Journal: Journal of Advanced Research

Article Title: Tussilagone attenuated cigarette smoke-induced chronic obstructive pulmonary disease through regulating Nrf2 and NF-κB/NLRP3 inflammasome via directly targeting cysteine 434 of KEAP1

doi: 10.1016/j.jare.2025.07.019

Figure Lengend Snippet: Discovery of TUS as a potent therapeutic agent for COPD. (A) High-throughput screening for CXCL1 inhibitory activity of compounds from FF. RAW 264.7 macrophages were treated with 1 μg/mL LPS and 20 μM diverse compounds from FF for 24 h, and the level of CXCL1 was detected with ELISA kit. (B) Structures of sesquiterpenoids from FF. (C) The H&E or Masson’s trichrome staining images of lung tissues of mice in different groups. (D) The protein levels of iNOS, COX-2, and IL-1β in the homogenate of lung tissues of mice in different groups. (E-F) The levels of MUC5AC and SP-D in the homogenate of lung tissues of mice in different groups. (G) The percentage of neutrophil in white blood cells of mice in different groups. (H-I) The levels of TNF-α and IL-1β in BALF of mice in different groups. The results were expressed as mean ± SD (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs CS group.

Article Snippet: The primary antibodies for KEAP1 (10503-2-AP, 1:5000), Nrf2 (16396-1-AP, 1:2000), glutamate-cystine ligase, modifier subunit (GCLM, 14241-1-AP, 1:2000), cyclooxygenase-2 (COX-2, 27308-1-AP, 1:1000), IL-1β (16806-1-AP, 1:500), β-actin (20536-1-AP, 1:30000), inducible Nitric Oxide Synthase (iNOS, 22226-1-AP, 1:2000), HA (51064-2-AP, 1:2000), inhibitor of NF-κB (IκB, 10268-1-AP, 1:2000), Caspase-1 (22915-1-AP, 1:2000), NLRP3 (27458-1-AP, 1:2000), α-tubulin (11224-1-AP, 1:10000), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (66031-1-Ig, 1:10000) and HRP-conjugated goat anti-rabbit IgG (66031-2-Ig, 1:10000) were purchased from Proteintech Group (Wuhan, China).

Techniques: High Throughput Screening Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Journal of Advanced Research

Article Title: Tussilagone attenuated cigarette smoke-induced chronic obstructive pulmonary disease through regulating Nrf2 and NF-κB/NLRP3 inflammasome via directly targeting cysteine 434 of KEAP1

doi: 10.1016/j.jare.2025.07.019

Figure Lengend Snippet: TUS inhibited NF-κB by activating Nrf2. (A) The protein levels of p -NF-κB, IκB, iNOS, and COX-2 in the RAW 264.7 cells treated with 1 μg/mL LPS and TUS at 2.5, 5, 10, and 20 μM. (B) The regulation of TUS on p -NF-κB levels in the cytoplasm and nucleus. RAW 264.7 cells treated with 1 μg/mL LPS and TUS at 10 and 20 μM for 8 h, and the protein level of p -NF-κB in the cytoplasm and nucleus were detected using Western blot. (C) Immunofluorescence indicated that TUS inhibited p -NF-κB translocation into the nucleus. RAW 264.7 cells were treated with 1 μg/mL LPS and TUS at 10 and 20 μM for 8 h, followed by incubation with p -NF-κB primary antibody and Alex 594 secondary antibody. The immunofluorescence of p -NF-κB was observed and imaged. (D-E) The regulatory effects of TUS on protein levels of p -NF-κB, IκB, iNOS, and COX-2 in the WT and Nrf2-silenced RAW 264.7 cells. WT and Nrf2-silenced RAW 264.7 cells were treated with 10 μM TUS, and the protein levels of p -NF-κB, iNOS, and COX-2 were detected using Western blot. (F) The regulatory effects of TUS on protein levels of iNOS and COX-2 in the WT or C434A mutant RAW 264.7 cells. WT and C434A mutant RAW 264.7 cells were treated with 10 μM TUS, and the protein levels of p -NF-κB, iNOS, and COX-2 were detected using Western blot. The results were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs LPS group.

Article Snippet: The primary antibodies for KEAP1 (10503-2-AP, 1:5000), Nrf2 (16396-1-AP, 1:2000), glutamate-cystine ligase, modifier subunit (GCLM, 14241-1-AP, 1:2000), cyclooxygenase-2 (COX-2, 27308-1-AP, 1:1000), IL-1β (16806-1-AP, 1:500), β-actin (20536-1-AP, 1:30000), inducible Nitric Oxide Synthase (iNOS, 22226-1-AP, 1:2000), HA (51064-2-AP, 1:2000), inhibitor of NF-κB (IκB, 10268-1-AP, 1:2000), Caspase-1 (22915-1-AP, 1:2000), NLRP3 (27458-1-AP, 1:2000), α-tubulin (11224-1-AP, 1:10000), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (66031-1-Ig, 1:10000) and HRP-conjugated goat anti-rabbit IgG (66031-2-Ig, 1:10000) were purchased from Proteintech Group (Wuhan, China).

Techniques: Western Blot, Immunofluorescence, Translocation Assay, Incubation, Mutagenesis

Journal: Journal of Advanced Research

Article Title: Tussilagone attenuated cigarette smoke-induced chronic obstructive pulmonary disease through regulating Nrf2 and NF-κB/NLRP3 inflammasome via directly targeting cysteine 434 of KEAP1

doi: 10.1016/j.jare.2025.07.019

Figure Lengend Snippet: TUS inhibited lung inflammation by activating Nrf2. (A) The images of lung tissues stained by H&E in indicated groups. (B-E) The levels of TNF-α and IL-1β in BALF of different groups. (F) The protein levels of Nrf2, iNOS, p -NF-κB, and IL-1β in the homogenate of lung tissues in indicated groups. (G) The mRNA levels of NF-κB, iNOS, NLRP3, COX-2, TNF-α, and IL-1β in the homogenate of lung tissues in indicated groups. The results were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs LPS group.

Article Snippet: The primary antibodies for KEAP1 (10503-2-AP, 1:5000), Nrf2 (16396-1-AP, 1:2000), glutamate-cystine ligase, modifier subunit (GCLM, 14241-1-AP, 1:2000), cyclooxygenase-2 (COX-2, 27308-1-AP, 1:1000), IL-1β (16806-1-AP, 1:500), β-actin (20536-1-AP, 1:30000), inducible Nitric Oxide Synthase (iNOS, 22226-1-AP, 1:2000), HA (51064-2-AP, 1:2000), inhibitor of NF-κB (IκB, 10268-1-AP, 1:2000), Caspase-1 (22915-1-AP, 1:2000), NLRP3 (27458-1-AP, 1:2000), α-tubulin (11224-1-AP, 1:10000), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (66031-1-Ig, 1:10000) and HRP-conjugated goat anti-rabbit IgG (66031-2-Ig, 1:10000) were purchased from Proteintech Group (Wuhan, China).

Techniques: Staining