Journal: Molecules

Article Title: Anti-Inflammatory Effects of Auranamide and Patriscabratine—Mechanisms and In Silico Studies

doi: 10.3390/molecules27154992

Figure Lengend Snippet: The effect of auranamide and patriscabratine on ( A ) NRF2 protein expression; ( B ) HO-1 protein expression; ( C ) NQO1 activity in nuclear extracts of Hepa-1c1c7 cells. The results are expressed as mean ± SD (n = 3). **, p ≤ 0.01; ****, p ≤ 0.0001 compared to LPS Ec + 0.1% DMSO treatment.

Article Snippet: NRF2 ELISA kit (#OKCD09342) was purchased from Aviva Systems Biology, San Diego, CA 92121, USA.

Techniques: Expressing, Activity Assay

Journal: Antioxidants

Article Title: Punicalagin Protects Human Retinal Pigment Epithelium Cells from Ultraviolet Radiation-Induced Oxidative Damage by Activating Nrf2/HO-1 Signaling Pathway and Reducing Apoptosis

doi: 10.3390/antiox9060473

Figure Lengend Snippet: Pre-treatment with 10 µM PUN for 24 h modulates gene expression ( A ), protein synthesis ( B ), and nuclear activation ( C ) of Nrf2 in ARPE-19 cells exposed to UV-A radiation. ( A ) The levels of mRNA expression were measured by RT-PCR. Values are expressed as the mean percentage relative to unirradiated and not pre-treated cells (control 100%) ± SEM of four independent experiment performed in single. ( B ) Nrf2 protein levels were detected by colorimetric cell-based ELISA kit (see “Materials and Methods” section) and were expressed as the mean percentage relative to unirradiated and not pre-treated cells (control 100%) ± SEM of three independent experiment performed in triplicate. ( C ) To quantify Nrf2 activation in the nuclear extract, was utilized a colorimetric assay kit (see “Materials and Methods” section). All OD values were mathematically expressed as a percentage relative to unirradiated and not pre-treated cells (control 100%) ± SEM of three independent experiment performed in duplicate. Differences between mean values (shown in ) were assessed by one-way ANOVA analysis followed by post hoc Newman–Keuls. * = p < 0.05 and *** = p < 0.001 vs. control; °° = p < 0.01 and °°° = p < 0.001 vs. PUN alone; §§ = p < 0.01 and §§§ = p < 0.001 vs. UV-A alone at the same experimental time.

Article Snippet: Intracellular Nrf-2 levels were determined by colorimetric cell-based ELISA kit (LSBio, LifeSpan Biosciences; Seattle, WA, USA).

Techniques: Expressing, Activation Assay, Reverse Transcription Polymerase Chain Reaction, In-Cell ELISA, Colorimetric Assay

Journal: Antioxidants

Article Title: Punicalagin Protects Human Retinal Pigment Epithelium Cells from Ultraviolet Radiation-Induced Oxidative Damage by Activating Nrf2/HO-1 Signaling Pathway and Reducing Apoptosis

doi: 10.3390/antiox9060473

Figure Lengend Snippet: Protective effect of PUN on UV-A-induced apoptosis in ARPE-19 cells. Cells were pre-treated with 10 μM PUN and incubated for 24 h, and then exposed to UV-A radiations for 1, 3, and 5 h. Protein levels of apoptotic hallmarks, Bax ( left-panel ) and Bcl-2 ( right-panel ), were determined by colorimetric cell-based ELISA kits (details in “Materials and Methods” section). Values obtained, normalized to GAPDH, were expressed as a percentage relative to unirradiated and not pre-treated cells (control 100%). Results are from two independent experiments, each including three replicates per experimental group; they are presented as the means ± SEM. One-way ANOVA analysis followed by post hoc Newman–Keuls was carried out. ** = p < 0.01 and *** = p < 0.001 vs. control; °° = p < 0.01 vs. PUN alone; § = p < 0.05 and §§§ = p < 0.001 vs. UV-A alone at the same experimental time.

Article Snippet: Intracellular Nrf-2 levels were determined by colorimetric cell-based ELISA kit (LSBio, LifeSpan Biosciences; Seattle, WA, USA).

Techniques: Incubation, In-Cell ELISA

Journal: Scientific reports

Article Title: Daeshiho-tang attenuates inflammatory response and oxidative stress in LPS-stimulated macrophages by regulating TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways.

doi: 10.1038/s41598-023-46033-y

Figure Lengend Snippet: Figure 8. Effect of DSHT on the expression of Nrf2/HO-1 pathway in LPS-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentration of DSHT (100 and 400 μg/mL) and stimulated with LPS (1 μg/mL) for 18 h. Data are presented as mean ± SD (n = 3). ###p < 0.001 vs. control; *p < 0.05 vs. LPS.

Article Snippet: The following materials were purchased for the study: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin and streptomycin (P/S) from Gibco BRL (Grand Island, NY, USA); LPS (Escherichia coli; O111:B4); L-NMMA, indomethacin and radioimmunoprecipitation assay lysis buffer (RIPA) from Sigma-Aldrich (St. Louis, MO, USA); CCK-8 from Dojindo (Kumamoto, Japan); Griess reagent from Promega (Madison, WI, USA); 2,2′-Azino-bis-3-ethyl benzothiozoline-6-sulfonic acid (ABTS)-based antioxidant kit and PGE2 enzyme-linked immunosorbent assay (ELISA) detection kit from Cayman (Ann Arbor, MI, USA); Primary antibodies from Cell Signaling Technology (Beverly, MA, USA) against iNOS (#13120), COX-2, NF-κB p65, IκBα, phospho-p38 (Thr180/Tyr182), p38, phospho-ERK (Thr202/Tyr204), ERK, phospho-JNK (Thr183/ Tyr185), JNK, β-actin, GAPDH, Lamin B1, MyD88, Nrf2, and HO-1, and TLR4 from Santa Cruz Biotechnology (Dallas, TX, USA); horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit and mouse from Jackson ImmunoResearch (West Grove, PA, USA); Halt protease & phosphatase inhibitor cocktail and NE-PER nuclear and cytoplasmic extraction reagents from Thermo Fisher Scientific (Rockford, IL, USA); ECL chemiluminescence from GE Healthcare (Chicago, IL, USA).

Techniques: Expressing, Concentration Assay, Control

Journal: Scientific reports

Article Title: Daeshiho-tang attenuates inflammatory response and oxidative stress in LPS-stimulated macrophages by regulating TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways.

doi: 10.1038/s41598-023-46033-y

Figure Lengend Snippet: Figure 6. Effect of DSHT on TLR4/MyD88 pathways in LPS-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentration of DSHT (100 and 400 μg/mL) and stimulated with LPS (1 μg/mL) for 8 h. Data are presented as mean ± SD (n = 3). ###p < 0.001 vs. control; *p < 0.05, **p < 0.01, and ***p < 0.001 vs. LPS.

Article Snippet: The following materials were purchased for the study: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin and streptomycin (P/S) from Gibco BRL (Grand Island, NY, USA); LPS (Escherichia coli; O111:B4); L-NMMA, indomethacin and radioimmunoprecipitation assay lysis buffer (RIPA) from Sigma-Aldrich (St. Louis, MO, USA); CCK-8 from Dojindo (Kumamoto, Japan); Griess reagent from Promega (Madison, WI, USA); 2,2′-Azino-bis-3-ethyl benzothiozoline-6-sulfonic acid (ABTS)-based antioxidant kit and PGE2 enzyme-linked immunosorbent assay (ELISA) detection kit from Cayman (Ann Arbor, MI, USA); Primary antibodies from Cell Signaling Technology (Beverly, MA, USA) against iNOS (#13120), COX-2, NF-κB p65, IκBα, phospho-p38 (Thr180/Tyr182), p38, phospho-ERK (Thr202/Tyr204), ERK, phospho-JNK (Thr183/ Tyr185), JNK, β-actin, GAPDH, Lamin B1, MyD88, Nrf2, and HO-1, and TLR4 from Santa Cruz Biotechnology (Dallas, TX, USA); horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit and mouse from Jackson ImmunoResearch (West Grove, PA, USA); Halt protease & phosphatase inhibitor cocktail and NE-PER nuclear and cytoplasmic extraction reagents from Thermo Fisher Scientific (Rockford, IL, USA); ECL chemiluminescence from GE Healthcare (Chicago, IL, USA).

Techniques: Concentration Assay, Control

Journal: Scientific reports

Article Title: Daeshiho-tang attenuates inflammatory response and oxidative stress in LPS-stimulated macrophages by regulating TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways.

doi: 10.1038/s41598-023-46033-y

Figure Lengend Snippet: Figure 5. Effect of DSHT on the phosphorylation of p38 (A), ERK (B), and JNK (C) pathways in LPS- stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentration of DSHT (100 and 400 μg/mL) and stimulated with LPS (1 μg/mL) for 30 min. Data are presented as mean ± SD (n = 3). ##p < 0.01 and ###p < 0.001 vs. control; *p < 0.05 and ***p < 0.001 vs. LPS.

Article Snippet: The following materials were purchased for the study: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin and streptomycin (P/S) from Gibco BRL (Grand Island, NY, USA); LPS (Escherichia coli; O111:B4); L-NMMA, indomethacin and radioimmunoprecipitation assay lysis buffer (RIPA) from Sigma-Aldrich (St. Louis, MO, USA); CCK-8 from Dojindo (Kumamoto, Japan); Griess reagent from Promega (Madison, WI, USA); 2,2′-Azino-bis-3-ethyl benzothiozoline-6-sulfonic acid (ABTS)-based antioxidant kit and PGE2 enzyme-linked immunosorbent assay (ELISA) detection kit from Cayman (Ann Arbor, MI, USA); Primary antibodies from Cell Signaling Technology (Beverly, MA, USA) against iNOS (#13120), COX-2, NF-κB p65, IκBα, phospho-p38 (Thr180/Tyr182), p38, phospho-ERK (Thr202/Tyr204), ERK, phospho-JNK (Thr183/ Tyr185), JNK, β-actin, GAPDH, Lamin B1, MyD88, Nrf2, and HO-1, and TLR4 from Santa Cruz Biotechnology (Dallas, TX, USA); horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit and mouse from Jackson ImmunoResearch (West Grove, PA, USA); Halt protease & phosphatase inhibitor cocktail and NE-PER nuclear and cytoplasmic extraction reagents from Thermo Fisher Scientific (Rockford, IL, USA); ECL chemiluminescence from GE Healthcare (Chicago, IL, USA).

Techniques: Phospho-proteomics, Concentration Assay, Control

Journal: Scientific reports

Article Title: Daeshiho-tang attenuates inflammatory response and oxidative stress in LPS-stimulated macrophages by regulating TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways.

doi: 10.1038/s41598-023-46033-y

Figure Lengend Snippet: Figure 6. Effect of DSHT on TLR4/MyD88 pathways in LPS-stimulated RAW 264.7 macrophages. Cells were treated with the indicated concentration of DSHT (100 and 400 μg/mL) and stimulated with LPS (1 μg/mL) for 8 h. Data are presented as mean ± SD (n = 3). ###p < 0.001 vs. control; *p < 0.05, **p < 0.01, and ***p < 0.001 vs. LPS.

Article Snippet: The following materials were purchased for the study: Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin and streptomycin (P/S) from Gibco BRL (Grand Island, NY, USA); LPS (Escherichia coli; O111:B4); L-NMMA, indomethacin and radioimmunoprecipitation assay lysis buffer (RIPA) from Sigma-Aldrich (St. Louis, MO, USA); CCK-8 from Dojindo (Kumamoto, Japan); Griess reagent from Promega (Madison, WI, USA); 2,2′-Azino-bis-3-ethyl benzothiozoline-6-sulfonic acid (ABTS)-based antioxidant kit and PGE2 enzyme-linked immunosorbent assay (ELISA) detection kit from Cayman (Ann Arbor, MI, USA); Primary antibodies from Cell Signaling Technology (Beverly, MA, USA) against iNOS (#13120), COX-2, NF-κB p65, IκBα, phospho-p38 (Thr180/Tyr182), p38, phospho-ERK (Thr202/Tyr204), ERK, phospho-JNK (Thr183/ Tyr185), JNK, β-actin, GAPDH, Lamin B1, MyD88, Nrf2, and HO-1, and TLR4 from Santa Cruz Biotechnology (Dallas, TX, USA); horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit and mouse from Jackson ImmunoResearch (West Grove, PA, USA); Halt protease & phosphatase inhibitor cocktail and NE-PER nuclear and cytoplasmic extraction reagents from Thermo Fisher Scientific (Rockford, IL, USA); ECL chemiluminescence from GE Healthcare (Chicago, IL, USA).

Techniques: Concentration Assay, Control