Journal: Journal of ethnopharmacology

Article Title: Artichoke water extract protects against Lead-induced hepatotoxicity by activating Nrf2 signaling and inhibiting NLRP3/caspase-1/GSDMD-mediated pyroptosis.

doi: 10.1016/j.jep.2025.119654

Figure Lengend Snippet: Fig. 6. Effects of AWE on Pb induced hepatic inflammation in mice (A-C) Liver relative mRNA expression of IL-1β (A), IL-6 (B), and TNF-α (C) was quantified by qPCR in each group. mRNA levels of target genes were normalized to GAPDH. (D–F) Serum protein levels of IL-1β (D), IL-6 (E), and TNF-α (F) were determined in each group. (G) Representative Western blot images of NLRP3, Caspase- 1, GSDMD, and GAPDH of the liver sample from each group. (H–J) Quantitative analysis of NLRP3 (H), Caspase-1 (I), and GSDMD (J). GAPDH is used as a loading control for protein normalization. The data are expressed as mean ± SD. n = (3–6) per group. **P < 0.01 compared with the blank group; #P < 0.05, ##P < 0.01 compared with the Pb group. $P < 0.05, $$P < 0.01 compared with the Pb + AWE (0.8) group.

Article Snippet: The primary antibodies against NLRP3 (HY-P80766), Nrf2 (HYP81051), SOD2 (HY-P80329), and GPX4 (HY-P80450) were purchased from MedChemExpress (South Brunswick, NJ, USA).

Techniques: Expressing, Western Blot, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium

doi: 10.1167/iovs.63.8.15

Figure Lengend Snippet: Gene-Specific Primers Used in the qPCR

Article Snippet: To determine the effects of NLRP3 and IL-1β on the development of corneal fibrosis, the model mice received sub-conjunctival injections of 5 μL NLRP3 inhibitor (MCC950, 10 mg/mL; MCE, Houston, TX), IL-1β neutralizing antibody (a-IL-1β antibody, 100 ng/mL; R & D Systems, Minneapolis, MN), IL-1R antagonist (anakinra, 10 mg/mL; MCE), or PBS (control) on days 0, 3, and 6 after injury.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium

doi: 10.1167/iovs.63.8.15

Figure Lengend Snippet: Corneal scarring is associated with the activation of the NLRP3. Corneal scarring model was established by mechanical-injury in WT mice. The corneas were gathered on days 1, 3, 5, and 7 after injury. ( A , Representative images of injured cornea under bright-field microscopic (top) and corneal epithelial defect area with fluorescein sodium staining (bottom). ( B , The quantification of corneal scarring ( n = 10). ( C , The quantification of corneal epithelial wound healing rate ( n = 10). ( D , The mRNA levels of fibrosis markers (α-SMA, collagen I, and fibronectin) in the corneal tissue of an established corneal scarring model. ( E , The mRNA levels of NLRP3-associated inflammatory markers (NLRP3, ASC, caspase1, IL-1β, and IL-18) in the corneal tissue of an established corneal scarring model. ( F , G ) Western blot and statistical analysis for the protein expression of NLRP3, caspase1, ASC, pro-caspase1, c-caspase1, pro-IL-1β, c-IL-β, and α-SMA in corneas. All experiments were repeated three times independently. Data are presented as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001. ASC, apoptosis-associated speck-like protein containing a CARD; c-caspase1, cleaved caspase 1; C-IL-β, cleaved IL-1 beta.

Article Snippet: To determine the effects of NLRP3 and IL-1β on the development of corneal fibrosis, the model mice received sub-conjunctival injections of 5 μL NLRP3 inhibitor (MCC950, 10 mg/mL; MCE, Houston, TX), IL-1β neutralizing antibody (a-IL-1β antibody, 100 ng/mL; R & D Systems, Minneapolis, MN), IL-1R antagonist (anakinra, 10 mg/mL; MCE), or PBS (control) on days 0, 3, and 6 after injury.

Techniques: Activation Assay, Staining, Western Blot, Expressing, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium

doi: 10.1167/iovs.63.8.15

Figure Lengend Snippet: Small inhibitor MCC950 inhibits corneal scar formation. Established corneal scarring models were caused by mechanical injury in WT mice. MCC950 (NLRP3 inhibitor) or PBS (control group) was treated in mice via subconjunctival injection. ( A ) Bright field microscopy of corneas on days 3, 5, and 7 after injury. ( B ) The score of corneal scarring was rated on a scale ( n = 10). ( C ) Relative mRNA level of fibrotic associated genes (α-SMA, collagen I) in corneal tissue on day 7 after injury. ( D ) Representative fluorescence staining images and relative protein level of α-SMA and in corneas on day 7 after injury. Scale bar, 50 µm. All experiments were repeated three times independently. Data are presented as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To determine the effects of NLRP3 and IL-1β on the development of corneal fibrosis, the model mice received sub-conjunctival injections of 5 μL NLRP3 inhibitor (MCC950, 10 mg/mL; MCE, Houston, TX), IL-1β neutralizing antibody (a-IL-1β antibody, 100 ng/mL; R & D Systems, Minneapolis, MN), IL-1R antagonist (anakinra, 10 mg/mL; MCE), or PBS (control) on days 0, 3, and 6 after injury.

Techniques: Control, Injection, Microscopy, Fluorescence, Staining, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium

doi: 10.1167/iovs.63.8.15

Figure Lengend Snippet: The NLRP3 KO reduces corneal scarring. Mechanical corneal injury was established in NLRP3 − / − mice and NLRP3 loxp mice. ( A ) Macroscopic images of corneas under bright field microscopy on days 3, 5, and 7 after wounding. ( B ) The quantitative analysis of corneal scarring ( n = 10). ( C ) A qRT-PCR analysis for the mRNA expression of fibrosis genes (α-SMA, collagen I) in corneal tissue on day 7 after injury. ( D ) Representative images of fluorescence staining targeting α-SMA in corneas on day 7 after injury. Scale bar, 50 µm. ( E ) The protein level of α-SMA in corneas on day 7 after injury. All experiments were repeated three times independently. Data are presented as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To determine the effects of NLRP3 and IL-1β on the development of corneal fibrosis, the model mice received sub-conjunctival injections of 5 μL NLRP3 inhibitor (MCC950, 10 mg/mL; MCE, Houston, TX), IL-1β neutralizing antibody (a-IL-1β antibody, 100 ng/mL; R & D Systems, Minneapolis, MN), IL-1R antagonist (anakinra, 10 mg/mL; MCE), or PBS (control) on days 0, 3, and 6 after injury.

Techniques: Microscopy, Quantitative RT-PCR, Expressing, Fluorescence, Staining, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium

doi: 10.1167/iovs.63.8.15

Figure Lengend Snippet: Macrophage infiltration is associated with corneal scar formation. Mechanical injury induced corneal scarring in WT mice and the corneas were removed at different time points. ( A ) Gating strategy for single cells (irregular quadrilateral), blood leukocytes (CD45 + , rectangle), neutrophils (CD45 + /Ly6G + , rectangle), macrophages (CD45 + /F4/80 + , rectangle). ( B , D ) The number of blood cells, neutrophils and macrophages in the normal corneas and corneas on days 1, 3, 5, and 7 after injury ( n = 6). ( E ) Immunofluorescence staining displayed the colocalization of F4/80 with NLRP3 (top) or IL-1β (bottom) in corneas on day 7 after injury. Scale bar, 50 µm. All experiments were repeated three times independently. Data are presented as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To determine the effects of NLRP3 and IL-1β on the development of corneal fibrosis, the model mice received sub-conjunctival injections of 5 μL NLRP3 inhibitor (MCC950, 10 mg/mL; MCE, Houston, TX), IL-1β neutralizing antibody (a-IL-1β antibody, 100 ng/mL; R & D Systems, Minneapolis, MN), IL-1R antagonist (anakinra, 10 mg/mL; MCE), or PBS (control) on days 0, 3, and 6 after injury.

Techniques: Immunofluorescence, Staining, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium

doi: 10.1167/iovs.63.8.15

Figure Lengend Snippet: Macrophage-specific NLRP3 KO reduces corneal scarring. Mechanical corneal injury was established in NLRP3 Lyz-KO ( LyzCre + NLRP3 loxp/loxp ) mice and Lyz-Cre mice. (A) The photograph of corneas under bright field microscope of corneas on days 3, 5, and 7 after injury. ( B ) The quantitative analysis of corneal scarring ( n = 10). ( C ) Representative fluorescence staining images showing α-SMA expression in corneas on day 7 after injury. Scale bar, 50 µm. ( D ) The relative protein level of α-SMA in corneas on day 7 after injury. All experiments were repeated three times independently. Data are presented as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To determine the effects of NLRP3 and IL-1β on the development of corneal fibrosis, the model mice received sub-conjunctival injections of 5 μL NLRP3 inhibitor (MCC950, 10 mg/mL; MCE, Houston, TX), IL-1β neutralizing antibody (a-IL-1β antibody, 100 ng/mL; R & D Systems, Minneapolis, MN), IL-1R antagonist (anakinra, 10 mg/mL; MCE), or PBS (control) on days 0, 3, and 6 after injury.

Techniques: Microscopy, Fluorescence, Staining, Expressing, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium

doi: 10.1167/iovs.63.8.15

Figure Lengend Snippet: The NLRP3 KO blocks TGF-β1 synthesis in corneal epithelial cells. In vivo, mechanical corneal injury was established in NLRP3 loxp (control group), NLRP3 − / − and NLRP3 Lyz-KO mice respectively. In vitro, peritoneal macrophage extracted from WT mice and NLRP3 − / – mice. Macrophages were treated with or without LPS and Nigercin (classic NLRP3 inflammasome inducer). Cell precipitation and culture supernatant was collected. Cell supernatant from peritoneal macrophage was cocultured with human corneal epithelial cell (HCECs). ( A ) Representative images of immunofluorescent staining targeting TGF-β1 in corneal tissue post injury. Scale bar, 50 µm. ( B , C ) Western blotting analysis comparing protein levels of TGF-β1 between unwound corneas and injured corneas on day 7. ( D ) The protein level of NLRP3 in peritoneal macrophage with or without stimulation. ( E ) Quantitative assay detecting release of IL-1β in cellular supernatant after stimulation with PBS (control group) or LPS with Nigercin (LPS + Nigercin). ( F ) qRT-PCR assay showing TGF-β1 mRNA level in HCECs with or without the macrophage (derived from WT mice and NLRP3 − / − mice) supernatants. ( G ) The mRNA level of TGF-β1 in HCECs with or without macrophage (derived from WT mice) supernatants. Cell supernatants were exposed to LPS + Nigercin or LPS + Nigercin +_α-IL-1β antibody (IL-1β neutralizing antibody). All experiments were repeated three times independently. Data are presented as the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To determine the effects of NLRP3 and IL-1β on the development of corneal fibrosis, the model mice received sub-conjunctival injections of 5 μL NLRP3 inhibitor (MCC950, 10 mg/mL; MCE, Houston, TX), IL-1β neutralizing antibody (a-IL-1β antibody, 100 ng/mL; R & D Systems, Minneapolis, MN), IL-1R antagonist (anakinra, 10 mg/mL; MCE), or PBS (control) on days 0, 3, and 6 after injury.

Techniques: In Vivo, Control, In Vitro, Staining, Western Blot, Quantitative RT-PCR, Derivative Assay, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-β1 Expression in the Corneal Epithelium

doi: 10.1167/iovs.63.8.15

Figure Lengend Snippet: Schematic diagram showing the NLRP3 activation in infiltrating macrophages contributes to corneal fibrosis by inducing TGF-β1 expression in the corneal epithelium.

Article Snippet: To determine the effects of NLRP3 and IL-1β on the development of corneal fibrosis, the model mice received sub-conjunctival injections of 5 μL NLRP3 inhibitor (MCC950, 10 mg/mL; MCE, Houston, TX), IL-1β neutralizing antibody (a-IL-1β antibody, 100 ng/mL; R & D Systems, Minneapolis, MN), IL-1R antagonist (anakinra, 10 mg/mL; MCE), or PBS (control) on days 0, 3, and 6 after injury.

Techniques: Activation Assay, Expressing

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: Identification of CPX as a potential inhibitor of NLRP3 Inflammasome. (A) Screening workflow of 190 FDA‐approved drugs. The THP‐1 cells (pretreated with 200 ng/mL PMA overnight) were primed with LPS, and incubated with drugs (10 µ m ) for 1 h prior to nigericin stimulation. Then, IL‐1β secretion was quantified by ELISA. (B) IL‐1β inhibition rate in culture supernatants (Sup) from THP‐1 cells. (C) The chemical structure of CPX. (D) IC 50 of CPX on the NLRP3 inflammasome‐dependent IL‐1β secretion (0–80 µ m ). (E,F) LPS‐primed THP‐1 cells were treated with various doses of CPX or DMSO for 1 h and then stimulated with nigericin. (E) ELISA of IL‐1β in Sup from THP‐1 cells. (F) Immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup, and NLRP3, pro‐IL‐1β, pro‐caspase‐1, and ASC in lysates (Lys) from THP‐1 cells. (G) The cytotoxicity of CPX on THP‐1 cells was measured using the CCK‐8 kit. Data from three independent experiments (E. Values are mean ± SD) or are representative of three independent experiments(F). Statistical analyses were carried out via one‐way ANOVA for (E), ** p < 0.01, *** p < 0.001.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Inhibition, Western Blot, CCK-8 Assay

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX blocks NLRP3 inflammasome activation. (A,B) THP‐1 cells were first primed with LPS (3 h) and then treated with or without 20 µ m CPX for 1 h, followed by stimulation with ATP for 40 min, nigericin for 1 h, or MSU for 6 h. ELISA of IL‐1β in Sup (A), or immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup. (C,D) LPS‐primed BMDMs were treated with 20 µ m CPX and then stimulated with ATP for 40 min, nigericin for 1 h, or MSU for 6 h. ELISA of IL‐1β in Sup (C), or immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup (D). (E‐F) THP‐1 cells were primed with LPS (3 h) and then treated with or without 20 µ m CPX for 1 h, followed by stimulation with Alum for 6 h, imiquimod for 3 h, or SiO2 for 6 h. ELISA of IL‐1β in Sup (E), or immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup(F). (G,H) THP‐1 cells were treated and primed with LPS (3 h) and then treated with or without 20 µ m CPX for 1 h, followed by stimulation with poly(dA:dT) for 4 h, FLA‐ST for 4 h, or TcdB for 1 h. ELISA of IL‐1β in Sup (G) or immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup (H). (I) Representative microscopic images of LPS‐primed THP‐1 cells upon nigericin stimulation with or without CPX pre‐treatment (red arrows mark pyroptotic cells). Scale bars correspond to 100 µm. (J) PI (red fluorescence) and Hoechst (blue fluorescence) staining of THP‐1 cells revealed the dead cells. Scale bars correspond to 100 µm. (K) The release of LDH from LPS‐primed THP‐1 cells, pre‐treated with or without CPX and then stimulated with ATP, nigericin, or MSU. (L) Immunoblot analysis of GSDMD cleavage from THP‐1 cells, primed with LPS before treatment with or without CPX, followed by treatment with nigericin. Data from three independent experiments (A,C,E,G,K. Values are mean ± SD) or are representative of three independent experiments with similar results (B,D,F,H–J). Statistical analyses were carried out via two‐way ANOVA for (A,C,E,G,K), *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, Staining

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX inhibits NLRP3 inflammasome activation in vivo and in patient cells. (A–J) C57BL/6 mice were intraperitoneally injected with vehicle, CPX (10 mg/kg), or CPX (20 mg/kg) for 1 h prior to LPS injection to induce sepsis. (A) Schematic diagram of the LPS‐induced sepsis model. (B) Survival analysis of mice pretreated with vehicle or CPX (20 mg/kg) for 1 h before being intraperitoneally injected with LPS ( n = 10). (C,D) The activity of plasma ALT and AST ( n = 6). (E) Representative images of H&E‐stained liver sections. Scale bars, 50 µm. (F–I) ELISA of IL‐1β and IL‐6 in serum and in Sup from cultured liver tissues ( n = 6). (F) IL‐1β and (G) IL‐6 levels in serum. (H) IL‐1β and (I) IL‐6 levels in liver tissues. (J) Immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in liver tissues. (K–R) C57BL/6 mice were treated with 3% DSS in drinking water for 6 days, followed by 4 days of normal drinking water. CPX or vehicle was administered daily ( n = 6). (K) Schematic diagram of DSS‐induced colitis model. (L) Body weights, (M) the disease activity index (DAI), (N) representative colon images, and (O) colon length were measured. (P) ELISA of IL‐1β in colon tissues. (Q) H&E‐stained colon tissues. (R) Immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in colon tissues. (S,T) PBMCs primed with LPS and treated with 20 µ m CPX for 1 h, and then stimulated with nigericin or MSU. ELISA of IL‐1β in Sup of PBMCs isolated from healthy individuals (S) or gout patients (T). Date from three independent experiments (S,T. Values are mean ± SD). Statistical analyses were carried out via two‐way ANOVA for (B–D, F–L, L,M, O,P, S,T), * p < 0.05, ** p < 0.01, *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Activation Assay, In Vivo, Injection, Activity Assay, Clinical Proteomics, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Isolation

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX inhibits NLRP3 inflammasome assembly. (A) ELISA of TNF‐α in Sup from THP‐1 cells treated with various doses of CPX and then stimulated with 500 ng/mL LPS for 3 h. (B) ELISA of TNF‐α and (C) immunoblot analysis of NLRP3, pro‐caspase‐1, pro‐IL‐1β, and ASC in Lys from THP‐1 cells treated with 20 µ m CPX before or after stimulation with 500 ng/mL LPS for 3 h. (D,E) Qualification of potassium and chloride ions in LPS‐primed THP‐1 cells pretreated with or without 20 µ m CPX and then stimulated with nigericin. (F,G) Qualification of mtROS in THP‐1 cells was primed with LPS, pre‐treated with or without CPX, and subsequently stimulated with nigericin. (H) Immunoblot analysis of ASC oligomerization. (I) Representative images of ASC specks detected by immunofluorescence. DAPI shows nuclei, and white arrows show an example of ASC specks formed. Scale bar corresponds to 50 µm. (J) Percentage of ASC speck‐positive cells. (K) IP and immunoblot analysis to evaluate the NLRP3‐ASC interaction in LPS‐primed THP‐1 cells pretreated with CPX and then stimulated with nigericin. (L) IP and immunoblot analysis of HEK‐293T cells transfected with FLAG‐NLRP3, HIS‐ASC plasmids as indicated and treated with CPX. (M) IP and immunoblot analysis to evaluate the NLRP3‐NEK7 interaction in LPS‐primed THP‐1 cells pretreated with CPX and then stimulated with nigericin. (N) IP and immunoblot analysis of HEK‐293T cells transfected with FLAG‐NLRP3, MYC‐NEK7 plasmids as indicated, and treated with the CPX Data from three independent experiments(A,B,D,E,G,J. Values are mean ± SD) or are representative of three independent experiments with similar results(F,H,I,K–N). Statistical analyses were carried out via two‐way ANOVA for (A,B,D,E,G,J), *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Transfection

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX directly targets NLRP3 and inhibits its oligomerization. (A) The schematic shows the steps for identifying the target of CPX in THP‐1 cells using drug affinity responsive target stability (DARTS) and mass spectrometry analysis. (B) Coomassie blue staining of DARTS assay using THP‐1 cell lysates treated with or without CPX. (C) The target protein of CPX was identified by mass spectrogram. (D) DARTS in THP‐1 cells incubation with CPX or DMSO. (E) DARTS in HEK‐293T cells transfected with the FLAG‐NLRP3 plasmid under incubation with CPX or DMSO. (F) CESTA of NLRP3 in THP‐1 cells incubated with CPX or DMSO. (G) The melting curve generated from CETSA. (H) The oligomerization of NLRP3 in THP‐1 cells treated with CPX was analyzed by native‐PAGE. (I) NLRP3 oligomerization in HEK‐293T cells expressing FLAG‐NLRP3 plasmids and treated with CPX. (J) IP and immunoblot analysis of HEK‐293T cells transfected with FLAG‐NLRP3 and MYC‐NLRP3 plasmids as indicated and treated with CPX. (K) ELISA of IL‐1β in Sup and immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup from WT and NLRP3 −/− BMDMs were first primed with LPS and then treated with CPX, followed by stimulation with nigericin. (L) ELISA of IL‐1β in Sup and immunoblot analysis in Lys from HEK‐293T cells transfected with HA‐IL‐1β, HA‐pro‐caspase‐1, HIS‐ASC, with or without FLAG‐NLRP3 plasmids as indicated and treated with CPX. Data from three independent experiments (G,K,L. Values are mean ± SD) or are representative of three independent experiments with similar results (D,F,H–L). Statistical analyses were carried out via two‐way ANOVA for (K,L), ** p < 0.01, *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Mass Spectrometry, Staining, Incubation, Transfection, Plasmid Preparation, Generated, Clear Native PAGE, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: CPX binds to tyrosine 381 of the NACHT domain. (A,B) LPS‐primed THP‐1 cells were treated with CPX for 30 min and washed out, then stimulated with nigericin. (A) ELISA of IL‐1β in Sup. (B) Immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup. (C) Schematic of the protein domains of NLRP3. (D–F) DARTS in HEK293T transfected with (D) FLAG‐NACHT domain, (E) FLAG‐PYD domain, and (F) FLAG‐LRR domain plasmids under incubation with CPX or DMSO. (G,H) The binding affinity of CPX to the NLRP3‐NACHT domain was measured by SPR assay. (I) Molecular docking analysis of CPX bound to NLRP3‐NACHT domain. CPX (yellow, stick model) in the binding site of NLRP3‐NACHT domain (lavender, cartoon, and PDB ID: 7ALV). (J) Structural comparison of the complex at five moments (0, 25, 50, 75, 100 ns) during a 100 ns molecular dynamics simulation. (K) Energy contribution of amino acid residues involved in binding. (L) DARTS in HEK293T transfected with R167A NACHT domain or Y381A NACHT domain plasmids under incubation with CPX or DMSO. (M) IP and immunoblot analysis of HEK293T cells transfected with MYC‐NEK7, FLAG‐NLRP3, or Y381A mutant FLAG‐NLRP3 plasmids and treated with 20 µ m CPX. (N) IP and immunoblot analysis of HEK293T cells transfected with HIS‐ASC, FLAG‐NLRP3, or Y381A mutant FLAG‐NLRP3 plasmids and treated with 20 µ m CPX. (O) NLRP3 oligomerization in HEK‐293T cells transfected with FLAG‐NLRP3 or Y381A mutant FLAG‐NLRP3 plasmids and treated with CPX. (R) LPS‐primed NLRP3‐/‐ BMDMs reconstituted with WT or Y381A mutant NLRP3, were treated with CPX and then stimulated with nigericin. ELISA of IL‐1β in Sup, immunoblot analysis of matured‐IL‐1β and cleaved caspase‐1 in Sup. (Q) ATPase activity assay for purified human NLRP3 in the presence of different concentrations of CPX. Data from three independent experiments (A, P, Q. Values are mean ± SD) or are representative of three independent experiments with similar results (B, D–F, L–P). Statistical analyses were carried out via two‐way ANOVA for (A, P, Q), ** p < 0.01, *** p < 0.001, NS not significant.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Incubation, Binding Assay, SPR Assay, Comparison, Mutagenesis, Activity Assay, Purification

Journal: Advanced Science

Article Title: Ciclopirox Olamine Inhibits the NLRP3 Inflammasome to Alleviate Inflammatory Diseases

doi: 10.1002/advs.75704

Figure Lengend Snippet: The mechanism of CPX blocking NLRP3 inflammasome activation.

Article Snippet: Ciclopirox olamine, imiquimod, TcdB toxin, and recombinant human NLRP3 were purchased from MedChemExpress (New Jersey, USA); Phorbol‐12‐myristate‐13‐acetate(PMA), LPS, nigericin, LDH activity assay kit, MitoTracker, MitoSOX, RIPA, human IgG, penicillin/streptomycin, NP40, and protein A+G agarose were supplied by Beyotime (Shanghai, China); MSU, SiO 2 , Alum, poly(dA:dT), pronase, disuccinimidyl suberate, and anti‐MYC were purchased from Sigma (St, Louis, USA); ATP, lipofectamine 2000, and Salmonella typhimurium (FLA‐ST) were supplied by InvivoGen (New Jersey, USA); and the ADP‐Glo Kinase assay kit was purchased from Promega (WI, USA).

Techniques: Blocking Assay, Activation Assay