Journal: Regenerative Therapy

Article Title: Prednisolone attenuates the immunomodulatory capacity and reshapes the functional phenotype of canine adipose-derived mesenchymal stromal cells

doi: 10.1016/j.reth.2026.101184

Figure Lengend Snippet: Effects of prednisolone on macrophage polarization and regulatory T (Treg) cell induction mediated by cADSCs. (a) Representative flow cytometry plots showing cluster designation (CD) 80 and CD206 expression in CD14 + DH82 cells. Cells cultured alone under control conditions or stimulated with interleukin (IL)-4/IL-13 or lipopolysaccharide (LPS)/interferon (IFN)-γ are shown, together with DH82 cells co-cultured with cADSCs pretreated with prednisolone at the indicated concentrations (0–100 μM) under LPS/IFN-γ stimulation. (b) Quantitative analysis of macrophage subsets, defined as CD14 + CD80 + CD206 - , CD14 + CD80 − CD206 + , CD14 + CD80 + CD206 + , and CD14 + CD80 − CD206 - cells. IL-4/IL-13 stimulation was included as an M2-polarizing control, whereas LPS/IFN-γ stimulation induced M1-like polarization. (c) Representative flow cytometry plots showing CD25 and Foxp3 expression in CD4 + T cells after co-culture of PBMCs with cADSCs pretreated with prednisolone. Red boxes indicate CD25 + Foxp3 + Treg cells. (d) Quantification of CD25 + Foxp3 + Treg cells within the CD4 + T-cell population. Data are presented as mean ± SD from three independent experiments using cADSCs derived from three independent donors. * p < 0.05 vs. control or PBMCs alone; † p < 0.05 vs. LPS/IFN-γ.

Article Snippet: To induce macrophage polarization, either a combination of 50 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) and 20 ng/mL recombinant canine interferon (IFN)-γ (R&D Systems), or a combination of 40 ng/mL recombinant human interleukin (IL)-4 (Thermo Fisher Scientific) and 40 ng/mL recombinant human IL-13 (Thermo Fisher Scientific), was added to the cultures, followed by incubation at 37 °C in 5% CO 2 for 72 h. At the end of the co-culture period, cells were collected, washed with FACS buffer, and incubated on ice with canine Fc receptor binding inhibitor.

Techniques: Flow Cytometry, Expressing, Cell Culture, Control, Analysis, Co-culture Assay, Derivative Assay

Journal: iScience

Article Title: IL-4 and arginine synergy accelerates early bone regeneration via enhanced efferocytosis and ameliorated microenvironment

doi: 10.1016/j.isci.2026.117739

Figure Lengend Snippet: IL-4 and Arg remodel macrophages and promote their migration (A) Schematic illustration of the regulatory role of IL-4 on macrophage phenotype. (B) Volcano plot displaying differentially expressed genes (DEGs) between the control and IL-4-treated macrophages. (C) Heatmap demonstrating the expression of key macrophage polarization markers. (D) Multi-pathway Gene Set Enrichment Analysis (GSEA) demonstrating the integrated enrichment of indicated pathways regulating macrophage functions. (E) Schematic illustration of macrophage priming for efferocytosis mediated by IL-4 and Arg. (F) Relative cell viability of macrophages treated with different concentrations of Arg at 24, 48, and 72 h. (G and H) Macrophage proliferation evaluated by 5-ethynyl-2′-deoxyuridine (EdU) assay, showing representative fluorescence images of EdU and 4',6-diamidino-2-phenylindole (DAPI) staining at 24, 48, and 72 h (G) and quantification of EdU-positive cells treated with 2 mM Arg (H). (I) Schematic illustration of the Transwell migration assay. (J and K) Macrophage recruitment evaluated by Transwell assay, showing representative crystal violet staining images of migrated macrophages after 48 h (J) and quantification of the number of migrated macrophages (K). Error bars represent SD. Data were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test (F and H) or one-way ANOVA followed by Tukey’s post hoc test (K). Sample sizes: n = 3 biological replicates (B–D and F); n = 5 biological replicates, each performed with 3 technical replicates (G, H, J, and K). Scale bars, 25 μm (G), 500 μm (J, top), and 100 μm (J, bottom).

Article Snippet: The cumulative release of Arg was quantified using an Arg Content Assay Kit (Solarbio, Beijing, China), and the release of IL-4 was determined using a Mouse IL-4 ELISA Kit (Elabscience, Wuhan, China).

Techniques: Migration, Control, Expressing, Analysis, EdU Assay, Fluorescence, Staining, Transwell Migration Assay, Transwell Assay

Journal: iScience

Article Title: IL-4 and arginine synergy accelerates early bone regeneration via enhanced efferocytosis and ameliorated microenvironment

doi: 10.1016/j.isci.2026.117739

Figure Lengend Snippet: Simultaneous application of IL-4 and Arg promotes efferocytosis and improves the pro-regenerative microenvironment (A) Schematic illustration of the in vitro efferocytosis assessment. (B and C) Macrophage efferocytosis assessment via flow cytometry, showing representative scatterplots of macrophages engulfing apoptotic cells (B) and quantification of the efferocytosis rate after 24 h (C). (D) Morphological evaluation of macrophage efferocytosis, showing representative cell tracking images of macrophages (labeled with CMFDA) engulfing apoptotic cells (labeled with CM-Dil) after 24 h of co-culture. (E) Quantification of the efferocytosis rate after 6, 24, and 48 h determined by flow cytometry under Control and IL-4 + Arg conditions. (F) Experimental design for detecting microenvironmental changes following efferocytosis. (G) ROS quantification following efferocytosis. (H) Enzyme-linked immunosorbent assay (ELISA) quantification of TGF-β1 and IL-10 in the culture supernatants. Error bars represent SD. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test (C, G, and H) or two-way ANOVA followed by Sidak’s multiple comparisons test (E). Sample sizes: n = 3 biological replicates (B–E); n = 5 biological replicates, each performed with 3 technical replicates (G) and n = 3 biological replicates, each performed with 3 technical replicates (H). Scale bars, 100 μm (D) and 20 μm (inset).

Article Snippet: The cumulative release of Arg was quantified using an Arg Content Assay Kit (Solarbio, Beijing, China), and the release of IL-4 was determined using a Mouse IL-4 ELISA Kit (Elabscience, Wuhan, China).

Techniques: In Vitro, Flow Cytometry, Cell Tracking Assay, Labeling, Co-culture Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: IL-4 and arginine synergy accelerates early bone regeneration via enhanced efferocytosis and ameliorated microenvironment

doi: 10.1016/j.isci.2026.117739

Figure Lengend Snippet: Transcriptomic changes underlying synergistic regulation of macrophages by IL-4 and Arg (A) RT-qPCR analysis of M2-related marker gene expression ( Tgfb1 , Il10 , Arg1 , and Cd206 ) in macrophages. (B) Schematic illustration of the bone marrow-derived macrophages (BMDMs) isolation, total RNA extraction, and transcriptomic sequencing workflow. (C) Functional annotation of upregulated genes using Gene Ontology (GO) enrichment analysis. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis emphasizing significant signaling pathways. (E) RT-qPCR analysis of Tgfb1 and Il10 mRNA expression levels of macrophage after treatment with the IL-4 + Arg combination in the presence or absence of nor-NOHA. (F) ELISA quantification of secreted TGF-β1 and IL-10 protein concentrations in the culture supernatants under nor-NOHA inhibition. Error bars represent SD. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test (A, E, and F). Sample sizes: n = 3 biological replicates (C and D); n = 3 (A and F) and 4 (E) biological replicates, each performed with 3 technical replicates.

Article Snippet: The cumulative release of Arg was quantified using an Arg Content Assay Kit (Solarbio, Beijing, China), and the release of IL-4 was determined using a Mouse IL-4 ELISA Kit (Elabscience, Wuhan, China).

Techniques: Quantitative RT-PCR, Analysis, Marker, Gene Expression, Derivative Assay, Isolation, RNA Extraction, Sequencing, Functional Assay, Protein-Protein interactions, Expressing, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: iScience

Article Title: IL-4 and arginine synergy accelerates early bone regeneration via enhanced efferocytosis and ameliorated microenvironment

doi: 10.1016/j.isci.2026.117739

Figure Lengend Snippet: Simultaneous application of IL-4 and Arg promotes bone regeneration in vivo (A) Schematic illustration of the surgical procedure and timeline. (B and C) Evaluation of early macrophage polarization in vivo , showing representative immunofluorescence images of macrophages (F4/80, red) and M2 marker (Arg1, green) at days 1 and 3 post-surgery (B) and quantification of the percentage of M2 macrophages (Arg1 + /F4/80 + ) (C). (D–F) Bone regeneration assessment via micro-CT, showing representative 3D reconstruction images of the bone defect area at day 7 and day 14 post-surgery (D) and quantification of BV (E) and BV/TV (F). (G and H) Histological evaluation of bone repair, showing representative images of H&E and Masson’s trichrome staining at day 7 and day 14 (G) and quantification of new bone area (%) (H). Error bars represent SD. Data were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test (C, E, F, and H). Sample sizes: n = 4 animals per group. Scale bars, 50 μm (B) and 10 μm (inset), 0.5 mm (D) and 200 μm (inset), 250 μm (G).

Article Snippet: The cumulative release of Arg was quantified using an Arg Content Assay Kit (Solarbio, Beijing, China), and the release of IL-4 was determined using a Mouse IL-4 ELISA Kit (Elabscience, Wuhan, China).

Techniques: In Vivo, Immunofluorescence, Marker, Micro-CT, Staining

Journal: iScience

Article Title: IL-4 and arginine synergy accelerates early bone regeneration via enhanced efferocytosis and ameliorated microenvironment

doi: 10.1016/j.isci.2026.117739

Figure Lengend Snippet:

Article Snippet: The cumulative release of Arg was quantified using an Arg Content Assay Kit (Solarbio, Beijing, China), and the release of IL-4 was determined using a Mouse IL-4 ELISA Kit (Elabscience, Wuhan, China).

Techniques: Cell-Signaling, Recombinant, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Software

Journal: Food Science & Nutrition

Article Title: Ginsenoside Rb1 From the Functional Food Panax ginseng Suppresses Pancreatic Cancer Progression via Macrophage Reprogramming Through the PI3K ‐ AKT Pathway

doi: 10.1002/fsn3.72468

Figure Lengend Snippet: Food‐derived bioactive compound GRB1 inhibits M2 macrophage polarization and pancreatic cancer progression via PI3K–AKT signaling. (A) Schematic of macrophage differentiation and co‐culture workflow. PBMCs or THP‐1 cells were induced to M2 macrophages with IL‐4, with or without GRB1 treatment. GRB1‐treated macrophages were co‐cultured with pancreatic cancer organoids or co‐injected with ASPC1 cells in vivo. (B) qRT–PCR showing reduced expression of M2 markers (CD206, TGFB1, CD115) after GRB1 treatment. (C) Western blot showing that GRB1 inhibited PI3K and AKT phosphorylation in macrophages. (D) Bright‐field images of organoids co‐cultured with control, M2, or GRB1‐treated M2 macrophages, showing reduced growth in the GRB1 group. (E–G) Representative liver images, H&E/IHC staining for CD163 and CD206, and quantification showing that GRB1 decreased M2 macrophage infiltration and liver metastases. Data are presented as mean ± SEM. ns, no significance; * p < 0.01, ** p < 0.001, *** p < 0.0001.

Article Snippet: Gemcitabine (Solarbio, China, G8970), Ginsenoside Rb1 (Macklin, Shanghai, G742231), Mouse Ki67 (Abclonal, China, A20018); CK19 (Abclonal, China, A25546); SOX2 (Abclonal, China, A19118); Vimentin (Abclonal, China, A19607); Zeb‐1 (Abclonal, China, A5600); E‐cadherin (Abcam, USA, ab40772); AKT (Abclonal, China, AP0637); P‐AKT (Abclonal, China, AP0637); PI3K (Abclonal, China, A27717); P‐PI3K (Abclonal, China, A27717); GAPDH (Abclonal, China, A19056); CD45 (BioLegend, USA, 103106); CD3 (BioLegend, USA, 100219); CD11b (BioLegend, USA, 101224); CD206 (Abclonal, China, A21014); CD206 (BioLegend, USA, 141721); CD163 (BioLegend, USA, 155327); ARG1 (BioLegend, USA, 165804); CD86 (BioLegend, USA, 105013); M‐CSF (MCE, USA, HY‐ P70488 ); IL4 (MCE, USA, HY‐139092); PMA (MCE, USA, HY‐18739); collagenase Type IV (Sigma, USA, C5138); deoxyribonuclease I (Thermo, USA, EN0521); Matrigel (Corning, USA, 354234); Organoid culture medium (Omabio, China, OM12).

Techniques: Derivative Assay, Co-culture Assay, Cell Culture, Organoids, Injection, In Vivo, Quantitative RT-PCR, Expressing, Western Blot, Phospho-proteomics, Control, Immunohistochemistry