Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome regulates ASC speck formation during S. aureus . Fluorescence microscopy images of MAC-T cells immunoassayed for ASC (red) with or without MCC950 after 4 h of S. aureus treatment.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Fluorescence, Microscopy

Journal: Veterinary Research

Article Title: Staphylococcus aureus mediates pyroptosis in bovine mammary epithelial cell via activation of NLRP3 inflammasome

doi: 10.1186/s13567-022-01027-y

Figure Lengend Snippet: NLRP3 inflammasome activation by S. aureus is essential for the generation of GSDMD-N and the release of IL-1β and IL-18. A Activated caspase-1 and B GSDMD-N or released C IL-1β and D IL-18 of MAC-T cells treated with or without NLRP3 inhibitor MCC950 or caspase-1 inhibitor VX765 for 1.5 h prior to treatment with S. aureus for 4 h. E Levels of IL-1β and F IL-18 released after S. aureus treatment of MAC-T cells for 4 h in the presence or absence of 25, 50, or 75 mM KCl.

Article Snippet: Caspase-1 inhibitor VX765 and NLRP3 inflammasome inhibitor MCC950 were obtained from Glpbio (USA) and used at final concentrations of 100 and 15 μM.

Techniques: Activation Assay

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Sequences of primers.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques:

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Elevated expression of molecules in TMJ synovium. (a, b) Western blotting results of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD in the sham and OA synovitis rats. (c) mRNA levels of NLRP3, ASC, caspase-1, IL-1 β , and GSDMD expressed in TMJ synovium between the sham and OA rats. (d) Immunohistochemistry of TMJ slices from the sham or OA rats using anti-NLRP3, anti-caspase-1, anti-GSDMD, and anti-IL-1 β antibodies, scale bar = 50 μ m. Positive cells are mainly around synovial membrane and vessels (black arrow). (e) Percentage of positive cells. Data in this figure are analyzed by independent samples t -test and presented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Expressing, Western Blot, Immunohistochemistry, Membrane

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: The biological effect of AYC and MCC950 on inflammasome- and pyroptosis-related molecules. (a, b) Western blotting results showing the protein levels of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD among the sham, OA, and AYC groups. (a, c) Levels of the above proteins among the sham, OA, and MCC950 groups. (d, e) TUNEL assay indicated that positive nuclei were increased markedly in the OA synovial tissue compared with the sham-treated group. Positive cells were reduced in the Ac-YVAD-cmk and MCC950 groups. Data in this figure are presented as mean ± SD and analyzed by parametric test. Statistical significance is determined with one-way ANOVA followed by Bonferroni's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Western Blot, TUNEL Assay

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: LPS+ATP-treated macrophages. (a) Altered morphology of THP-1 cells under PMA stimulation. Scale bar = 50 μ m. (b, c) Western blotting results of NLRP3, ASC, cleaved caspase-1, IL-1 β , and GSDMD changes on protein levels in LPS-treated macrophages. (d) The results of qRT-PCR showed changes on mRNA levels among the control, LPS+ATP, LPS+ATP+MCC950, and LPS+ATP+AYC groups. Data are presented as mean ± SD and analyzed with one-way ANOVA followed by Bonferroni's post hoc test. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Western Blot, Quantitative RT-PCR, Control

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: LPS+ATP stimulated NLRP3 inflammasome- and pyroptosis-related molecules in the cultured FLS cells alone and cocultured system. (a, b) When FLS cells were cultured alone, lysate was collected for western blot after being treated with LPS+ATP. (c) Total RNAs of FLS cells were extracted for detecting mRNAs of NLRP3, ASC, caspase-1, GSDMD, and IL-1 β . Statistical analysis is run by independent samples t -test. (d, e) Secretion of IL-1 β and IL-18 in the supernate increased when FLS cell was cocultured with active THP-1 cell. (f–h) Western blotting and qRT-PCR results of cell lysate indicated the growing expression of NLRP3, ASC, caspase-1, GSDMD, and IL-1 β on protein and mRNA levels. Both the upregulation trend of the above tests after LPS+ATP intervention could be inhibited by MCC950 and AYC in the coculture system. Data are presented as mean ± SD; one-way ANOVA followed by Bonferroni's post hoc test was adopted for analysis. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Cell Culture, Western Blot, Quantitative RT-PCR, Expressing

Journal: Mediators of Inflammation

Article Title: Targeting NLRP3 Inflammasome Alleviates Synovitis by Reducing Pyroptosis in Rats with Experimental Temporomandibular Joint Osteoarthritis

doi: 10.1155/2022/2581151

Figure Lengend Snippet: Detection of pyroptosis in fibroblast-like synovial cells of coculture system. Cocultured FLS cells were challenged with LPS for 12 h and ATP for 2 h. Cell exposed to saline was as the control group. The MCC950 group and Ac-YVAD-cmk group were simultaneously added inhibitors to FLS cells. (a) PI- and Hoechst 33342-stained FLS cells in the control and LPS+ATP groups. (b) Quantification of PI-positive cells. Independent samples t -test. (c) The release of LDH was measured among the control, LPS+ATP, LPS+ATP+MCC950, and LPS+ATP+AYC groups. (d, e) Cells double stained by PI and kFluor488 for flow cytometry. One-way ANOVA followed by Bonferroni's post hoc test was used. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (f) Representative transmission electron micrographs of FLS cells. Compared with control group, FLS cells exhibited pyroptotic features: extensive membrane bubbles and pores (red arrow); chromatin margination and condensation (yellow arrow); and swollen mitochondria with collapsed cristae (blue arrow).

Article Snippet: The NLRP3 inhibitor MCC950 (MedChemExpress, Monmouth, NJ, USA) was diluted to 2 mg/ml with sterile saline and injected 10 mg/kg intraperitoneally.

Techniques: Saline, Control, Staining, Flow Cytometry, Transmission Assay, Membrane

Journal: Indian Journal of Pharmaceutical Sciences

Article Title: Effects of Propofol Anesthesia on Pyroptosis and the Nod-Like Receptor Protein 3/Caspase-1 Pathway in the Neonatal Rat Hippocampus

doi: 10.36468/pharmaceutical-sciences.1098

Figure Lengend Snippet: Fig. 2: The protein expression of NLRP3, ASC and caspase-1 in the hippocampal tissues of rats in each group

Article Snippet: Main reagents: Propofol (Cat No.: T1300) was purchased from target molecule Corp, United States of America (USA); the NLRP3 inhibitor MCC950 (Cat No: T6887) was purchased from TargetMol, USA; Transferase dUTP Nick End Labeling (TUNEL) kit (Cat No.: AT2190-50T) was purchased from Shanghai ACMEC Biochemical Technology Co., Ltd.; Interleukin-1 beta (IL-1β), Interleukin-18 (IL-18) detection kit (Cat No.: YAD1545 and YAD1758) was purchased from Beijing Jiehui Bogao Biotechnology Co., Ltd.; Bicinchoninic Acid (BCA) protein assay kit (Cat No.: 701780-480) was purchased from Cayman, USA; primary antibodies NLRP3 antibody, ASC antibody, caspase-1 antibody, Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) antibody and secondary antibodies Immunoglobulin G (IgG) antibody (Cat No.: ab263899, ab175449, ab138483, ab59164 and ab6721) were purchased from Abcam.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: Both NLRP3- and NLRC4-inflammasome activation contributes to rFlaA:Betv1 induced IL-1β and pro-inflammatory cytokine secretion from THP-1 macrophages. PMA-differentiated wild-type (WT), NLRP3-, NLRC4-, or ASC-deficient THP-1 macrophages were stimulated with either LPS as a positive control, or equimolar amounts of rFlaA + rBet v 1, rFlaA:Betv1, rFlaA *D1 :Betv1, or rFlaA ΔDC0 :Betv1 for 24 h (A) . Supernatants were collected and checked for the secretion of IL-1β, IL-6, IL-12, and TNF-α by ELISA (B) . Data are the mean results of three independent experiments ± SD, and statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 ( Invivo Gen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 ( Invivo Gen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, Invivo Gen), SB202190 (p38α/β MAPK inhibitor, Invivo Gen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in .

Techniques: Activation Assay, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages

doi: 10.3389/fimmu.2023.1136669

Figure Lengend Snippet: NFκB- and SAP/JNK MAP kinase-signaling pathways contribute to inflammasome activation by regulating pro-Caspase-1, pro-IL-1β-, and NLRP3-expression in rFlaA:Betv1-stimulated THP-1 macrophages. PMA-differentiated THP-1 macrophages were pre-treated with the indicated inhibitors (500 nM TPCA-1, 5 µM BMS-345541, 25 µM SP600125, or 10 µM SB202190) for 90 minutes, followed by stimulation with 27.4 µg/mL rFlaA:Betv1 for additional 24 h (A) . Proteins in supernatant (Sup) and cell lysate (Lysate) were examined by Western Blot (B) . The intensity of Western Blot bands from three independent experiments was analyzed, first normalized to the loading control β-Tubulin and then again normalized to the unstimulated group (shown as value of “1”) (C) . Data are either representative (B) or mean results of three independent experiments ± SD (C) . Statistical significances are indicated as ns: p-value > 0.05, *: p-value < 0.05, **: p-value < 0.01, ***: p-value < 0.001.

Article Snippet: PMA-differentiated, THP-1 macrophages were pre-incubated with the indicated amounts of either the IKK-β inhibitors TPCA-1 (Abcam, Cambridge, UK) or BMS-345541 (Abcam), the unspecific inflammasome inhibitor VX-765 ( Invivo Gen), which inhibits caspase-1 activity, the specific NLRP3-inflammasome inhibitor MCC950 ( Invivo Gen), or the MAPK inhibitors SP600125 (SAP/JNK MAPK inhibitor, Invivo Gen), SB202190 (p38α/β MAPK inhibitor, Invivo Gen), or U0126 (ERK MAPK inhibitor, Cell Signaling Technologies, Leiden, The Netherlands) for 90 min and subsequently stimulated with rFlaA:Betv1 for 24 h. The target molecules of the used inhibitors are summarized in .

Techniques: Activation Assay, Expressing, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A , B , Representative immunoblots ( A ) and quantification ( B ) of NLRP3, ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 in the heart of WT mice subjected to either ND or HFD consumption for 24 wks (n=6 per group). ( C ) Representative images of wheat germ agglutinin staining (green) and immunofluorescence staining of NLRP3 (red) in the heart of WT mice fed an HFD or ND for 24 wks. D , E , Protein of IL‐1β ( D ) and IL‐18 ( E ) in the heart of WT mice fed an HFD or ND for 24 wks. IL‐1β and IL‐18 protein were detected by ELISA. Values were normalized to total protein level (n=6 per group). F , Representative images of immunohistochemical staining of IL‐1β in the heart of WT mice subjected to either ND or HFD consumption for 24 wks. ( G ) Representative immunoblots of NLRP3, ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 protein in NRVMs treated with BSA or BSA+PA for 24 h. H , Representative images of immunofluorescence staining of NLRP3 (red) and F‐Actin (green) in NRVMs treated with BSA or BSA+PA. Significance was assessed by Student t test. Data are shown as the mean±SEM. ASC indicates apoptosis‐associated speck like protein; HFD, high‐fat diet; BSA, Bovine Serum Albumin; IL, interleukin; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; PA, palmitic acid; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Western Blot, Staining, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A , Representative M‐mode echocardiographic images of left ventricle from WT or NLRP3 ‐/‐ mice subjected to ND or HFD feeding for 24 wks. B , C , LV ejection fraction and fractional shortening were measured by echocardiography (n=12 per group). D , E , Representative images of transmitral flow obtained by Doppler echocardiography and ratio between mitral E wave and A wave in WT or NLRP3 ‐/‐ mice subjected to ND or HFD feeding for 24 wks. E wave DT is indicated by red bars (n=10 per group). F , E wave DT, a marker of diastolic function, evaluated by transmitral flow Doppler echocardiography in indicated mice (n=10 per group). G , LV mass was measured by echocardiography in WT or NLRP3 ‐/‐ mice subjected to ND or HFD feeding for 24 wks (n=10 per group). H , Heart weight normalized by tibia length, a marker of cardiac hypertrophy. I , Representative images of cardiac H&E, WGA staining, Oil Red O staining and Masson trichrome staining in perivascular and interstitial area. J , Quantification of cardiomyocyte cross‐sectional area by WGA in indicated hearts (n=5 per group). K , Quantification of left ventricle collagen volume by Masson trichrome staining (n=6 per group). L , Quantification of myocardial lipid accumulation by Oil Red O staining (n=5 per group). DT indicates deceleration time; H&E, hematoxylin and eosin; HFD, high‐fat diet; LV, left ventricular; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; ORO, Oil Red O; WGA, wheat germ agglutinin; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Marker, Staining, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through L , Wild‐type mice treated with NS or MCC950 for 12 wks were subjected to ND or HFD feeding for 24 wks. A , Representative M‐mode echocardiographic images of left ventricle from the indicated mice. B , C , LV ejection fraction and fractional shortening were measured by echocardiography (n=12 per group). D , E , Representative images of transmitral flow obtained by Doppler echocardiography and ratio between mitral E wave and A wave. E wave DT is indicated by red bars (n=10 per group). F , E wave DT, a marker of diastolic function, evaluated by transmitral flow Doppler echocardiography in indicated mice (n=10 per group). G , LV mass was measured by echocardiography in indicated mice subjected to NS or MCC950 for 12 wks (n=10 per group). H , Heart weight normalized by tibia length, a marker of cardiac hypertrophy (n=10 per group). I , Representative images of cardiac H&E, WGA staining, Oil Red O staining and Masson trichrome staining in perivascular and interstitial area. J , Quantification of cardiomyocyte cross‐sectional area by WGA in indicated hearts (n=6 per group). K , Quantification of LV collagen volume by Masson trichrome staining (n=6 per group). L , Quantification of myocardial lipid accumulation by Oil Red O staining (n=6 per group). M , N , Mitochondrial oxidative capacity was measured in real time after MCC950 treatment or BSA+PA incubation in NRVMs. Quantification of maximal respiration in ( L ) (n=6 independent experiments). DT indicates deceleration time; FCCP, Trifluoromethoxy carbonylcyanide phenylhydrazone; H&E, hematoxylin and eosin; HFD, high‐fat diet; BSA, Bovine Serum Albumin; HW/TL, heart weight/tibia length ratio; ND, normal diet; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; NS, normal saline; OCR, oxygen consumption rate; ORO, Oil Red O; PA, palmitic acid; and WGA, wheat germ agglutinin.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Marker, Staining, Incubation, Binding Assay, Saline

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through D , Wild‐type mice treated with NS or MCC950 for 12 wks were subjected to ND or HFD feeding for 24 wks. A , qRT‐PCR detection of indicated genes related to NLRP3 inflammasome components and the proinflammatory cytokine IL‐1β and IL‐18 in hearts from indicated mice (n=5 per group). B , Immunohistochemical staining for NF‐κB in indicated murine heart. C , D , Representative images of immunoblot and quantitative analysis of NF‐κB in heart tissues from indicated mice. E through F , NRVMs treated with NS or MCC950 were incubated with BSA or BSA+PA medium for 24 h. E , Representative images of immunofluorescence staining of NF‐κB (red) and F‐Actin (green) in NRVMs. F , Immunoblotting analysis of NF‐κB protein in whole‐cell lysates and cytoplasmic and nuclear components in the indicated NRVMs. HFD indicates high‐fat diet; IL, interleukin; ND, normal diet; NF‐κB, nuclear factor kappa B; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; ns, not significant; NS, normal saline; p‐NF‐κB, phosphorylated nuclear factor kappa B; PA, palmitic acid; and qRT‐PCR, quantitative real‐time polymerase chain reaction.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Quantitative RT-PCR, Immunohistochemical staining, Staining, Western Blot, Incubation, Immunofluorescence, Binding Assay, Saline, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A , Representative immunoblots of TXNIP in the whole‐cell lysate of the heart at different time points following HFD consumption in mice. B , qRT‐PCR detection of TXNIP in hearts of mice fed an ND or an HFD for different durations (n=5 per group). C , Protocol and schematics of establishment of TXNIP overexpressing mouse model in WT or NLRP3 ‐/‐ mice subjected to ND or HFD feeding for 24 wks. D through N , WT or NLRP3 ‐/‐ mice received rAAV9‐cTnT‐null or rAAV9‐cTnT‐TXNIP were subjected to ND or HFD feeding for 24 wks. D , Representative images of M‐model echocardiography (upper) and transmitral flow obtained by Doppler echocardiography (lower). E wave deceleration time is indicated by red bars. E , F , LV ejection fraction ( E ) and fractional shortening ( F ) were measured by echocardiography in indicated mice (n=10 per group). G , H , Histogram of ratio between mitral E wave and A wave and E wave deceleration time, evaluated by transmitral flow Doppler echocardiography in indicated mice (n=10 per group). I , J , Histogram of LV mass ( I ) and HW/TL ( J ) in indicated mice (n=10 per group). K , Representative images of cardiac WGA staining, Oil Red O staining and Masson trichrome staining in perivascular and interstitial area (n=10 per group). L , Quantification of cardiomyocyte cross‐sectional area by WGA (n=5 per group). M , Quantification of LV collagen volume in interstitial area by Masson trichrome staining (n=5 per group). N , Quantification of myocardial lipid accumulation by Oil Red O staining (n=5 per group). cTnT indicates cardiac troponin T; HFD, high‐fat diet; HW/TL, heart weight/tibia length ratio; LV, left ventricular; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; ORO, Oil Red O; qRT‐PCR, quantitative real‐time polymerase chain reaction; rAAV9, recombinant adeno‐associated virus serotype 9 encoding TXNIP; TXNIP, thioredoxin‐interacting protein; WGA, wheat germ agglutinin; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Western Blot, Quantitative RT-PCR, Staining, Binding Assay, Real-time Polymerase Chain Reaction, Recombinant, Virus

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through H , NRVMs treated with PBS or MitoTEMPO were incubated with BSA or BSA+PA medium for 24 h. A , Schematic diagram showing the procedure of NRVMs. B , Intracellular total ROS using DCFH‐DA staining (upper) and mitochondrial ROS using MitoSOX staining (lower) were determined in NRVMs. C , Cell lysates of NRVMs were immunoprecipitated with TXNIP antibody, and immunoblot assays were performed using NLRP3, TXNIP, and TRX antibodies. NRVMs were treated with siTXNIP to knockdown of TXNIP. D , Triple immunofluorescence staining for TXNIP (red), NLRP3 (green), and nuclei (DAPI, blue) was performed in indicated NRVMs. E , Representative immunoblots of ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 protein in NRVMs from indicated groups. F , Oil Red O staining of NRVMs from indicated groups. Red indicates lipid droplets, blue indicates nuclei. G , qRT‐PCR detection of indicated genes related to fatty acid transport genes Cd36 and fatty acid oxidation genes Cpt1b , Acadl , and Acadvl in hearts from indicated mice (n=6 independent experiments). H , Expression level of hypertrophic marker gene BNP was determined by RT‐PCR and normalized to that of GAPDH (n=6 independent experiments). ASC indicates apoptosis‐associated speck like protein; BNP, B‐type natriuretic peptide; BSA, Bovine Serum Albumin; IL, interleukin; IP:TXNIP, immunoprecipitation:thioredoxin‐interacting protein; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; PA, palmitic acid; qRT‐PCR, quantitative real‐time polymerase chain reaction; and ROS, reactive oxygen species.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Incubation, Staining, Immunoprecipitation, Western Blot, Knockdown, Immunofluorescence, Quantitative RT-PCR, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through L , WT mice received NS or MitoTEMPO were subjected to ND or HFD feeding for 24 wks. A , Experimental schematic diagram showing the process of HFD‐induced obesity cardiomyopathy. After 12 wks of HFD feeding, MitoTEMPO was injected into obese mice at a dose of 10 mg/kg per day for 12 wks. B , C , LV fractional shortening ( B ) and ratio between mitral E wave and A wave ( C ) were measured by echocardiography in indicated mice (n=12 per group). D , Histogram of HW/TL in indicated mice. HW indicated wet weight (n=12 per group). E , Representative images of cardiac WGA staining, Oil Red O staining, and Masson trichrome staining in perivascular and interstitial area in indicated heart. F , Quantification of cardiomyocyte cross‐sectional area by WGA (n=6 per group). G , Quantification of LV collagen volume in interstitial area by Masson trichrome staining (n=6 per group). H , Quantification of myocardial lipid accumulation by Oil Red O staining (n=6 per group). I , Tissue lysate in indicated heart were immunoprecipitated with TXNIP antibody, and immunoblot assays were performed using NLRP3, TXNIP, and thioredoxin antibodies. J , K , Protein of IL‐1β ( J ) and IL‐18 ( K ) in the heart of WT mice fed an HFD or ND for 24 wks with or without MitoTEMPO treatment. IL‐1β and IL‐18 protein were detected by ELISA. Values were normalized to total protein level (n=6 per group). L , Representative immunoblots of ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 in the indicated heart. ASC indicates apoptosis‐associated speck like protein; HFD, high‐fat diet; HW/TL, heart weight/tibia length ratio; IL, interleukin; IP:TXNIP, immunoprecipitation:thioredoxin‐interacting protein; LV, left ventricular; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; ORO, Oil Red O; NS, normal saline; WGA, wheat germ agglutinin; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Injection, Staining, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Saline

Journal: Cell communication and signaling : CCS

Article Title: P2X7 receptor activation leads to NLRP3-independent IL-1β release by human macrophages.

doi: 10.1186/s12964-023-01356-1

Figure Lengend Snippet: Fig. 2 P2X7-mediated IL-1β release is NLRP3 independent. A THP-1 macrophages were primed with Pam3CSK4 and then stimulated with ATP, BzATP or nigericin for 3 h. Antagonists of P2X7 receptor (A-804598, oxATP), P2X4 receptor (5-BDBD) or P2X receptors (PPADS) were added 1 h before stimulation. IL-1β concentration in the supernatants was analyzed by ELISA. ATP, BzATP or nigericin induced IL-1β release was set to 100%. Mean + SEM (n = 3). One-sample t test against 100%, ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. B THP-1 macrophages were primed with Pam3CSK4. Potassium chloride was added together with ATP, BzATP or nigericin for 3 h. IL-1β concentration in the supernatants was analyzed by ELISA. ATP, BzATP or nigericin induced IL-1β release was set to 100%. Mean + SEM (n = 3). One-sample t test against 100%, *P ≤ 0.05, ****P ≤ 0.0001. C THP-1 macrophages were primed with Pam3CSK4 and then stimulated with ATP, BzATP or nigericin for 3 h. NLRP3 inhibitors MCC950 or Bay 11–7082 were added 1 h before stimulation. IL-1β concentration in the supernatants was analyzed by ELISA. ATP, BzATP or nigericin induced IL-1β release was set to 100%. Mean + SEM (n = 3—4). One-sample t test against 100%, ns ≥ 0.05, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 (D) NLRP3 KO THP-1 macrophages were primed with Pam3CSK4. Cells were then stimulated with ATP, BzATP or nigericin for 3 h. IL-1β concentration in the supernatants was analyzed by ELISA. Mean + SEM (n = 3). Two-tailed two sample t test, ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001

Article Snippet: In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM, 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP, 300 μM, 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD (25 μM, SML0450-5MG, SigmaAldrich, Taufkirchen, Germany), P2X receptor antagonist PPADS (100 μM, 0625, Tocris Bioscience, Bristol, United Kingdom), NLRP3 inhibitor MCC950 (10 μM, 5479, Tocris Bioscience, Bristol, United Kingdom) or Bay 11–7082 (20 μM, B5556, Sigma-Aldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM, 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VADfmk (40 μM, tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM, 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM, 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM, 2936, Carl Roth, Karlsruhe, Germany) 1 h before stimulation.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell communication and signaling : CCS

Article Title: P2X7 receptor activation leads to NLRP3-independent IL-1β release by human macrophages.

doi: 10.1186/s12964-023-01356-1

Figure Lengend Snippet: Fig. 4 Influence of priming with different TLR ligands on IL-1β release. A THP-1 macrophages were primed with LPS or Pam2CSK4. Cells were then stimulated with ATP, BzATP or nigericin for 3 h. IL-1β concentration in the supernatants was analyzed by ELISA. Mean + SEM (n = 3). Two-tailed two-sample t test, ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. B THP-1 macrophages were primed with Pam3CSK4, Pam2CSK4 or LPS. Gene expression of IL18 or IL1B was normalized to GAPDH. Mean + SEM (n = 3). One-way ANOVA followed by Tukey´s post-test, ns ≥ 0.05. C THP-1 macrophages were primed with Pam3CSK4, Pam2CSK4 or LPS and then stimulated with ATP, BzATP or nigericin. NLRP3 inhibitor (MCC950) was added 1 h before stimulation. IL-1β concentration in the supernatants was analyzed by ELISA. ATP, BzATP or nigericin induced IL-1β release was set to 100%. Mean + SEM (n = 3—5). One-way ANOVA followed by Tukey´s post-test, ns ≥ 0.05, *P ≤ 0.05, **P ≤ 0.01

Article Snippet: In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM, 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP, 300 μM, 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD (25 μM, SML0450-5MG, SigmaAldrich, Taufkirchen, Germany), P2X receptor antagonist PPADS (100 μM, 0625, Tocris Bioscience, Bristol, United Kingdom), NLRP3 inhibitor MCC950 (10 μM, 5479, Tocris Bioscience, Bristol, United Kingdom) or Bay 11–7082 (20 μM, B5556, Sigma-Aldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM, 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VADfmk (40 μM, tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM, 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM, 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM, 2936, Carl Roth, Karlsruhe, Germany) 1 h before stimulation.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Gene Expression

Journal: Cell communication and signaling : CCS

Article Title: P2X7 receptor activation leads to NLRP3-independent IL-1β release by human macrophages.

doi: 10.1186/s12964-023-01356-1

Figure Lengend Snippet: Fig. 5 Proposed mechanism for NLRP3-independent IL-1β release by human macrophages after P2X7 receptor activation. Activation of TLR2/1 by Pam3CSK4 leads to NF-κB mediated production of pro-IL-1β and components of NLRP3 inflammasome. Nigericin-mediated K+ efflux leads to NLRP3 inflammasome oligomerization causing serine protease- and caspase-dependent IL-1β release. In contrast, P2X7 receptor stimulation by BzATP potentially initiates two distinct IL-1β releasing mechanisms that are both NLRP3-independent. One mechanism relies on K+ efflux and requires activation of caspase-1 and serine proteases. The other mechanism is independent of K+ efflux, caspases, and serine-, cysteine-, and aspartic proteases, suggesting the involvement of other mechanisms for proteolytic processing of IL-1β

Article Snippet: In selected experiments, cells were preincubated with the non-competitive P2X7 receptor antagonist A804598 (1 μM, 4473, Tocris Bioscience, Bristol United Kingdom), irreversible P2X7 receptor antagonist oxidized ATP (oxATP, 300 μM, 505758, Merck, Darmstadt, Germany), P2X4 receptor antagonist 5-BDBD (25 μM, SML0450-5MG, SigmaAldrich, Taufkirchen, Germany), P2X receptor antagonist PPADS (100 μM, 0625, Tocris Bioscience, Bristol, United Kingdom), NLRP3 inhibitor MCC950 (10 μM, 5479, Tocris Bioscience, Bristol, United Kingdom) or Bay 11–7082 (20 μM, B5556, Sigma-Aldrich, Taufkirchen, Germany), Caspase-1 inhibitor Ac-YVAD-cmk (40 μM, 10014, Biomol, Hamburg, Germany), pan-caspase inhibitor Z-VADfmk (40 μM, tlrl-vad, Invivogen, Toulouse, France), serine protease inhibitor AEBSF (300 μM, 50985.100, Biomol, Hamburg, Germany), cysteine protease inhibitor E64 (10 μM, 324890, Merck, Darmstadt, Germany), aspartic protease inhibitor pepstatin A (50 μM, 2936, Carl Roth, Karlsruhe, Germany) 1 h before stimulation.

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Andrade-Oliveira Salvianolic Acid B Modulates Caspase-1–Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via Nrf2 Pathway

doi: 10.3389/fphar.2020.541426

Figure Lengend Snippet: Salvianolic acid B (SalB) promotes Nrf2 nuclear activation and inhibits NLR family pyrin domain-containing 3 (NLRP3)/thioredoxin-interacting protein-thioredoxin1 (TXNIP) expression in ischemia-reperfusion (I/R) mice. (A–C) The expression of NLRP3 and TXNIP. (D) Immunofluorescence images (magnification ×200) showing the nuclear expression and localization of Nrf2 in the Sham, I/R, SalB-L, SalB-M, SalB-H groups. Blue: nuclear staining (DAPI); red: Nrf2; staining. Scale bar: 20 μm. (E) Representative western blots and (F–H) quantification of relative protein expression for nuclear Nrf2, keap1 and HO-1. (I–K) Superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) detected by a microplate reader. Data are represented as images or expressed as the mean ± SEM of each group from three separate experiments. *p < 0.05, **p < 0.01 vs. sham group; # p < 0.05, ## p < 0.01 vs. I/R group.

Article Snippet: After two–four passages, cells were cultured for 24 h and then were randomly divided into three groups: Control group: cells were cultured under normal conditions (5% CO2, 21% O2, 74% N2); Model group: the cells were incubated with different concentrations of SalB(purity>98%; 358153; Nanjing DASF Biotechnology Co.Ltd.) for 24 h; Positive control group: cells were incubated in certain concentration of NLRP3 inhibitor MCC950(S7809;Selleck), caspase-1 specific inhibitor VX-765(S2228;Selleck) for 2 h, and were then placed in a hypoxia incubator chamber (27310, stemcell) hypoxia (5% CO2, 95% N2) for 6 h and then reoxygenated for an additional 1 h (5% CO2, 21% O2, 74% N2).

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Andrade-Oliveira Salvianolic Acid B Modulates Caspase-1–Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via Nrf2 Pathway

doi: 10.3389/fphar.2020.541426

Figure Lengend Snippet: Nuclear factor erythroid-2 related factor 2 (Nrf2) nuclear expression is upregulated and NLR family pyrin domain-containing 3 (NLRP3)/thioredoxin-interacting protein-thioredoxin1 (TXNIP) is down-regulated after SalB treatment in H/R (A–C) NLRP3 and TXNIP expression were examined by western blot. (D) Immunofluorescence results (magnification ×400) showing the expression of Nrf2 under normal conditions (control), SalB treatment and H/R-treated HK-2 cells. Blue, nuclear staining (DAPI); red, Nrf2 staining. Scale bar: 20 μm. (E) Representative western blots and (F–H) quantification of relative protein expression for nuclear Nrf2, keap1 and HO-1. (I) Representative images of fluorescence of ROS probed by DCFH-DA. Data are represented as images or expressed as the mean ± SEM of each group from three separate experiments. **p < 0.01 vs. control group; # p < 0.05, ## p < 0.01 vs. H/R group.

Article Snippet: After two–four passages, cells were cultured for 24 h and then were randomly divided into three groups: Control group: cells were cultured under normal conditions (5% CO2, 21% O2, 74% N2); Model group: the cells were incubated with different concentrations of SalB(purity>98%; 358153; Nanjing DASF Biotechnology Co.Ltd.) for 24 h; Positive control group: cells were incubated in certain concentration of NLRP3 inhibitor MCC950(S7809;Selleck), caspase-1 specific inhibitor VX-765(S2228;Selleck) for 2 h, and were then placed in a hypoxia incubator chamber (27310, stemcell) hypoxia (5% CO2, 95% N2) for 6 h and then reoxygenated for an additional 1 h (5% CO2, 21% O2, 74% N2).

Techniques: Expressing, Western Blot, Immunofluorescence, Control, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Andrade-Oliveira Salvianolic Acid B Modulates Caspase-1–Mediated Pyroptosis in Renal Ischemia-Reperfusion Injury via Nrf2 Pathway

doi: 10.3389/fphar.2020.541426

Figure Lengend Snippet: The pyroptosis of acute kidney injury (AKI) signaling and protective effect of SalB through the nuclear factor erythroid-2 related factor 2 (Nrf2)/NLR family pyrin domain-containing 3 (NLRP3) pathway. I/R and H/R trigger pyroptotic cell death signaling, including upregulation of thioredoxin-interacting protein-thioredoxin1 (TXNIP) and suppression of Nrf2 expression, leads to NLRP3 oligomerization, ASC recruitment and subsequent caspase-1 activation. Fortunately, SalB treatment ameliorates AKI by regulating the Nrf2/NLRP3 pathway. The arrows represent promotion, while the inverted T represent inhibition. The effect of Salvianolic acid B (SalB) is shown in red.

Article Snippet: After two–four passages, cells were cultured for 24 h and then were randomly divided into three groups: Control group: cells were cultured under normal conditions (5% CO2, 21% O2, 74% N2); Model group: the cells were incubated with different concentrations of SalB(purity>98%; 358153; Nanjing DASF Biotechnology Co.Ltd.) for 24 h; Positive control group: cells were incubated in certain concentration of NLRP3 inhibitor MCC950(S7809;Selleck), caspase-1 specific inhibitor VX-765(S2228;Selleck) for 2 h, and were then placed in a hypoxia incubator chamber (27310, stemcell) hypoxia (5% CO2, 95% N2) for 6 h and then reoxygenated for an additional 1 h (5% CO2, 21% O2, 74% N2).

Techniques: Expressing, Activation Assay, Inhibition