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TUS inhibited the activation of NLRP3 inflammasome by activating Nrf2. (A) TUS at 2.5, 5, and 10 μM inhibited the activation of NLRP3 inflammasome. J774 A.1 cells were treated with 2.5, 5, and 10 μM TUS and induced by 200 ng/μL LPS and 3 μM nigericin, and the protein levels of IL-1β, Caspase-1, NLRP3, pro-caspase-1, and pro-IL-1β were detected. (B) TUS at 20 μM inhibited the activation of NLRP3 inflammasome induced by nigericin, MSU, and ATP. J774 A.1 cells were treated with 20 μM TUS and induced by 200 ng/μL LPS, nigericin, MSU, and ATP, and the protein levels of IL-1β, Caspase-1, NLRP3, pro-caspase-1, and pro-IL-1β were detected. (C) TUS inhibited the production and secretion of IL-1β. J774 A.1 cells were treated with 5, 10, and 20 μM TUS and induced by 200 ng/μL LPS and 3 μM nigericin, and the IL-1β levels were detected with ELISA kit. (D) TUS inhibited the protein–protein interaction b4etween NLRP3, ASC, and NEK7. (E) TUS inhibited ASC oligomerization. (F) The ROS scavenger NAC inhibited NLRP3 inflammasome activation. (G) The effect of TUS on NLRP3 inflammasome activation in WT and Nrf2-silenced J774 A.1 cells. (H) The effect of TUS on NLRP3 inflammasome activation in WT and C434A mutant J774 A.1 cells. The results were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs LPS + nigericin group.

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 the activation of NLRP3 inflammasome by activating Nrf2. (A) TUS at 2.5, 5, and 10 μM inhibited the activation of NLRP3 inflammasome. J774 A.1 cells were treated with 2.5, 5, and 10 μM TUS and induced by 200 ng/μL LPS and 3 μM nigericin, and the protein levels of IL-1β, Caspase-1, NLRP3, pro-caspase-1, and pro-IL-1β were detected. (B) TUS at 20 μM inhibited the activation of NLRP3 inflammasome induced by nigericin, MSU, and ATP. J774 A.1 cells were treated with 20 μM TUS and induced by 200 ng/μL LPS, nigericin, MSU, and ATP, and the protein levels of IL-1β, Caspase-1, NLRP3, pro-caspase-1, and pro-IL-1β were detected. (C) TUS inhibited the production and secretion of IL-1β. J774 A.1 cells were treated with 5, 10, and 20 μM TUS and induced by 200 ng/μL LPS and 3 μM nigericin, and the IL-1β levels were detected with ELISA kit. (D) TUS inhibited the protein–protein interaction b4etween NLRP3, ASC, and NEK7. (E) TUS inhibited ASC oligomerization. (F) The ROS scavenger NAC inhibited NLRP3 inflammasome activation. (G) The effect of TUS on NLRP3 inflammasome activation in WT and Nrf2-silenced J774 A.1 cells. (H) The effect of TUS on NLRP3 inflammasome activation in WT and C434A mutant J774 A.1 cells. The results were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs LPS + nigericin 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: Activation Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis