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: 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: Acta Biochimica et Biophysica Sinica

Article Title: AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation

doi: 10.3724/abbs.2023039

Figure Lengend Snippet: Effect of AICAR treatment on NLRP3, NLRP1, NLRC4, and AIM2 inflammasome components (A,C) Representative immunofluorescence staining images showing the expressions of NLRP3 and caspase-1 in the spinal dorsal horn of sham and CIBP rats. Scale bar: 20 μm. (B,D) Quantitative analysis of fluorescence intensity in (A) and (C). Data are expressed as the mean±SD ( n=3). * P<0.05 vs sham group. (E) Western blot analysis of the levels of NLRP3, caspase-1 and activated caspase-1 in the spinal cord of the sham, CIBP and CIBP+AICAR groups. β-Actin was used as a loading control. (F) Quantification of the relative protein levels in (E). The relative level of activated caspase-1 was normalized to caspase-1. Data are presented as the mean±SD ( n=3). * P<0.05 vs sham group, # P<0.05 vs CIBP group. (G) Western blot analysis of the levels of NLRP1, NLRC4, and AIM2 in the spinal cord of the sham, CIBP and CIBP+AICAR groups. (H) Quantification of the relative protein levels in (G). Data are presented as the mean±SD ( n=3). * P<0.05 vs sham group, # P<0.05 vs CIBP group.

Article Snippet: The AMPK activator AICAR (S1802), NLRP3 inhibitor MCC950 (S8930), and Drp1 inhibitor Mdivi-1 (S7162) were purchased from Selleck Chemicals (Houston, USA).

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

Journal: Acta Biochimica et Biophysica Sinica

Article Title: AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation

doi: 10.3724/abbs.2023039

Figure Lengend Snippet: Effect of AICAR and dorsomorphin treatment on mitochondrial membrane potential and mitochondrial ROS in C6 cells (A) Western blot analysis of the levels of phosphorylated AMPK, AMPK, phosphorylated Drp1, Drp1 and NLRP3 in AICAR- or dorsomorphin-treated C6 cells. (B) Quantification of the relative protein levels in (A). The relative level of phosphorylated AMPK was normalized to total AMPK; phosphorylated Drp1 was normalized to total Drp1. Data are presented as the mean±SD ( n=3). * P<0.05 vs Control group, # P<0.05 vs AICAR group. (C) Representative fluorescence images of JC-1 in the control, IL-1β, IL-1β+AICAR, IL-1β+Mdivi-1, and IL-1β+MCC950 groups. Red fluorescence indicates JC-1 aggregates representing an increase in mitochondrial membrane potential. The green fluorescence indicates the JC-1 monomer, representing a reduction in mitochondrial membrane potential. CCCP was used as a positive control. (D) Representative fluorescence images of Mito-ROS in the control, IL-1β, IL-1β+AICAR, IL-1β+Mdivi-1, and IL-1β+MCC950 groups. (E) Quantitative analysis of the ratio of red/green fluorescent intensity. Data are shown as the mean±SD ( n=3). * P<0.05 vs control group, # P<0.05 vs IL-1β group. Scale bar: 20 μm. (F) Quantitative analysis of fluorescence intensity in D. Data are shown as the mean±SD ( n=3). * P<0.05 vs control group, # P<0.05 vs IL-1β. Scale bar: 20 μm.

Article Snippet: The AMPK activator AICAR (S1802), NLRP3 inhibitor MCC950 (S8930), and Drp1 inhibitor Mdivi-1 (S7162) were purchased from Selleck Chemicals (Houston, USA).

Techniques: Membrane, Western Blot, Control, Fluorescence, Positive Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation

doi: 10.3724/abbs.2023039

Figure Lengend Snippet: Schematic representation of the potential mechanisms by which AICAR treatment reduces spinal inflammation and alleviates cancer-induced bone pain Activation of AMPK by AICAR inhibits mitochondrial fission and NLRP3 inflammasome activity and consequently reduces IL-1β expression in the spinal cord of CIBP rats. The decreased expression of proinflammatory cytokine is relevant to the downregulated pain sensitivity in CIBP rats. Accordingly, AICAR treatment has an analgesia effect on CIBP rats.

Article Snippet: The AMPK activator AICAR (S1802), NLRP3 inhibitor MCC950 (S8930), and Drp1 inhibitor Mdivi-1 (S7162) were purchased from Selleck Chemicals (Houston, USA).

Techniques: Activation Assay, Activity Assay, Expressing

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