Journal: International Journal of Molecular Sciences

Article Title: Hydrogen Mitigated Doxorubicin-Induced Liver Injury via Nrf2/HO-1 Pathway Activation

doi: 10.3390/ijms27062774

Figure Lengend Snippet: Hydrogen activated the Nrf2/HO-1 pathway to attenuate liver injury in DOX mice. ( A , B ) Nfe2l2 and Hmox1 gene expression in liver tissue (n = 3). ( C – E ) IHC staining and statistics of Nrf2 and HO-1 protein (scale bar = 100 µm, n = 3). ( F – I ) Expression and statistics of Keap1, Nrf2, and HO-1 protein levels measured by Western blot (n = 4). The results are presented as the mean ± SEM. * p < 0.05 vs. Con group. # p < 0.05 vs. DOX group.

Article Snippet: Antibodies: Bcl-2 ( GB153375 , Servicebio, Wuhan, China), Bax (GB11007, Servicebio, Wuhan, China), Caspase 3 (#14220, Cell Signaling Technology, Danvers, MA, USA), MDA (ab243066, Abcam, Cincinnati, OH, USA), 4-HNE ( ARG23717 , Arigo Biolaboratories, Hsinchu, Taiwan), IL-6 (DF6087, Affinity, Cincinnati, OH, USA), NLRP3 (BA3677, Boster, Wuhan, China), Nrf2 ( GB115673 , Servicebio, Wuhan, China), HO-1 (GB12104, Servicebio, Wuhan, China), Keap1 (bs-4900R, BIOSS, Shanghai, China), and α-Tubulin (GB11200, Servicebio, Wuhan, China).

Techniques: Gene Expression, Immunohistochemistry, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Hydrogen Mitigated Doxorubicin-Induced Liver Injury via Nrf2/HO-1 Pathway Activation

doi: 10.3390/ijms27062774

Figure Lengend Snippet: The schematic diagram of hydrogen protection against DOX-induced liver injury. DOX has been observed to provoke biochemical alterations and pathological abnormalities in the liver. Specifically, DOX facilitates the generation of ROS and promotes mitochondria-dependent cell apoptosis. Additionally, DOX impedes the reduction in the expression of Keap1 and Nrf2, thereby inhibiting the downstream antioxidant signaling pathways of Nrf2, including HO-1, CAT, and T-SOD. Concurrently, it enhances the expression of lipid peroxidation products such as MDA and 4-HNE. Furthermore, DOX instigates inflammatory processes and augments the release of pro-inflammatory cytokines. In contrast, hydrogen exerts a protective effect on DOX-induced liver dysfunction by modulating Nrf2, which mitigates oxidative stress and inflammatory responses.

Article Snippet: Antibodies: Bcl-2 ( GB153375 , Servicebio, Wuhan, China), Bax (GB11007, Servicebio, Wuhan, China), Caspase 3 (#14220, Cell Signaling Technology, Danvers, MA, USA), MDA (ab243066, Abcam, Cincinnati, OH, USA), 4-HNE ( ARG23717 , Arigo Biolaboratories, Hsinchu, Taiwan), IL-6 (DF6087, Affinity, Cincinnati, OH, USA), NLRP3 (BA3677, Boster, Wuhan, China), Nrf2 ( GB115673 , Servicebio, Wuhan, China), HO-1 (GB12104, Servicebio, Wuhan, China), Keap1 (bs-4900R, BIOSS, Shanghai, China), and α-Tubulin (GB11200, Servicebio, Wuhan, China).

Techniques: Expressing, Protein-Protein interactions

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: International Journal of Clinical and Experimental Pathology

Article Title: Prognostic and predictive values of Nrf2, Keap1, p16 and E-cadherin expression in ovarian epithelial carcinoma

doi:

Figure Lengend Snippet: Baseline characteristics of 108 cases with ovarian carcinomas

Article Snippet: The primary antibodies used were Nrf2 (courtesy of Professor Huang of Academia Sinica; 1: 100), Keap1 (clone: 1G2; Origene; 1: 100), E-cadherin (clone: NCH-38; DAKO Carpinteria CA; 1: 100), p53 (clone: DO-7; Ventena; prediluted), p16 (clone: E6H4; Ventena; prediluted) and estrogen receptor (clone: 6F11; Novocastra; prediluted).

Techniques:

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Prognostic and predictive values of Nrf2, Keap1, p16 and E-cadherin expression in ovarian epithelial carcinoma

doi:

Figure Lengend Snippet: Ovarian serous carcinoma with corresponding (A), Nrf2 nuclear staining (original magnification × 100) (B), Keap1 cytoplasmic staining (original magnification × 100) and (C), E-cadherin membranous staining (original magnification × 200).

Article Snippet: The primary antibodies used were Nrf2 (courtesy of Professor Huang of Academia Sinica; 1: 100), Keap1 (clone: 1G2; Origene; 1: 100), E-cadherin (clone: NCH-38; DAKO Carpinteria CA; 1: 100), p53 (clone: DO-7; Ventena; prediluted), p16 (clone: E6H4; Ventena; prediluted) and estrogen receptor (clone: 6F11; Novocastra; prediluted).

Techniques: Staining

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Prognostic and predictive values of Nrf2, Keap1, p16 and E-cadherin expression in ovarian epithelial carcinoma

doi:

Figure Lengend Snippet: Univariate analyses showing HRs for patient OS and DFS conferred age, FIGO stage, histological subtypes, p53, p16, ER, Nrf2, Keap1 and E-cadherin expression (n=108)

Article Snippet: The primary antibodies used were Nrf2 (courtesy of Professor Huang of Academia Sinica; 1: 100), Keap1 (clone: 1G2; Origene; 1: 100), E-cadherin (clone: NCH-38; DAKO Carpinteria CA; 1: 100), p53 (clone: DO-7; Ventena; prediluted), p16 (clone: E6H4; Ventena; prediluted) and estrogen receptor (clone: 6F11; Novocastra; prediluted).

Techniques: Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Oxidative Stress and Ultrastructural Changes in Laminar Tissue of Dairy Cows with Acute Laminitis Induced by Oligofructose Overload

doi: 10.3390/ani16060980

Figure Lengend Snippet: RT-qPCR results of mRNA concentration of oxidative stress-related genes including, Keap1 , Nrf2 , Ho1 , and Nqo1 in laminar tissue of the both groups. Data were analyzed using Student’s t -test and are presented as mean ± SD. “*” show ( p < 0.05); “**” show ( p < 0.01).

Article Snippet: The resulting fragments were incubated with bovine serum albumin (20 min, room temperature), primed overnight (4 °C) with a primary antibody mixture (1: 200) dilution against Keap1 and Nrf2 (Novus Biologicals, Littleton, CO, USA), and then incubated with streptavidin-conjugated horseradish peroxidase (30 min, room temperature).

Techniques: Quantitative RT-PCR, Concentration Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: Oxidative Stress and Ultrastructural Changes in Laminar Tissue of Dairy Cows with Acute Laminitis Induced by Oligofructose Overload

doi: 10.3390/ani16060980

Figure Lengend Snippet: Western blot results of oxidative stress-related proteins expression including Keap1 , Nrf2 , Ho1 , and Nqo1 , in the laminar tissue of both groups. Data were analyzed using Student’s t -test and are presented as mean ± SD. “*” show ( p < 0.05); “**” show ( p < 0.01).

Article Snippet: The resulting fragments were incubated with bovine serum albumin (20 min, room temperature), primed overnight (4 °C) with a primary antibody mixture (1: 200) dilution against Keap1 and Nrf2 (Novus Biologicals, Littleton, CO, USA), and then incubated with streptavidin-conjugated horseradish peroxidase (30 min, room temperature).

Techniques: Western Blot, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Oxidative Stress and Ultrastructural Changes in Laminar Tissue of Dairy Cows with Acute Laminitis Induced by Oligofructose Overload

doi: 10.3390/ani16060980

Figure Lengend Snippet: Immunohistochemical staining of Keap1 in laminar tissues: Scale = 100 μm, 200×, ( A – C ) control cows; ( D – F ) OF-treated cows. EC, epidermal cels of epidermal lamellae; DC, dermal cells of dermal lamellae. Data were analyzed using Student’s t -test and are presented as mean ± SD. “*” show ( p < 0.05).

Article Snippet: The resulting fragments were incubated with bovine serum albumin (20 min, room temperature), primed overnight (4 °C) with a primary antibody mixture (1: 200) dilution against Keap1 and Nrf2 (Novus Biologicals, Littleton, CO, USA), and then incubated with streptavidin-conjugated horseradish peroxidase (30 min, room temperature).

Techniques: Immunohistochemical staining, Staining, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Oxidative Stress and Ultrastructural Changes in Laminar Tissue of Dairy Cows with Acute Laminitis Induced by Oligofructose Overload

doi: 10.3390/ani16060980

Figure Lengend Snippet: Schematic diagram illustrating the proposed oxidative stress pathway in laminar tissue during OF-induced acute laminitis in dairy cows. Following OF-overload, rumen fermentation changes, leading to rumen acidosis and systemic inflammation. Consequently, the pathway begins with the activation of the oxidative stress response and the accumulation of ROS, a key driver of acute laminitis. Elevated ROS levels increase the expression of Keap1 , which sequesters and promotes the degradation of the master antioxidant regulator Nrf2 . With Nrf2 activity suppressed, the expression of cytoprotective antioxidant genes is diminished, disabling the tissue’s intrinsic defense mechanisms. This weakened cellular protection leads to laminar tissue damage, characterized by collagen fiber disruption (including reduced hemidesmosomes), separation of epidermal layers, thickening of the lamina densa, and eventual detachment of basal cells from the basement membrane. This cascade elucidates the molecular link between systemic oxidative insult and the structural failure characteristic of laminitis. The figure was created with BioRender.com.

Article Snippet: The resulting fragments were incubated with bovine serum albumin (20 min, room temperature), primed overnight (4 °C) with a primary antibody mixture (1: 200) dilution against Keap1 and Nrf2 (Novus Biologicals, Littleton, CO, USA), and then incubated with streptavidin-conjugated horseradish peroxidase (30 min, room temperature).

Techniques: Activation Assay, Expressing, Activity Assay, Disruption, Membrane

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Hydrogen improves the efficacy of tetrandrine in the treatment of silicosis by inhibiting vascular endothelial mesenchymal transition caused by oxidative stress

doi: 10.3389/fbioe.2025.1668524

Figure Lengend Snippet: Tetrandrine concentration decreased in lung tissue and oxidative stress excessive activated in silicosis. (A,B) , HE staining and quantitative analysis of the degree of vascular stenosis of each group of mice (n = 3), scale bar, 20 μm; (C,D) , Masson staining and collagen volume fraction of each group of mice (n = 3), scale bar, 20 μm; (E,F) , Immunofluorescence and semi-quantitative of the expression of the endothelial to mesenchymal transition (EndMT)-related proteins platelet endothelial cell adhesion molecule-1 (CD31) and alfa-smooth muscle actin (α–SMA) in the lung tissues of different mouse groups (n = 3). Scale bar, 10 μm and 2 μm, Pearson’s R is the correlation coefficient—a statistical measure that indicates both the strength and direction of a linear relationship between two continuous variables, ranging from −1 (strong negative) to +1 (strong positive); (G) , The concentration of tetrandrine in lung tissue was detected by LC/MS-MS (n = 3); (H,I) , Changes of ROS levels in the lung tissues (n = 3). Scale bar, 20 μm, ROS: reactive oxygen species, DAPI: 4′,6-Diamidino-2-phenylindole dihydrochloride, Merge: ROS combines with the cell nucleus; (J) , Western blotting of Keap1 and Nrf2 expression in the lung tissues (n = 3); (K,L) , Semi-quantitative of the expression of Keap1 and Nrf2 in the lung tissues (n = 3). a P < 0.05; aa P < 0.01 vs. the control group. Data are expressed as mean ± SD.

Article Snippet: The membranes were incubated with the primary antibodies, namely, Pink1 (1:1000 dilution, Proteintech, United States, 23274-1-AP), LC3 (1:1000 dilution, Cell Signaling Technology, United States, 12741T), sequestosome 1 (SQSTM1/p62) (1:1000 dilution, Cell Signaling Technology, United States, 5114T), Parkin (1:2000 dilution, Proteintech, United States, 14060-1-AP), Kelch-like ECH-associated protein 1 (Keap1) (1:1000 dilution, Proteintech, United States, 60027-1-Ig), nuclear factor E2-related factor 2 (Nrf2) (1:2000 dilution, Proteintech, United States, 16396-1-AP), B- cell lymphoma - 2 (Bcl-2) (1:2000 dilution, Proteintech, United States, 26593-1-AP), Bcl - 2-associated X protein (Bax) (1:5000 dilution, Proteintech, United States, 50599-2-Ig), Cytochrome C (Cyto C) (1:5000, Proteintech, United States, 10993-1-AP), Caspase 3 (1:1000 dilution, Proteintech, United States, 19677-1-AP) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:500000 dilution, Proteintech, United States, 60004-1-Ig).

Techniques: Concentration Assay, Staining, Immunofluorescence, Expressing, Liquid Chromatography with Mass Spectroscopy, Western Blot, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Hydrogen improves the efficacy of tetrandrine in the treatment of silicosis by inhibiting vascular endothelial mesenchymal transition caused by oxidative stress

doi: 10.3389/fbioe.2025.1668524

Figure Lengend Snippet: Inhibiting oxidative stress can improve the concentration of tetrandrine in lung tissue in mice with silicosis. (A,B) , Changes in the ROS levels in the lung tissues (n = 3). Scale bar, 20 μm, ROS: reactive oxygen species, DAPI: 4′,6-Diamidino-2-phenylindole dihydrochloride, Merge: ROS combines with the cell nucleus; (C) , Western blotting of Keap1, Nrf2, Pink1, Parkin, Bcl-2 and Bax expression in the lung tissues (n = 3); (D,E) , Semi-quantitative of the expression of Keap1 and Nrf2 in the lung tissues of different mouse groups (n = 3); (F) , Mitochondrial injury observed under transmission electron microscopy (TEM) (n = 3). Scale bar, 2.0 (×4.0k) and 1.0 (×8.0k) μm; (G–J) , Semi-quantitative of the expression of Pink1, Parkin, Bcl-2 and Bax in the lung tissues of different mouse groups (n = 3); (K,L) , Immunofluorescence and semi-quantitative of the expression of the endothelial to mesenchymal transition (EndMT)-related proteins platelet endothelial cell adhesion molecule-1 (CD31) and alfa-smooth muscle actin (α–SMA) in the lung tissues of different mouse groups (n = 3). Scale bar, 10 μm and 2 μm; (M) , The concentration of tetrandrine in lung tissue was detected by LC/MS-MS (n = 3 biologically independent experiments). a P < 0.05; aa P < 0.01; aaa P < 0.001 vs. the control group. b P < 0.05; bb P < 0.01 vs. the model group. Data are expressed as mean ± SD.

Article Snippet: The membranes were incubated with the primary antibodies, namely, Pink1 (1:1000 dilution, Proteintech, United States, 23274-1-AP), LC3 (1:1000 dilution, Cell Signaling Technology, United States, 12741T), sequestosome 1 (SQSTM1/p62) (1:1000 dilution, Cell Signaling Technology, United States, 5114T), Parkin (1:2000 dilution, Proteintech, United States, 14060-1-AP), Kelch-like ECH-associated protein 1 (Keap1) (1:1000 dilution, Proteintech, United States, 60027-1-Ig), nuclear factor E2-related factor 2 (Nrf2) (1:2000 dilution, Proteintech, United States, 16396-1-AP), B- cell lymphoma - 2 (Bcl-2) (1:2000 dilution, Proteintech, United States, 26593-1-AP), Bcl - 2-associated X protein (Bax) (1:5000 dilution, Proteintech, United States, 50599-2-Ig), Cytochrome C (Cyto C) (1:5000, Proteintech, United States, 10993-1-AP), Caspase 3 (1:1000 dilution, Proteintech, United States, 19677-1-AP) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:500000 dilution, Proteintech, United States, 60004-1-Ig).

Techniques: Concentration Assay, Western Blot, Expressing, Transmission Assay, Electron Microscopy, Immunofluorescence, Liquid Chromatography with Mass Spectroscopy, Control