alx fpr2 inhibitor wrw4 hyp1119  (MedChemExpress)

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: Intestinal epithelial Fpr2 deficiency in mice impairs intestinal epithelium homeostasis. (a and b) Expression of Fpr2 mRNA in tissues and organs of IEC-specific Fpr2 knockout mice ( Fpr2 VKO ) and control mice (Fpr2 f/f ) determined by RT-qPCR. n = 3 mice/group. (c) Expression of FPR2 protein in ileal organoids derived from Fpr2 f/f and Fpr2 VKO mice determined by western blot analysis. n = 3 mice/group. (d) Body weight of Fpr2 f/f and Fpr2 VKO mice after birth. n = 10 mice/group. (e and f) Length of the small intestine (e) and colon (f) in Fpr2 f/f and Fpr2 VKO mice. n = 8 mice/group. (g) Representative H&E staining images (left) of jejunal, ileal, and colonic sections from Fpr2 f/f and Fpr2 VKO mice, with quantification of villus height and crypt depth (right). n = 6 mice/group. Scale bar, 50 μm. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, by unpaired two-tailed Student’s t test.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Expressing, Knock-Out, Control, Quantitative RT-PCR, Derivative Assay, Western Blot, Staining, Two Tailed Test

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: Alteration of IECs in intestinal epithelial Fpr2 -deficient mice. (a) Representative PAS staining images of jejunal, ileal, and colonic sections from Fpr2 f/f and Fpr2 VKO mice, with quantification of positive cells. (b–d) Representative images of immunofluorescence staining for CHGA (b) and LYZ (c), and immunohistochemical staining for OLFM4 (d) in sections of jejunum, ileum, and colon from Fpr2 f/f and Fpr2 VKO mice, with quantification of positive cells. (e–j) The expression of various IEC type marker genes and the cell proliferation marker gene Mki67 determined by RT-qPCR in small intestinal tissues of Fpr2 f/f and Fpr2 VKO mice. n = 6 mice/group. Scale bar, 50 μm. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, by unpaired two-tailed ­Student’s t test.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Staining, Immunofluorescence, Immunohistochemical staining, Expressing, Marker, Quantitative RT-PCR, Two Tailed Test

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: Fpr2 deficiency in mouse intestinal epithelium impairs cell proliferation in intestinal crypts. (a) Representative images of immunohistochemical staining for Ki67 in sections of jejunum, ileum, and colon from Fpr2 f/f and Fpr2 VKO mice, with quantification of positive cells. n = 6 mice/group. (b) Representative images of immunofluorescence staining for BrdU in sections of jejunum, ileum, and colon after intraperitoneal injection of BrdU for 2 h in Fpr2 f/f and Fpr2 VKO mice, with quantification of positive cells. n = 6 mice/group. (c) Representative images of TUNEL assays of jejunal, ileal, and colonic sections from Fpr2 f/f and Fpr2 VKO mice, with quantification of TUNEL + apoptotic cells (green). n = 4 mice/group. Scale bar, 50 μm. Data are presented as mean ± SEM. *** P < 0.001, by unpaired two-tailed Student’s t test.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Immunohistochemical staining, Staining, Immunofluorescence, Injection, TUNEL Assay, Two Tailed Test

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: scRNA-seq of IECs from small intestinal crypts reveals aberrant ISC proliferation and differentiation in intestinal epithelial Fpr2 -deficient mice. (a and b) t -SNE visualization of clustering of single epithelial cells isolated from small intestinal crypts (23,078 cells from Fpr2 f/f mice, n = 3; 23,444 cells from Fpr2 VKO mice, n = 3) (a), and expression of IEC marker genes in each cluster (b). (c) Pseudotime trajectory analysis of all IECs. (d and e) t -SNE visua­lization of IC subclusters in intestinal crypts from Fpr2 f/f and Fpr2 VKO mice (d), and expression of IEC marker genes in each subcluster (e). (f) Pseudotime trajectory analysis of subclusters from (d). (g and h) t -SNE visualization of clustering of IECs in Fpr2 f/f and Fpr2 VKO mice (g) and percentage of each cluster (h). (i and j) t -SNE visualization of stem cell subclusters in Fpr2 f/f and Fpr2 VKO mice (i) and expression of stem cell marker genes and cell proliferation marker Mki67 in each subcluster (j). (k and l) t -SNE visualization of cell cycle phase distribution in the stem cell cluster from Fpr2 f/f and Fpr2 VKO mice (k) and percentage of cells in G1, G2/M, and S phases in each stem cell subcluster (l). (m and n) t -SNE visualization of stem cell subclusters in Fpr2 f/f and Fpr2 VKO mice (m) and percentage of each subcluster (n). (o) Analysis of cell cycles and quantification for cell cycle distribution in small intestinal ISCs from Lgr5-Fpr2 f/f and Lgr5 - Fpr2 KO mice. IECs were isolated from the small intestinal crypts of Lgr5-Fpr2 f/f and Lgr5 - Fpr2 KO mice. GFP high ISCs were gated to detect DNA content via flow cytometry and quantified for cell cycle distribution. Stem, intestinal stem cells; TA, transit-amplifying cells; IC, immature cells; E, enterocyte cells; EE, enteroendocrine cells; Tuft, tuft cells.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Isolation, Expressing, Marker, Flow Cytometry

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: Activation of intestinal epithelial FPR2 promotes the growth and budding of organoids derived from small intestinal crypts. Crypts were isolated from the ileum of Fpr2 f/f and Fpr2 VKO mice, followed by culturing with or without the following: 1 µmol/L MMK-1, 10 µmol/L fMLF, 1 µmol/L MMK-1, and 10 µmol/L WRW4, or 10 µmol/L fMLF and 1 µmol/L CyH. (a–f) Representative images of ileal organoids cultured for 3 and 5 days (a and d), and quantification of the number of buds per organoid (b, c, e, and f). Scale bar, 100 μm. n = 5/group. More than 130 organoids were counted in each group. (g–i) Representative images of immunofluorescence staining for Ki67 (green) and LYZ (red) in ileal organoids cultured for 5 days. Cell nuclei were stained with DAPI. Scale bar, 50 μm. CyH, cyclosporine H. Each group of organoids was cultured from the ileal crypts of three mice, and more than 30 organoids were counted in each group. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, by one-way ANOVA.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Activation Assay, Derivative Assay, Isolation, Cell Culture, Immunofluorescence, Staining

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: Activation of FPR2 promotes the proliferation and differentiation of ISCs by activating the Wnt, Hippo, and Notch signaling pathways via PKC-ERK. Ileal organoids derived from mice were treated with 1 μmol/L MMK-1 and examined for gene expression via RNA-seq. (a) Volcano plot showing DEGs in MMK-1-treated versus control organoids. n = 3/group. (b) KEGG pathway enrichment analysis of genes upregulated by MMK-1. (c) Heatmaps showing MMK-1-regulated genes involved in cell proliferation and ISC differentiation, as well as genes in the Wnt, Notch, Hippo, and MAPK signaling pathways, as detected via RNA-seq. (d) RT-qPCR analysis to verify the expression of MMK-1-regulated genes in organoids. (e) Representative images of immunofluorescence staining for active β-catenin (green) in MMK-1-treated organoids, along with a quantification of β-catenin-positive nuclei per organoid. Scale bar, 50 μm. (f−h) Expression analysis of MMK-1-induced genes treated with the Gβγ inhibitor Gallein, the PKC inhibitor Go6983, or the MEK inhibitor PD98059. The impact of Gallein (10 μmol/L), Go6983 (1 μmol/L), and PD98059 (10 μmol/L) on the aforementioned gene expression in organoids was examined via RT-qPCR analysis. (i) A schematic diagram illustrating the signaling pathways by which FPR2 activation regulates ISC proliferation and differentiation. n = 3/group. Each sample contains organoids derived from three mice. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, by unpaired two-tailed Student’s t -test.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Activation Assay, Protein-Protein interactions, Derivative Assay, Gene Expression, RNA Sequencing, Control, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Two Tailed Test

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: Intestinal epithelial Fpr2 deficiency in mice impairs small intestinal epithelium regeneration after irradiation-induced injury. (a) Schematic diagram of the experiments. Fpr2 f/f and Fpr2 VKO mice were exposed to 12 Gy X-rays and small intestines were harvested for analysis at different time points after IR. (b and c) Body weight (b) and small intestine length (c) of Fpr2 f/f and Fpr2 VKO mice after IR. (d) Representative H&E staining images of small intestine sections from Fpr2 f/f and Fpr2 VKO mice after IR, with quantification of villus height, crypt depth, and number of crypts per millimeter. (e) Representative images of immunohistochemical staining for OLFM4 in small intestine sections from Fpr2 f/f and Fpr2 VKO mice after IR, with quantification of OLFM4 + stem cells at the indicated time after IR. (f) Representative images of BrdU immunohistochemical staining of small intestine sections after intraperitoneal injection of BrdU for 2 h at the indicated time after IR in Fpr2 f/f and Fpr2 VKO mice, with quantification of positive cells. n = 3 mice/group on days 0, 1, 2 and 4; n = 5 mice/group on day 3 after IR. Scale bar, 50 μm. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, by unpaired two-tailed Student’s t -test.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Irradiation, Staining, Immunohistochemical staining, Injection, Two Tailed Test

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: Intestinal epithelial Fpr2 deficiency in mice impairs small intestinal epithelium regeneration after 5-FU-induced injury. (a) Schematic diagram of the experiments. Fpr2 f/f and Fpr2 VKO mice were injected intraperitoneally with 5-FU (50 mg/kg body weight/day) or equal volume of PBS, and small intestines were harvested at different time points for analysis. (b–d) Body weight (b), small intestine length (c), and colon length (d) of Fpr2 f/f and Fpr2 VKO mice during and after 5-FU or PBS treatment. (e) Representative H&E staining images of small intestine sections from Fpr2 f/f and Fpr2 VKO mice after 5-FU or PBS treatment, with quantification of villus height and crypt depth. (f) Representative OLFM4 immunohistochemical staining images of small intestine sections from 5-FU- or PBS-treated Fpr2 f/f and Fpr2 VKO mice, with quantification of OLFM4 + stem cells. (g) Representative BrdU immunohistochemical staining images of small intestine sections after intraperitoneal injection of BrdU for 2 h at the indicated time points after 5-FU or PBS treatment in Fpr2 f/f and Fpr2 VKO mice, with quantification of BrdU + cells. n = 6 mice/group. Scale bar, 50 μm. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, by two-way ANOVA.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Injection, Staining, Immunohistochemical staining

Journal: Life Metabolism

Article Title: Intestinal epithelial formyl peptide receptor 2 contributes to intestinal epithelium homeostasis and injury repair by regulating intestinal stem cell and transit-amplifying cell proliferation and differentiation

doi: 10.1093/lifemeta/loaf045

Figure Lengend Snippet: Activation of FPR2 promotes intestinal epithelium regeneration after irradiation in mice. Mice were administered either MMK-1 (10 mg/kg body weight/day, intraperitoneally) or an equal volume of PBS on the day of 10-Gy IR and for the subsequent 5 days. The small intestines were harvested at different time points after IR to examine histology. (a and b) Body weight (a) and survival rates (b) of mice after IR. (c–e) Representative H&E-stained images of small intestinal sections (c), and quantitative analyses of villus height (d) and crypt depth (e), at the indicated time points after IR. n = 6 mice/group on days 0, 3, 5, and 7 post-IR; n = 3 mice/group on day 1 post-IR. Scale bar, 50 μm. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001, by unpaired two-tailed Student’s t test.

Article Snippet: To determine whether the activation of FPR2 or FPR1 is involved in organoid growth and budding, the organoids were exposed to 1 μmol/L MMK-1 (MedChemExpress, Monmouth Junction, NJ, USA) in the presence or absence of FPR2 antagonist WRW4 (10 μmol/L, MedChemExpress); or to 10 μmol/L fMLF (MedChemExpress) in the presence or absence of the FPR1 anta gonist cyclosporine H (CyH, 1 μmol/L, MedChemExpress).

Techniques: Activation Assay, Irradiation, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Fumarate inhibits the formation of neutrophil extracellular traps (NETs) in a Nrf2-controlled and Annexin-A1-dependent manner associated with mitochondrial fusion

doi: 10.3389/fimmu.2026.1770063

Figure Lengend Snippet: Expression of Nrf2 and ANXA1 in relation to NET release by neutrophils treated with dimethyl fumarate. Neutrophils isolated from mice were either pre-treated with DMF (25 µM), ML385 (Nrf2 inhibitor), WRW4 (Fpr2 antagonist) for 1 h or their combination: DMF→ML385, WRW4→DMF incubated with lipopolysaccharide (LPS) at a concentration of 50 μg/mL (for 6 h) or left unstimulated (CTR). (A) Quantification of Nrf2 expression and (B) NETs (extDNA and citH3) quantification after Nrf2 inhibition. (Ci) Representative images and (Cii) area of NETs formed upon the above treatments with WRW4 and DMF. ExtDNA is shown in green and citH3 is shown in red. To visualize co-localization of extDNA with citH3 expression, the images from each channel were overlaid. (D) Levels of ANXA1 were measured in neutrophils supernatants upon the above treatments with DMF and ML385 by ELISA. The results are expressed as the mean values ± SD; n≥3. Values significantly different between the groups (p < 0.05) according to one-way ANOVA ( post hoc Bonferroni test) are designated by letters, where the same letter indicates no differences between groups (different letters indicate statistical differences). Explanation of abbreviations: Nrf2, nuclear factor erythroid 2-related factor 2; ANXA1, Annexin A1; extDNA, extracellular DNA; citH3, citrullinated histone H3.

Article Snippet: The following inhibitors were used: nuclear factor erythroid 2-related factor 2 (NRF2) inhibitor – ML385 (10 μM, MedChemExpress), formyl peptide receptor 2 (Fpr2) antagonist - WRW4 (10 μM, MedChemExpress), NADPH oxidase (NOX) inhibitor - Diphenyleneiodonium chloride (DPI, 10 μM, Merck), necroptosis inhibitor - Necrostatin-1 (50 μM, Torcis Bioscence, Bristol, UK), pyroptosis inhibitor - Disulfiram (30 μM, MedChemExpress) and mitochondrial complex I inhibitor - Rotenone (5 μM, Sigma-Aldrich, Saint Louis, MO, USA).

Techniques: Expressing, Isolation, Incubation, Concentration Assay, Inhibition, Enzyme-linked Immunosorbent Assay