Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin attenuated macrophage pyroptosis and reduced IL-1β release into the myocardium and serum during I/R injury. A-C The RNA sequencing data were extracted from GSE 126772. The mRNA levels of Nlrp3 , Pycard ( ASC ) and Il-1b in macrophages isolated from heart after MI were shown as fold change compared to the Pre group (control group, n = 2,3). D Macrophages were identified and isolated from the heart by flow cytometry. E–F Immunoblotting was performed to assess the expression levels of NLRP3, GSDMD and IL-1β in macrophages after I/R injury with or without bufalin treatment. Each target band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Sham + PBS group (control group, n = 3). G Representative IL-1β expression and localization in murine hearts are shown by IHC analysis in longitudinal sections of whole hearts. For each mouse, 5 representative zones in the left ventricle were chosen randomly to determine the percentage of IL-1β-positive ratio by ImageJ ( n = 3). H IL-1β levels in the peripheral blood of mice that were subjected to I/R injury and were treated with or without bufalin were measured by ELISA ( n = 6). IHC (immunohistochemistry). Data are expressed as the mean ± SEM. P values were determined by ordinary one-way ANOVA with Dunnett’s multiple comparisons test ( A - C ) and two-way ANOVA with Tukey’s multiple comparisons test ( F and H )

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: RNA Sequencing, Isolation, Control, Flow Cytometry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin attenuated H/R-treated macrophage pyroptosis in vitro. Macrophages were subjected to 1 h of hypoxia and 24 h of reoxygenation treated with PBS or bufalin. A-B Proteins were extracted from cells and immunoblotting analysis was performed. The expressions of NLRP3, GSDMD, and IL-1β were normalized to that of β-actin and then compared with the Sham + PBS group (control group, n = 3). C PI staining was used to examine the plasma membrane integrity of macrophages. Positive cells were counted and normalized to the total number of cells ( n = 5). D Cell viability was assessed by measuring the quantity of ATP in macrophages subjected to different treatments ( n = 3, 6). E IL-1β levels in the culture medium of macrophages were measured by ELISA ( n = 3). Data are presented as the mean ± SEM. P values were calculated by two-way ANOVA with Tukey’s multiple comparisons test ( B , C , and E ) or Šídák’s multiple comparisons test ( D )

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: In Vitro, Western Blot, Control, Staining, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cardiovascular Translational Research

Article Title: Bufalin Ameliorates Myocardial Ischemia/Reperfusion Injury by Suppressing Macrophage Pyroptosis via P62 Pathway

doi: 10.1007/s12265-024-10577-9

Figure Lengend Snippet: Bufalin restored autophagic flux by suppressing P62 pathway in H/R-induced macrophages. Macrophages were transfected with the p62-overexpression plasmid for 24 h and then subjected to H/R with or without bufalin. A - B The protein levels of P62 were measured by immunoblotting. The bands were normalized to the β-actin band, and the relative expressions were calculated as the fold changes compared to the control group ( n = 3). C The mRNA levels of p62 were measured by qRT‒PCR. The Cq results were normalized to Mus-18 s and then compared with the control group ( n = 3). D-E Proteins were extracted from H/R-treated macrophages that were infected with vector or OE-p62 plasmid in advance and treated with bufalin or PBS. Immunoblotting was performed to measure the expressions of NLRP3, GSDMD and IL-1β. Each band was normalized to the β-actin band and the relative expression was calculated as the fold change compared to the Con + PBS group (control group, n = 3). F PI staining was used to assess pyroptosis in macrophages. Positive ratios were calculated as the percentage of positive cells compared with total number of cells ( n = 5). G IL-1β levels in the culture medium of macrophages were measured by ELISA ( n = 3). Data are presented as the mean ± SEM. P values were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test

Article Snippet: For interleukin-1β (IL-1β) treatment, recombinant mouse IL-1β (HY-P7073, MedChemExpress, New Jersey, USA) was added to HL-1 cells at different concentrations (0, 100 pg/ml, 300 pg/ml, 1000 pg/ml) and incubated for 24 h. For anakinra treatment, the recombinant human interleukin-1 receptor (IL-1R) antagonist anakinra (HY-108841, MedChemExpress) was added to cardiomyocytes every 6 h for 24 h when they were cocultured with normoxia-treated or H/R-treated macrophages.

Techniques: Transfection, Over Expression, Plasmid Preparation, Western Blot, Control, Infection, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Scutellarein ameliorates dextran sulfate sodium-induced ulcerative colitis by inhibiting colonic epithelial cell proinflammation and barrier disruption

doi: 10.3389/fphar.2024.1479441

Figure Lengend Snippet: Scu attenuated colonic inflammation in DSS-treated mice. (A) MPO activity in colon tissues from Veh- and DSS-treated mice, administered with Scu and 5-ASA. (B–F) Quantitative RT-PCR analysis of mRNA levels of Tnf-α (B) , Il-1β (C) , Il-1α (D) , Cxcl1 (E) , and Il-6 (F) , respectively, in colon tissues from different groups of mice. Data are presented as the mean ± SEM. N = 6 mice; **, p < 0.01 vs. Veh; # , p < 0.05, ## , p < 0.01 vs. DSS. One-way ANOVA with Bonferroni’s multiple comparisons tests.

Article Snippet: BAY11-7085 (Cat# HY-10257), 5-aminosalicylic acid (5-ASA, Cat# HY-15027), and IL-1β (Cat# HY-P78459A) were obtained from MedChemExpress (Monmouth Junction, NJ, United States).

Techniques: Activity Assay, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Scutellarein ameliorates dextran sulfate sodium-induced ulcerative colitis by inhibiting colonic epithelial cell proinflammation and barrier disruption

doi: 10.3389/fphar.2024.1479441

Figure Lengend Snippet: Scu inhibited IL-1β-induced mRNA expression of inflammatory mediators and barrier damage of HT-29 cells. (A) HT-29 cell viability measured by the MTT assay after being exposed to different concentrations of Scu for 24 h (B, C) Quantitative RT-PCR analysis of mRNA levels of IL-6 (B) and IL-8 (C) in HT-29 cells treated with IL-1β (10 ng/mL) for 12 h in the absence and presence of Scu (10 μM). (D) Change in the TEER of HT-29 monolayer treated with IL-1β (10 ng/mL, 24 h) in the absence and presence of Scu (10 μM). (E) Representative immunofluorescence images of E-cadherin (upper panel), occludin (middle panel), and ZO-1 (lower panel) in HT-29 cells after being exposed to IL-1β (10 ng/mL) for 24 h in the absence and presence of Scu (10 μM). Nuclei were stained with Hoechst 33342. Scale bar = 10 μm. (F) Representative Western blots of E-cadherin, occludin, and ZO-1 in HT-29 cells exposed to IL-1β (10 ng/mL) for 24 h in the absence and presence of Scu (10 μM). (G) Quantification of E-cadherin, occludin, and ZO-1 protein expression in HT-29 cells after being exposed to IL-1β (10 ng/mL) for 24 h in the absence and presence of Scu (10 μM). Data are expressed as the mean ± SEM. N = 5–6; **, p < 0.01 vs. Veh (0.1% DMSO); # , p < 0.05, ## , p < 0.01 vs. IL-1β. One-way ANOVA with Bonferroni’s multiple comparisons tests.

Article Snippet: BAY11-7085 (Cat# HY-10257), 5-aminosalicylic acid (5-ASA, Cat# HY-15027), and IL-1β (Cat# HY-P78459A) were obtained from MedChemExpress (Monmouth Junction, NJ, United States).

Techniques: Expressing, MTT Assay, Quantitative RT-PCR, Immunofluorescence, Staining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Scutellarein ameliorates dextran sulfate sodium-induced ulcerative colitis by inhibiting colonic epithelial cell proinflammation and barrier disruption

doi: 10.3389/fphar.2024.1479441

Figure Lengend Snippet: Scu inhibited IL-1β-induced phosphorylation of NF-κB and its nuclear translocation in HT-29 cells. (A) Representative Western blots and (B) quantification of phosphorylated NF-κB in HT-29 cells. HT-29 cells were exposed to IL-1β (10 ng/mL) in the absence and presence of Scu (10 μM). (C) Representative immunofluorescence images of stained with NF-κB in HT-29 cells. Cells were treated with IL-1β (10 ng/mL) for 30 min in the absence and presence of Scu (10 μM). Nuclei were stained with Hoechst 33342. Scale bar = 10 μm. (D, E) Quantitative RT-PCR analysis of mRNA levels of IL-6 (D) and IL-8 (E) in HT-29 cells treated with IL-1β (10 ng/mL) for 12 h in the absence and presence of BAY11-7082 (3 μM). (F) Changes in the TEER of HT-29 monolayer treated with IL-1β (10 ng/mL) for 24 h in the absence and presence of BAY11-7082 (3 μM). (G) Representative immunofluorescence images of E-cadherin, occludin, and ZO-1 in HT-29 cells exposed to IL-1β (10 ng/mL) for 24 h in the absence and presence of BAY11-7085 (3 μM). Nuclei were stained with Hoechst 33342. Scale bar = 10 μm. N = 3–6. **, p < 0.01 vs. Veh; # , p < 0.05, ## , p < 0.01 vs. IL-1β. One-way ANOVA with Bonferroni’s multiple comparisons tests.

Article Snippet: BAY11-7085 (Cat# HY-10257), 5-aminosalicylic acid (5-ASA, Cat# HY-15027), and IL-1β (Cat# HY-P78459A) were obtained from MedChemExpress (Monmouth Junction, NJ, United States).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Journal of Immunology Research

Article Title: 12,13-diHOME Promotes Inflammatory Macrophages and Epigenetically Modifies Their Capacity to Respond to Microbes and Allergens

doi: 10.1155/2024/2506586

Figure Lengend Snippet: 12,13-diHOME promotes inflammatory macrophage polarization and reduces phagocytic capacity, (a) Representative flow cytograms of IL-1 β (CD68 + IL-1 β Hi ; upper panel) and CD206 (CD68 + CD206 Hi ; lower panel) THP-1 macrophages following treatment with 37.5 or 75 μ M 12,13-diHOME for 24 or 48 hr (gating strategy provided in Supplementary figure ). As controls, cells were stimulated 48 hr with 20 ng ml −1 IFN γ and 100 ng ml −1 LPS together to promote an IL-1 β phenotype or 20 ng ml −1 IL-4 to induce a CD206 phenotype. (b) IL-1 β + /CD206 + THP-1 macrophage ratio following treatment with 37.5 or 75 μ M 12,13-diHOME or vehicle (DMSO; control) for 24 or 48 hr (Brown–Forsythe and Welch ANOVA test). (c) Temporal gene expression (1, 2, 4, 8, 12, 24, and 48 hr) of macrophage cytokines associated with inflammatory functional states ( IL-1β , TNFα , IL-6 , and NFκB ( RELA )) and anti-inflammatory functional states ( IL-10 , TGFβ ) following exposure of primary human macrophages to 12,13-diHOME. Mean gene expression of triplicate reactions quantitated by qRT-PCR followed with 2 − ΔΔ CT calculation; each follow-up timepoint was compared to baseline. Significant ( p < 0.05) findings are indicated with an asterisk (linear mixed model, Supplementary table ). Dose-dependent reduction in phagocytosis of fluorescently labeled E. coli following exposure to 12,13-diHOME (50 or 100 μ M) exposure is observed in (d) Raw264.7 (blue) and THP-1 (red) macrophages and (e) confirmed using a gentamicin protection assay (Kruskal–Wallis test). All box plots indicate the interquartile range (IQR) and median.

Article Snippet: To quantify secreted cytokines in cell culture supernatants, human IL-1 β and IL-6 cytokine flex sets (558279 and 558276, BD Cytometric Bead Array, BD BioScience) were used.

Techniques: Control, Expressing, Functional Assay, Quantitative RT-PCR, Labeling

Journal: Journal of Immunology Research

Article Title: 12,13-diHOME Promotes Inflammatory Macrophages and Epigenetically Modifies Their Capacity to Respond to Microbes and Allergens

doi: 10.1155/2024/2506586

Figure Lengend Snippet: 12,13-diHOME enhances inflammatory macrophages, IgE production, and cytokine secretion in the presence of antigenic stimulation. (a) 12,13-diHOME enhances allergen-induced a skew in IL-1 β + /CD206 + cell ratio in primary human monocyte-derived macrophages from four adult donors (A, B, C, and D). Cells were treated with individual allergens, CRA, LPS, OVA, or peanut extract, in the absence or presence of 12,13-diHOME (37.5 µ M). Data represent the mean ± standard deviation. A linear mixed model was used to determine significant differences. In primary human macrophage/T/B cell (from two adult donors) coculture assays, macrophage stimulation with peanut allergen (f171) in the presence of 12,13-diHOME (50 µ M) increases (b) the elevated ratio of IL-1 β + /CD206 + macrophages, secreted (c) IL-1 β , and (d) IL-6 concentrations (pg ml −1 ) in the supernatant after 5 days of coculture (e) the ratio of memory-B to naïve-B cell populations, and (f) the ratio of secreted IgE to IgG (data listed in Supplementary table , and the bar plots of both absolute IgE and IgG concentrations listed as Supplementary figure ) in the coculture supernatant (all box plots indicate the IQR and median; linear mixed model).

Article Snippet: To quantify secreted cytokines in cell culture supernatants, human IL-1 β and IL-6 cytokine flex sets (558279 and 558276, BD Cytometric Bead Array, BD BioScience) were used.

Techniques: Derivative Assay, Standard Deviation

Journal: Journal of Immunology Research

Article Title: 12,13-diHOME Promotes Inflammatory Macrophages and Epigenetically Modifies Their Capacity to Respond to Microbes and Allergens

doi: 10.1155/2024/2506586

Figure Lengend Snippet: Gut microbial derived 12,13-diHOME exposure in context of airway allergen stimulation induces pathways related to inflammatory polarization and epigenetic modifications. (a) Principal components analysis of the normalized expression for all genes between mice treated with and without the combinatorial 3 epoxide hydrolase (3EH)-expressing E. coli and cockroach antigen (CRA) distinguishes the four treatment groups: no treatment (vehicle; red), 3EH alone (green), CRA alone (blue), and the combination of 3EH + CRA (purple). PC1 and PC2 were significantly associated with the four 3EH and CRA treatment groups (ANOVA, p = 4.9 × 10 −5 and 5.8 × 10 −4 , respectively). (b) Five upregulated GSEA pathways after treatment with combinatorial 3EH + CRA compared with vehicle-group. (c) IL-1 signaling pathway. (d) One-carbon metabolism pathway. Genes in orange and blue show relatively higher and lower expression in mice treated with 3EH + CRA combinatorial group, respectively. NES, normalized enrichment score.

Article Snippet: To quantify secreted cytokines in cell culture supernatants, human IL-1 β and IL-6 cytokine flex sets (558279 and 558276, BD Cytometric Bead Array, BD BioScience) were used.

Techniques: Derivative Assay, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Human Anti-Insulin IgG Autoantibody Apparently Arises Through Clonal Selection from an Insulin-Specific “Germ-Line” Natural Antibody Template

doi:

Figure Lengend Snippet: Binding of the recombinant VHDJH13/VκJκ13 (A), VH13G12-DJH13/Vκ13K20-Jκ13 (B), Lys52/VκJκ13 (C), and Arg58/VκJκ13 (D) IgG1 Abs to human recombinant insulin (●), ssDNA (△), tetanus toxoid (■), human recombinant IL-1β (▲), E. coli β-galactosidase (◇), and BSA (□). The Ag-binding activity of each Ab to solid-phase Ag is expressed as optical absorbance at 492 nm.

Article Snippet: Eluates were brought to pH 7.5 by addition of neutralizing buffer (pH 9.0), dialyzed against PBS, and stored in aliquots at 4°C. mAb concentration and binding to human recombinant insulin (Eli Lilly Research Laboratories, Indianapolis, IN), ssDNA, human recombinant IL-1 β (BASF Biotech Corp., Worcester, MA), tetanus toxoid, and Escherichia coli β -galactosidase were measured using appropriate ELISA, as previously described ( 12 , 20 , 21 ).

Techniques: Binding Assay, Recombinant, Activity Assay