3B-II (Red) expression level was detected by immunofluorescence analysis. The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group. DAPI: 4′,6-diamidino-2′-phenylindole; CQ: chloroquin.

Article Snippet: Interaction analysis of caffeic acid (CA) analogs with kelch-like ECH-associated protein 1 (Keap1) Kelch . (A–D) The affinity of Keap1 Kelch binding with different components was detected by Biacore. (A) CGA with Keap1 Kelch , (B) CA-derivative with Keap1 Kelch , (C) Protocatechuic acid with Keap1 Kelch , D) Gallic acid with Keap1 Kelch . (E-H) The CA analogs and Keap1 Kelch interaction diagram. (E) Molecular docking of CGA with Keap1 Kelch , (F) Molecular docking of CA-derivative with Keap1 Kelch , (G) Molecular docking of protocatechuic acid with Keap1 Kelch , (H) Molecular docking of gallic acid with Keap1 Kelch , This figure is exported from the Ligplot software.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Standard Deviation, Control

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: Caffeic acid (CA) directly interacts with kelch-like ECH-associated protein 1 (Keap1) in vit r o . (A) Structure of CA and photo-affinity labeling probe (PAL-CA). (B) PAL-CA probe target fishing flowchart. (C) Silver staining of the PAL-CA complex in H9c2 cells. (D) Validation of Keap1 pulled down from mitochondria of the H9c2 cells with PAL-CA by Western blot. (E) Cellular thermal shift assay (CETSA) experiments of CA with Keap1 protein. (F) Size-exclusion chromatography analysis. The black and red lines represent the ultraviolet absorption of the standard and Keap1 proteins at 280 nM, respectively. (G) Surface plasmon resonance (SPR) experiments of CA with Keap1 protein. (H) Chemical structure of CA (top) and isothermal titration calorimetry (ITC) experiments (bottom). (I, J) SDS-PAGE gel and line graph were used to analyze the in vitro digestive stability of Keap1 under the action of trypsin.. (K) Native mass spectrometry analysis of apo Keap1. The Keap1 protein exists as monomers, dimers, and hexamers in solution (top). Enlarged view of the Keap1 protein dimer, including the P 1 and P 2 peaks (bottom). (L) Native mass spectrometry analysis of Keap1 with CA. After CA binds to the Keap1 protein, the monomers, dimers and hexamers exist in solution (top). Enlarged view of the increased dimerization that occurs after CA binds to the Keap1 protein (P 1 and P 2 peaks) (bottom). The results were normalized and are expressed as the mean ± standard deviation (SD) ( n = 3). DMSO: dimethyl sulfoxide.

Article Snippet: Interaction analysis of caffeic acid (CA) analogs with kelch-like ECH-associated protein 1 (Keap1) Kelch . (A–D) The affinity of Keap1 Kelch binding with different components was detected by Biacore. (A) CGA with Keap1 Kelch , (B) CA-derivative with Keap1 Kelch , (C) Protocatechuic acid with Keap1 Kelch , D) Gallic acid with Keap1 Kelch . (E-H) The CA analogs and Keap1 Kelch interaction diagram. (E) Molecular docking of CGA with Keap1 Kelch , (F) Molecular docking of CA-derivative with Keap1 Kelch , (G) Molecular docking of protocatechuic acid with Keap1 Kelch , (H) Molecular docking of gallic acid with Keap1 Kelch , This figure is exported from the Ligplot software.

Techniques: Labeling, Silver Staining, Biomarker Discovery, Western Blot, Thermal Shift Assay, Size-exclusion Chromatography, SPR Assay, Isothermal Titration Calorimetry, SDS Page, In Vitro, Mass Spectrometry, Standard Deviation

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: The complexed crystal structure confirms the interaction sites of caffeic acid (CA) with kelch-like ECH-associated protein 1 (Keap1). (A) Schematic design of the experiments. (B) Gel filtration traces of Keap1 and Keap1 gel filtered with CA with a Superdex 200 10/300 Increase column and superimposed on the chromatogram of two standard protein markers (75 kDa and 44 kDa). (C) Kelch crystal diagram. (D) The overall structure of the complex of the Kelch domain with CA. Two orthogonal views are shown. (E) The CA molecule and surrounding residues responsible for its binding are shown in ball-and-stick representation. The M550 and N532 residues of the Kelch domain interact with CA. (F) Isothermal titration calorimetry (ITC) experiments of N532A with Keap1. (G) Comparison of the mouse Kelch domain (PDB ID: 1X2J ) and the Kelch domain bound to CA (PDB ID: 7YEN ). The Kelch apo and Kelch-CA complexes are colored wheat and purple, respectively. (H) Protein-ligand complex structure of molecular dynamics (MD) simulations for wild type 5 ns (a), wild type 100 ns (b), N532A mutant (c), and M550A mutant (d) of Keap1 kelch domain. The ligand CA is shown as yellow sticks. (I) Multiple sequence alignment of Keap1 from different species. The red background represents extremely conserved residues, and the red font represents relatively conserved residues. H. sapiens : Homo sapiens; M. musculus : Mus musculus ; C. toad : Caucasian toad ; D. rerio : Danio rerio .DP: ?.

Article Snippet: Interaction analysis of caffeic acid (CA) analogs with kelch-like ECH-associated protein 1 (Keap1) Kelch . (A–D) The affinity of Keap1 Kelch binding with different components was detected by Biacore. (A) CGA with Keap1 Kelch , (B) CA-derivative with Keap1 Kelch , (C) Protocatechuic acid with Keap1 Kelch , D) Gallic acid with Keap1 Kelch . (E-H) The CA analogs and Keap1 Kelch interaction diagram. (E) Molecular docking of CGA with Keap1 Kelch , (F) Molecular docking of CA-derivative with Keap1 Kelch , (G) Molecular docking of protocatechuic acid with Keap1 Kelch , (H) Molecular docking of gallic acid with Keap1 Kelch , This figure is exported from the Ligplot software.

Techniques: Filtration, Binding Assay, Isothermal Titration Calorimetry, Comparison, Mutagenesis, Sequencing

Journal: Journal of Pharmaceutical Analysis

Article Title: Caffeic acid alleviates myocardial ischemia-reperfusion injury by directly targeting Keap1 N532/M550 and promoting its degradation

doi: 10.1016/j.jpha.2025.101219

Figure Lengend Snippet: Interaction analysis of caffeic acid (CA) analogs with kelch-like ECH-associated protein 1 (Keap1) Kelch . (A–D) The affinity of Keap1 Kelch binding with different components was detected by Biacore. (A) CGA with Keap1 Kelch , (B) CA-derivative with Keap1 Kelch , (C) Protocatechuic acid with Keap1 Kelch , D) Gallic acid with Keap1 Kelch . (E-H) The CA analogs and Keap1 Kelch interaction diagram. (E) Molecular docking of CGA with Keap1 Kelch , (F) Molecular docking of CA-derivative with Keap1 Kelch , (G) Molecular docking of protocatechuic acid with Keap1 Kelch , (H) Molecular docking of gallic acid with Keap1 Kelch , This figure is exported from the Ligplot software. Hydrogen bonds are shown as green dotted lines, while the spoked arcs represent residues making nonbonded contacts with the ligand. (I) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays of the toxic effects of chlorogenic acid (CGA) on H 2 O 2 -treated H9c2 cells. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to H 2 O 2 group. (J) Western blot and gray value analysis of Keap1 protein expression after treated with different concentrations of CGA. (K) Western blot and gray value analysis of Keap1 protein expression treated with different times of CGA. (L) Western blot and gray value analysis of Keap1 protein expression treated with different concentrations of 60 μM CGA. The results are expressed as the mean ± standard deviation (SD) ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, compared to the control group.

Article Snippet: Interaction analysis of caffeic acid (CA) analogs with kelch-like ECH-associated protein 1 (Keap1) Kelch . (A–D) The affinity of Keap1 Kelch binding with different components was detected by Biacore. (A) CGA with Keap1 Kelch , (B) CA-derivative with Keap1 Kelch , (C) Protocatechuic acid with Keap1 Kelch , D) Gallic acid with Keap1 Kelch . (E-H) The CA analogs and Keap1 Kelch interaction diagram. (E) Molecular docking of CGA with Keap1 Kelch , (F) Molecular docking of CA-derivative with Keap1 Kelch , (G) Molecular docking of protocatechuic acid with Keap1 Kelch , (H) Molecular docking of gallic acid with Keap1 Kelch , This figure is exported from the Ligplot software.

Techniques: Binding Assay, Software, Western Blot, Expressing, Standard Deviation, Control

Journal: Antioxidants

Article Title: Ellagic Acid Alleviates Oxidative Stress by Mediating Nrf2 Signaling Pathways and Protects against Paraquat-Induced Intestinal Injury in Piglets

doi: 10.3390/antiox11020252

Figure Lengend Snippet: Primer sequences 1 .

Article Snippet: Membranes were sealed for 10 min with a rapid blocking solution (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature and incubated for 2 h with the primary antibodies including NQO1 (1:1000; Proteintech; Chicago, IL, USA), Nrf2 (1:600; Abcam; Cambridge, UK), HO-1(1:800; Abcam; Cambridge, UK), Recombinant Kelch Like ECH Associated Protein 1 (Keap1) (1:800; Proteintech; Chicago, IL, USA), and β-actin (1:1000; Cell Signaling Technology; Danvers, MA, USA), along with the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5000; ZSGB, Biological Technology, Beijing, China).

Techniques:

Journal: Antioxidants

Article Title: Ellagic Acid Alleviates Oxidative Stress by Mediating Nrf2 Signaling Pathways and Protects against Paraquat-Induced Intestinal Injury in Piglets

doi: 10.3390/antiox11020252

Figure Lengend Snippet: Protein abundances of Nrf2 signaling pathway of cytosol and nucleus of jejunal and ileal mucosa in piglets. ( A ) Nuclear factor erythroid 2-related factor 2 (Nrf2), Recombinant Kelch Like ECH Associated Protein 1 (Keap1), heme oxygenase-1 (HO-1), and quinone oxidoreductase 1 (NQO1) protein abundances in cytosol of jejunal and ileal mucosa. ( B ) Nrf2, HO-1, and NQO1 protein abundances in nucleus of jejunal and ileal mucosa. PQ = 4 mg/kg paraquet; EL = 0.005% ellagic acid + 4 mg/kg paraquet; EM = 0.01% ellagic acid + 4 mg/kg paraquet; EH = 0.02% ellagic acid + 4 mg/kg paraquet. n = 8. Data are shown as mean ± SEM. * means the difference was significant when compared to the control group. ns means the difference was not significant when compared to the control group. a–c Values with different lowercase letters are significantly different among PQ, EL, EM, and EH groups ( p < 0.05).

Article Snippet: Membranes were sealed for 10 min with a rapid blocking solution (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature and incubated for 2 h with the primary antibodies including NQO1 (1:1000; Proteintech; Chicago, IL, USA), Nrf2 (1:600; Abcam; Cambridge, UK), HO-1(1:800; Abcam; Cambridge, UK), Recombinant Kelch Like ECH Associated Protein 1 (Keap1) (1:800; Proteintech; Chicago, IL, USA), and β-actin (1:1000; Cell Signaling Technology; Danvers, MA, USA), along with the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:5000; ZSGB, Biological Technology, Beijing, China).

Techniques: Recombinant, Control

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: NRF2 differentially regulates gene expression and cellular behavior in ESCC cells. Isogenic NRF2 null -KYSE70 cells were compared with NRF2 W24C -KYSE70 cells, and KEAP1 null -KYSE450 cells were compared with NRF2 WT -KYSE450 cells. ( A ) Expression of squamous differentiation and basal cell markers assessed by Western blotting. ( B ) Cell-cycle distribution determined by flow cytometry. ( C ) Cell proliferation measured by IncuCyte live-cell imaging. ( D ) Apoptosis quantified by IncuCyte analysis. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Original western blots are presented in .

Article Snippet: A total of 4 μL aliquots of 100 μM PYR was injected from a syringe into the sample cell containing 200 μL of human recombinant KEAP1 (Gln 2 -Cys 624 , MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Gene Expression, Expressing, Western Blot, Flow Cytometry, Live Cell Imaging

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: PYR enhances KEAP1-dependent degradation of NRF2 W24C . ( A ) Dose-dependent reduction in NRF2 W24C protein levels in KYSE70 cells following 4 h PYR treatment. ( B ) Lack of NRF2 W24C reduction in KEAP1 null -KYSE70 cells following 4 h PYR treatment. ( C ) Co-IP analysis showing increased association between NRF2 W24C and KEAP1 following PYR treatment (10 μM, 2 h), but not between NRF2 WT and KEAP1. MTX (1 μM, 2 h) did not alter NRF2 W24C -KEAP1 association. ( D ) PLA showing increased NRF2 W24C -KEAP1 interactions following PYR treatment (10 μM, 4 h). Red dots represent PLA signal. ( E , F ) NRF2 protein levels following 72 h PYR treatment (10 μM) in KEAP1 null -KYSE70 and KEAP1 null -KYSE450 cells. Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Original western blots are presented in .

Article Snippet: A total of 4 μL aliquots of 100 μM PYR was injected from a syringe into the sample cell containing 200 μL of human recombinant KEAP1 (Gln 2 -Cys 624 , MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: PYR modestly enhances DLGW24C–Kelch interaction in SPR assays. SPR sensorgrams showing binding of ( A ) DLG WT peptide and ( B ) DLG W24C peptide to the KEAP1 Kelch domain. ( C ) PYR has minimal effect on DLG WT –Kelch interaction. ( D ) PYR modestly increases binding of DLG W24C peptide to the Kelch domain. The colors in an SPR sensorgrams distinguish different concentrations of the analyte (DLG peptide).

Article Snippet: A total of 4 μL aliquots of 100 μM PYR was injected from a syringe into the sample cell containing 200 μL of human recombinant KEAP1 (Gln 2 -Cys 624 , MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: Binding Assay

Journal: Cancers

Article Title: Pyrimethamine Restores KEAP1-Mediated Degradation of Select NRF2 Mutants in Esophageal Squamous Cell Carcinoma

doi: 10.3390/cancers18091354

Figure Lengend Snippet: Computational modeling suggests PYR may facilitate NRF2 W24C –KEAP1 interaction via the Kelch domain. ( A ) Predicted interaction interface between the DLG WT motif and the Kelch domain. ( B ) Predicted weakening of the DLG W24C –Kelch interaction. ( C ) Docking of PYR into a Kelch domain pocket increases predicted interaction surface area and shape complementarity for the DLG W24C –Kelch complex. ( D ) Close-up view of PYR positioned within the Kelch domain cavity. ( E ) Two-dimensional interaction map showing predicted contacts between PYR and Kelch domain residues.

Article Snippet: A total of 4 μL aliquots of 100 μM PYR was injected from a syringe into the sample cell containing 200 μL of human recombinant KEAP1 (Gln 2 -Cys 624 , MedChemExpress, Monmouth Junction, NJ, USA).

Techniques: