Journal: Pharmaceutics

Article Title: Colon-Specific Delivery of Probenecid Enhances Therapeutic Activity of the Uricosuric Agent Against Rat Colitis

doi: 10.3390/pharmaceutics17111454

Figure Lengend Snippet: PBN-GA is therapeutically superior to PBN and SSZ in rat colitis. Three days following colitis induction, rats were orally administered SSZ (50.0 mg/kg), PBN (30.0 mg/kg), PBN-GA (L) (21.8 mg/kg, equivalent to 15 mg/kg of PBN), or PBN-GA (H) (43.6 mg/kg, equivalent to 30 mg/kg of PBN) each dissolved in 1.0 mL PBS (pH 7.4), once daily. Animals were sacrificed 24 h after the sixth dose. ( A ) Left panel: Representative photographs of the serosal and luminal surfaces of the distal colon. Right panel: Colonic damage score (CDS) for each treatment group. ( B ) Hematoxylin and eosin (H&E) staining was conducted on colonic tissue sections from the different treatment groups. Upper panel: Representative images of 100× magnification. Lower panel: Higher-magnification images (200×) of the dotted areas in the upper panel. ( C ) Myeloperoxidase (MPO) activity was assessed in the inflamed distal colons (4.0 cm). ( D ) Levels of CINC-3 in the inflamed colon were quantified using an ELISA kit. ( E ) Expression of iNOS and COX-2 in the inflamed colon was analyzed by Western blotting. α-Tubulin was used as a loading control to normalize iNOS and COX-2 levels. The data are presented as mean ± SD ( n = 5). * p < 0.05, vs. DNBS control. # p < 0.05, vs. PBN. ## p < 0.05. NM: not measurable.

Article Snippet: PBN and DNBS were sourced from Tokyo Chemical Industry (Tokyo, Japan) and SSZ was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

Techniques: Staining, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control

Journal: Advanced Science

Article Title: Acod1 Promotes PAD4 Ubiquitination via UBR5 Alkylation to Modulate NETosis and Exert Protective Effects in Sepsis

doi: 10.1002/advs.202411652

Figure Lengend Snippet: Acod1 is closely associated with sepsis‐related NETs. A) Volcano plot of differentially expressed genes in PBNs from C57BL/6 wild‐type mice between the CLP and Sham groups; B) Volcano plot of differentially expressed metabolites; C) GO enrichment analysis of differentially expressed genes; D) KEGG pathway enrichment analysis of differentially expressed genes; E,F) Pearson correlation analysis was conducted on the levels of MPO‐DNA and CitH3‐DNA in peripheral blood and the Acod1 mRNA level of patients with sepsis; G,H) Pearson correlation analysis was carried out on the SOFA score and APACHE II score of patients with sepsis and the Acod1 mRNA level. n=3 per group (A‐D), n=20 per group (E‐H). Student's t ‐test is used to compare two groups of data affected by a single variable. For the comparison of multiple groups of data, one‐way ANOVA is adopted, and Dunnett's multiple comparisons test is used for post hoc analysis. All data are presented as mean ± standard deviation. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse peripheral blood neutrophils (PBNs) were isolated from the whole blood of 8‐week‐old male C57BL/6 mice using the Mouse Peripheral Blood Neutrophil Isolation Kit (Solarbio, Beijing, China).

Techniques: Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Acod1 Promotes PAD4 Ubiquitination via UBR5 Alkylation to Modulate NETosis and Exert Protective Effects in Sepsis

doi: 10.1002/advs.202411652

Figure Lengend Snippet: Acod1 knockout promotes NETs formation. Immunohistochemical staining of the neutrophil marker Ly6G in mouse lung tissue (A) shows representative images and (B) quantitative analysis (scale bar = 50 µm). C–F) display the concentration levels of NETs markers CitH3‐DNA and MPO‐DNA complexes in the peripheral blood of mice. G) shows representative images and (H,I) quantitative analysis of NETs markers (CitH3 and MPO) in mouse lung tissue (scale bar = 20 µm). J) presents representative scanning electron microscopy images of PBN cell morphology in mouse peripheral blood (scale bar = 5 µm). n = 5 per group. Student's t ‐test is used to compare two groups of data affected by a single variable. For the comparison of multiple groups of data, one‐way ANOVA is adopted, and Dunnett's multiple comparisons test is used for post hoc analysis. All data are presented as mean ± standard deviation. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse peripheral blood neutrophils (PBNs) were isolated from the whole blood of 8‐week‐old male C57BL/6 mice using the Mouse Peripheral Blood Neutrophil Isolation Kit (Solarbio, Beijing, China).

Techniques: Knock-Out, Immunohistochemical staining, Staining, Marker, Concentration Assay, Electron Microscopy, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Acod1 Promotes PAD4 Ubiquitination via UBR5 Alkylation to Modulate NETosis and Exert Protective Effects in Sepsis

doi: 10.1002/advs.202411652

Figure Lengend Snippet: Acod1 regulates NETosis through the itaconate pathway. In Acod1 ‐/‐ PBNs cells, a series of Acod1 plasmids and mutant plasmids were transfected. A,B) The concentrations of NETs markers CitH3‐DNA and MPO‐DNA complexes in the cell supernatants were detected by ELISA. C) The fluorescence intensity of CitH3 and MPO was assessed by immunofluorescence (scale bar = 20 µm). In WT PBNs cells, overexpression of Acod1 or addition of exogenous ITA was performed. D,E) The concentrations of NETs markers CitH3‐DNA and MPO‐DNA complexes in the cell supernatants were measured by ELISA. n = 5 per group. Student's t ‐test is used to compare two groups of data affected by a single variable. For the comparison of multiple groups of data, one‐way ANOVA is adopted, and Dunnett's multiple comparisons test is used for post hoc analysis. All data are presented as mean ± standard deviation. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse peripheral blood neutrophils (PBNs) were isolated from the whole blood of 8‐week‐old male C57BL/6 mice using the Mouse Peripheral Blood Neutrophil Isolation Kit (Solarbio, Beijing, China).

Techniques: Mutagenesis, Transfection, Enzyme-linked Immunosorbent Assay, Fluorescence, Immunofluorescence, Over Expression, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Acod1 Promotes PAD4 Ubiquitination via UBR5 Alkylation to Modulate NETosis and Exert Protective Effects in Sepsis

doi: 10.1002/advs.202411652

Figure Lengend Snippet: Acod1/ITA promotes the degradation of the NETosis hub protein PAD4. A–C) qRT‐PCR was used to measure the mRNA levels in cells transfected with H159Q Acod1 plasmid, which does not express ITA, and OE‐Acod1 plasmid, which overexpresses ITA, under LPS stimulation. D) Western blot was used to detect the protein levels of PAD4, MPO, NE, and Acod1 in PBNs at different time points of LPS stimulation. E–H) Western blot was employed to measure and quantify the protein levels of PAD4, MPO, and NE in cells transfected with H159Q Acod1 and OE‐Acod1 plasmids under LPS stimulation. I) Western Blot was used to assess the PAD4 protein levels in PBNs cells under 20 µg mL −1 CHX treatment. J) PBNs cells were treated with CHX and stimulated with MG132 (10 µM) for 9 h. The PAD4 protein was determined by Western blotting. n = 5 per group (A‐C), n = 3 per group (D–J). Student's t ‐test is used to compare two groups of data affected by a single variable. For the comparison of multiple groups of data, one‐way ANOVA is adopted, and Dunnett's multiple comparisons test is used for post hoc analysis. All data are presented as mean ± standard deviation. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse peripheral blood neutrophils (PBNs) were isolated from the whole blood of 8‐week‐old male C57BL/6 mice using the Mouse Peripheral Blood Neutrophil Isolation Kit (Solarbio, Beijing, China).

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Acod1 Promotes PAD4 Ubiquitination via UBR5 Alkylation to Modulate NETosis and Exert Protective Effects in Sepsis

doi: 10.1002/advs.202411652

Figure Lengend Snippet: Acod1/ITA regulates NETosis by promoting the K48‐linked ubiquitin‐dependent degradation of PAD4. A) Ubiquitination detection of PAD4 protein. B) Representative immunofluorescence images of CitH3 and MPO in mouse PBNs (scale bar = 20 µm). C,D) Concentration levels of CitH3‐DNA and MPO‐DNA complexes in the culture supernatants of mouse PBNs cells. E) Scanning electron microscopy image of mouse PBNs cells (scale bar = 5 µm). Sample sizes: n=3 for (A), n=5 for (B‐E). Student's t ‐test is used to compare two groups of data affected by a single variable. For the comparison of multiple groups of data, one‐way ANOVA is adopted, and Dunnett's multiple comparisons test is used for post hoc analysis. All data are presented as mean ± standard deviation. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse peripheral blood neutrophils (PBNs) were isolated from the whole blood of 8‐week‐old male C57BL/6 mice using the Mouse Peripheral Blood Neutrophil Isolation Kit (Solarbio, Beijing, China).

Techniques: Ubiquitin Proteomics, Immunofluorescence, Concentration Assay, Electron Microscopy, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Acod1 Promotes PAD4 Ubiquitination via UBR5 Alkylation to Modulate NETosis and Exert Protective Effects in Sepsis

doi: 10.1002/advs.202411652

Figure Lengend Snippet: Acod1 promotes PAD4 degradation in a UBR5‐dependent manner. A) Proteomic analysis identifies proteins that bind to PAD4 in PBNs cells, showing the proteins with the highest binding scores. B,C) Co‐IP assays demonstrate the interaction between PAD4 and UBR5. D) Schematic representation of the full‐length PAD4 protein and its truncated domains. E) Immunoprecipitation assays assess the interaction between full‐length PAD4 (aa 1–663), PAD4‐N (aa 1–118), PAD4‐M (aa 119–522), or PAD4‐C (aa 523–663) with UBR5. F) Effects of UBR5 knockdown on PAD4 protein levels in PBNs cells are assessed. G) Immunofluorescence analysis measures the fluorescence intensity of CitH3 and MPO in the supernatant of PBNs cell cultures (scale bar = 20 µm). H,I) Concentration levels of CitH3‐DNA and MPO‐DNA complexes in the supernatant of PBNs cell cultures are determined. n=3 per group (B, C, E, and F); n=5 per group (H‐I). Student's t ‐test is used to compare two groups of data affected by a single variable. For the comparison of multiple groups of data, one‐way ANOVA is adopted, and Dunnett's multiple comparisons test is used for post hoc analysis. All data are presented as mean ± standard deviation. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse peripheral blood neutrophils (PBNs) were isolated from the whole blood of 8‐week‐old male C57BL/6 mice using the Mouse Peripheral Blood Neutrophil Isolation Kit (Solarbio, Beijing, China).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Knockdown, Immunofluorescence, Fluorescence, Concentration Assay, Comparison, Standard Deviation

Journal: Advanced Science

Article Title: Acod1 Promotes PAD4 Ubiquitination via UBR5 Alkylation to Modulate NETosis and Exert Protective Effects in Sepsis

doi: 10.1002/advs.202411652

Figure Lengend Snippet: Acod1 enhances the enzymatic activity of UBR5 through alkylation modification, thereby promoting the ubiquitination and degradation of PAD4. A) Proteomic analysis revealed alkylation modification of UBR5 protein; B,C) PBNs cells were infected with wild‐type UBR5 (referred to as WT) and the indicated mutants. After 2 h of incubation with either ITalk probe or vehicle control, the probe‐labeled proteins were detected by Western blotting. D) Western blotting assessed the effect of the Cys2768 mutation in UBR5 on PAD4 protein ubiquitination. E) Interaction model between ITA and UBR5 (dashed lines represent hydrogen bonds). F) Interaction model between UBR5 and PAD4 (dashed lines represent hydrogen bonds). n=3 per group for (B‐D). Student's t‐test is used to compare two groups of data affected by a single variable. For the comparison of multiple groups of data, one‐way ANOVA is adopted, and Dunnett's multiple comparisons test is used for post hoc analysis. All data are presented as mean ± standard deviation. Differences were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Mouse peripheral blood neutrophils (PBNs) were isolated from the whole blood of 8‐week‐old male C57BL/6 mice using the Mouse Peripheral Blood Neutrophil Isolation Kit (Solarbio, Beijing, China).

Techniques: Activity Assay, Modification, Ubiquitin Proteomics, Infection, Incubation, Control, Labeling, Western Blot, Mutagenesis, Comparison, Standard Deviation