Journal: Food Science & Nutrition

Article Title: Panax notoginseng Saponins Accelerate Diabetic Wound Repair by Minimizing Mitochondrial Dysfunction and Inhibiting Pyroptosis of Macrophage via Regulating Ephx2/11,12‐ EET Axis

doi: 10.1002/fsn3.72494

Figure Lengend Snippet: TPPU reduces macrophage pyroptosis and mitochondrial homeostasis without enhancing the efficacy of PNS. (A) ELISA was used to determine the level of 11,12‐EET in cell culture supernatant. (B) Representative images show macrophage differentiation. (C, D) CD86 levels in macrophages were analyzed by flow cytometry, accompanied by quantitative analysis. (E–H) ELISA was used to determine the level of IL‐1β, IL‐6, IL‐18, and IL‐10 in cell supernatant. (I, J) Flow cytometry was used to detect Annexin V/PI double‐positive regions in RAW264.7 cells, followed by quantitative analysis. (K–M) Western blotting was performed to assess the intracellular expression levels of NLRP3, Casp1, Casp1 p20, FL‐GSDMD, and N‐GSDMD, followed by quantitative analysis. (N, O) Immunofluorescence staining was carried out to detect GSDMD expression in RAW264.7 cells, followed by quantitative analysis. Scale bar: 20 μm. (P, Q) Western blotting was performed to assess the intracellular expression levels of Mfn1, Mfn2, OPA1, p‐Drp1, and Drp1, followed by quantitative analysis. Values were expressed as means ± SEM, n = 4–6. ** p < 0.01, vs. Con; # p < 0.05, ## p < 0.01 vs. HL.

Article Snippet: Intracellular and mitochondrial ROS levels in RAW264.7 cells (1 × 10 6 cells/sample) were assessed using the fluorescent probes 5 μM MitoSOX (Med Chem Express, HY‐D1055), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Analysis, Western Blot, Expressing, Immunofluorescence, Staining

Journal: Food Science & Nutrition

Article Title: Panax notoginseng Saponins Accelerate Diabetic Wound Repair by Minimizing Mitochondrial Dysfunction and Inhibiting Pyroptosis of Macrophage via Regulating Ephx2/11,12‐ EET Axis

doi: 10.1002/fsn3.72494

Figure Lengend Snippet: 14,15‐EEZE weakens the protection of the PNS against mitochondrial homeostasis. (A) ELISA was used to measure the level of 11,12‐EET in the supernatant of RAW264.7 cells. (B, C) The detection of mitoSOX‐positive areas reflects the mtROS levels in RAW264.7 cells, followed by quantitative analysis. (D) Mitochondrial status was visualized using MitoTracker staining and immunofluorescence in RAW264.7 cells. (E, F) The OCR and ECAR in RAW264.7 cells was measured using ELISA. (G, H) Fluorescence in the MitoTracker CMXRos/MitoTracker Green assay was assessed by flow cytometry, accompanied by quantitative analysis. Values were expressed as means ± SEM, n = 4–6. ** p < 0.01, vs. Con; # p < 0.05, ## p < 0.01 vs. HL; & p < 0.05, && p < 0.05, vs. PNS.

Article Snippet: Intracellular and mitochondrial ROS levels in RAW264.7 cells (1 × 10 6 cells/sample) were assessed using the fluorescent probes 5 μM MitoSOX (Med Chem Express, HY‐D1055), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Analysis, Staining, Immunofluorescence, Fluorescence, Flow Cytometry

Journal: Food Science & Nutrition

Article Title: Panax notoginseng Saponins Accelerate Diabetic Wound Repair by Minimizing Mitochondrial Dysfunction and Inhibiting Pyroptosis of Macrophage via Regulating Ephx2/11,12‐ EET Axis

doi: 10.1002/fsn3.72494

Figure Lengend Snippet: Ephx2/11,12‐EET is a key metabolic axis in the PNS promoting wound healing in DW mice. (A, B) Volcano plot analysis of differential metabolites between the NW and DW groups, and between the DW and PNS groups. (C) Venn diagram identifying common differential metabolites between the NW vs. DW comparison and the DW vs. PNS comparison. (D) Heatmap displaying the common differential metabolites. (E) Quantitative values of 11,12‐EET. (F) UMAP plots present the cell clusters in foot skin tissues from the DFU and NDFU cohorts in GSE165816 dataset. (G) Bar plots compare the proportional abundance of these cell clusters between the DFU and NDFU groups. (H, I) Differential expression of EPHX1 and EPHX2 in macrophages from the NDFU and DFU groups in GSE165816 dataset. (J) Cell viability of RAW264.7 cells was measured using the CCK‐8 assay. (K) mRNA expression levels of Cyp2j5, Cyp2j6, Cyp2j9, Ephx1, and Ephx2 in RAW264.7 cells were detected via qPCR. (L, M) Western blotting detected protein expression of Ephx2 in RAW264.7 cells with PNS‐L (30 μg/mL), PNS‐M (100 μg/mL), or PNS‐H (200 μg/mL), followed by quantitative analysis. (N) ELISA was used to determine the level of 11,12‐EET in cell culture supernatant. Values were expressed as means ± SEM, n = 4–6. * p < 0.05, ** p < 0.01, vs. NW; # p < 0.05, ## p < 0.05, vs. DW.

Article Snippet: Intracellular and mitochondrial ROS levels in RAW264.7 cells (1 × 10 6 cells/sample) were assessed using the fluorescent probes 5 μM MitoSOX (Med Chem Express, HY‐D1055), respectively.

Techniques: Analysis, Comparison, Quantitative Proteomics, CCK-8 Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Journal of Clinical and Translational Hepatology

Article Title: Single-cell RNA Sequencing Analysis Reveals That Targeting PLG–PLGRKT Signaling-mediated Pro-fibrotic Scar-associated Macrophages Ameliorates Liver Fibrosis in Biliary Atresia

doi: 10.14218/JCTH.2026.00278

Figure Lengend Snippet: (A) Schematic illustration of Trem2 antibody–siRNA conjugates used for Plgrkt knockdown in SAMs. (B) Uptake of Cy5-labeled Trem2–siRNA conjugates by RAW264.7 macrophages observed by fluorescence microscopy. (C) In vivo localization of Cy5-labeled Trem2–siRNA conjugates with F4/80-Cy3-positive macrophages in liver tissue. (D) Schematic overview of the BDL mouse model and treatment groups. (E) Western blot analysis of Plg-RKT protein levels in liver tissues from different experimental groups. (F) H&E and Sirius Red staining of liver sections from each group. (G) Representative flow cytometry analysis of SAMs (Cd45 + F4/80 + Trem2 + Cd9 + ) in liver tissue. (H) Quantification of SAM frequencies across experimental groups. (I) mRNA expression levels of SAM-associated genes ( Trem2 and Cd9 ) measured by RT-qPCR. (J) mRNA expression levels of fibrosis-related genes ( Spp1 , Anxa2 , Gpnmb , Lgals1 , and Lgals3 ) measured by RT-qPCR. Data represent n = 5 per group. * P < 0.05, ** P < 0.01, and *** P < 0.001. BDL, bile duct ligation; H&E, hematoxylin and eosin; RT-qPCR, reverse transcription quantitative polymerase chain reaction; SAMs, scar-associated macrophages; siRNA, small interfering RNA; PLGRKT, plasminogen receptor with a C-terminal lysine.

Article Snippet: For in vitro uptake, RAW264.7 macrophages (ATCC, Manassas, VA, USA) were seeded onto coverslips, incubated overnight, and exposed to Cy5-labeled TREM2–siRNA conjugates for 2 h. Cells were fixed with 4% paraformaldehyde, mounted in DAPI-containing mounting medium, and examined by fluorescence microscopy, as previously described.

Techniques: Knockdown, Labeling, Fluorescence, Microscopy, In Vivo, Tissue, Western Blot, Analysis, Staining, Flow Cytometry, Expressing, Quantitative RT-PCR, Ligation, Reverse Transcription, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Frontiers in Pharmacology

Article Title: Isobavachalcone attenuates experimental periodontitis by suppressing PTGS2-associated macrophage ferroptosis and restoring periodontal immune homeostasis

doi: 10.3389/fphar.2026.1913548

Figure Lengend Snippet: IBC mitigates macrophage ferroptosis via the PTGS2-MAPK-ROS-GPX4 axis. (A–G) Representative Western blots and quantitative analysis of iNOS, PTGS2, p-JNK, JNK, p-p38, p38, GPX4, and IL-1β in LPS-stimulated RAW264.7 cells treated with IBC in a dose-dependent manner. (H, J) Detection and quantification of iNOS, PTGS2, MAPK signaling proteins, and GPX4 in gingival tissues collected from rat models of experimental periodontitis. (I, K) Representative Western blots and quantitative analysis of the PTGS2-MAPK-GPX4 signaling axis in macrophages following OE-PTGS2 plasmid transfection and IBC treatment to assess pathway rescue. (L–P) Microscopic visualization of MitoSOX and FerroOrange signals, accompanied by the measurement of fluorescence intensity and MDA levels in cells undergoing OE-PTGS2 rescue treatments. (Q–T) Representative C11-BODIPY fluorescence imaging, GSH/GSSG ratio, and MDA content assessment in macrophages treated with the ROS scavenger NAC. Data Note: Data are presented as mean ± SD from three independent biological replicates (n = 6) for in vitro experiments, and n = 6 per group for in vivo analysis. Scale bar = 50 μm.

Article Snippet: The RAW264.7 murine macrophage cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States of America).

Techniques: Western Blot, Analysis, Plasmid Preparation, Transfection, Fluorescence, Imaging, In Vitro, In Vivo