Journal: Inflammation

Article Title: Hepatocyte-Derived IL-25 Promotes Macrophage Extracellular Trap Formation and Drives Liver Fibrosis Progression

doi: 10.1007/s10753-026-02506-6

Figure Lengend Snippet: IL-25 to the IL-25 receptor on macrophages triggers ROS burst via NOX activation and mitophagy, consequently inducing lysosomal activation and MET formation. A Cell ROS assay of RAW264.7 by DCFH-DA with the semi-quantitative results of the processed images from each group ( n = 6 per group). B Observation of METs formation in RAW cells by SYTOX Green staining, with Hochest for nuclear counterstaining with the semi-quantitative results of the processed images from each group ( n = 6 per group). C Observation of lysosome and mitochondrial morphology in RAW cells under transmission electron microscopy, red arrows indicate activated lysosomes and autophagosome formation, and blue arrows indicate METs formation. ( n = 4 per group). D Mitochondrial & Lysosomes staining in RAW cell with the semi-quantitative results of the processed images from each group ( n = 6 per group). E WB analysis of CitH3 expression in RAW cells with the semi-quantitative results from the grayscale analysis of protein bands for each group, representative western blots from three independent experiments are shown, each lane represents a biological replicate, and a subset of samples is displayed for clarity ( n = 6 per group). F-G WB analysis of phosphorylated p47phox protein expression in RAW cells and liver tissues with the semi-quantitative results from the grayscale analysis of protein bands for each group, representative western blots from three independent experiments are shown, each lane represents a biological replicate, and a subset of samples is displayed for clarity ( n = 6 per group). Cells were pretreated with VAS2870 25 μM for 30 min prior to stimulation, NAC 10 μM for 1 h before stimulation, CQ 10 μM for 2 h before stimulation. Mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Student’s t-test

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked, and incubated with primary antibodies: α-SMA (Cat. No. A17910, ABclonal), COL1A1 (Cat. No. ab260043, Abcam), CitH3 (Cat. No. ab219407, Abcam), phospho-p47phox (Cat. No. AF3917, Affinity Biosciences), β-actin (Cat. No. GB15001, Servicebio) (all 1:1000).

Techniques: Activation Assay, ROS Assay, Staining, Transmission Assay, Electron Microscopy, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Inhaled bovine lactoferrin modulates the p47phox–MPO–NETosis axis in acute lung injury: implications for bioengineered nanomedicine in respiratory infections

doi: 10.3389/fimmu.2026.1760949

Figure Lengend Snippet: GSEA was performed on the entire list of captured proteins from all four time points, regardless of their differential expression status, using the KEGG pathway database. A Venn diagram analysis of all enriched pathways across the time points (A) identified five pathways uniquely and commonly enriched at both D1 and D3, including “NET formation”, “ECM-receptor interaction”, “Circadian entrainment”, “Hypertrophic cardiomyopathy”, “Arrhythmogenic right ventricular cardiomyopathy”, and “Retrograde endocannabinoid signaling”. Notably, “NET formation” was the only pathway that overlapped with those identified in previous analyses. The proteins enriched in this pathway and LTF are displayed in a heatmap (B, C) , showing the upregulation of proteins such as MPO, p47phox, PADI4, NCF2, and NCF4. The GSEA enrichment score plots for the “NET formation” pathway at the D1 and D3 time points are shown in (D, E) , respectively.

Article Snippet: Antibodies used in western blotting were: LTF (Proteintech, China, #31267-1-AP), MPO (Immunoway, USA, YM8618), p47phox (Proteintech, China, #28187-1-AP), p-p47phox (Immunoway, China, #YP0828) and β-actin (QuaYad, China, #QYA10734A).

Techniques: Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Inhaled bovine lactoferrin modulates the p47phox–MPO–NETosis axis in acute lung injury: implications for bioengineered nanomedicine in respiratory infections

doi: 10.3389/fimmu.2026.1760949

Figure Lengend Snippet: Exogenous bLF attenuates LPS-induced acute lung injury and oxidative stress response at D1. Mice were divided into three groups ( n = 8 per group): CON_D1 (saline control), LPS_D1 (5 mg/kg LPS), and bLF_D1 (bLF administered via pulmonary delivery 1-2 h before LPS). (A) ELISA of lung homogenates ( n = 8) showing that LPS-induced elevations in IL-1β, IL-6, and TNF-α were reduced by bLF treatment, while the decreased anti-inflammatory cytokine IL-10 was restored and increased after bLF administration. (B) H&E staining ( n = 8) demonstrating that alveolar hemorrhage, neutrophil infiltration, and interstitial thickening in LPS_D1 lungs were alleviated in the bLF_D1 group. (C, D) Immunofluorescence staining ( n = 8) of Ly6G and MPO revealed increased fluorescence intensity following LPS stimulation, which was suppressed by bLF treatment. (E, F) qPCR analysis of lung tissue ( n = 8) indicating that LPS upregulates the mRNA expression of Ltf, Mpo, Ncf1, while bLF treatment reversed these changes. (G, H) Western blot analysis ( n = 4) confirmed that protein levels of MPO, p47phox, and p-p47phox were elevated in the LPS_D1 group and downregulated after bLF treatment. * p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant.

Article Snippet: Antibodies used in western blotting were: LTF (Proteintech, China, #31267-1-AP), MPO (Immunoway, USA, YM8618), p47phox (Proteintech, China, #28187-1-AP), p-p47phox (Immunoway, China, #YP0828) and β-actin (QuaYad, China, #QYA10734A).

Techniques: Saline, Control, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Fluorescence, Expressing, Western Blot

Journal: Materials Today Bio

Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102697

Figure Lengend Snippet: Lentivirus mediated p47phox knockdown or miR-19b-3p overexpression attenuated ALF, but with limited synergistic biological effects compared with miR-19-EXO. (A) Pattern diagram of ALF model and lentivirus pre-treatment. (B) 24 h-survival rate of Sham rats and ALF rats pre-treated with indicated lentivirus(2.5 × 10 8 U/kg)were recorded using Kaplan–Meier survival analysis (n = 8). (C) Hepatic function indicators ALT, AST, ALB, LDH, TBIL and ALP level in rat serum were detected using Full-automatic chemistry analyzer (n = 8). (D) ROS level rat liver tissues were measured using DCFH-DA probe method in indicated group. (E) Representative images showing the morphology in liver sections stained with H&E, scale bar = 100 μm. (F) Pro-inflammation associated genes of Tnfα , Il-6 , Il-1β , IFN-γ , IL1α and (G) anti-inflammation associated genes Il-10 and Il1ra mRNA level were detected using RT-PCR in rat liver tissues. Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01).

Article Snippet: The primary antibodies used for IHC staining were rabbit anti-p47phox antibody (1:100, Abbkine, ABP52116 , USA), rabbit anti-iNOS1 antibody (1:50, Abclonal, A14031, China), rabbit anti-Arginase1 antibody (1:100, CST, 93668S, USA), rabbit anti-CD68 antibody (1:100, Absin, abs120102, China), rabbit anti-CD206 antibody (1:200, CST, #24595S, USA).

Techniques: Knockdown, Over Expression, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: Materials Today Bio

Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102697

Figure Lengend Snippet: The ROS-scavenging drug NAC exerted finite ameliorated effect in ALF rats relative to miR-19-EXO. (A) Pattern diagram of ALF model and NAC treatment strategy. SD rats were divided to Sham group or subjected to D-GalN/LPS induced ALF. For ALF + NAC group, NAC treatment (200 mg/kg) were administrated 3 h after intraperitoneal injection of D-GalN/LPS in ALF rat model. (B) 24h-survival rate of Sham group,ALF group and ALF treated NAC (20 mg/kg) group were recorded using Kaplan–Meier survival analysis (n = 8). (C) H&E staining of liver sections, scale bar = 50 μm.(D) Hepatic function indicators ALT, AST, ALB, LDH, TBIL and ALP level in rat serum were measured using Full-automatic chemistry analyzer (n = 8). (E) p47phox and NF-κB P65 and p-P65 protein level change in rat liver were determined using western blot. (F) ROS level in liver tissues were examined using DCFH-DA probe method. (G) Pro-inflammation associated genes of Tnfα , Il-6 , Il-1β , IFN-γ , IL1α , Icam , Mcp1 , Cxcl1 and (H) anti-inflammation associated genes Il-10 and Il1ra mRNA level were detected using RT-PCR in rat liver tissues (I) Representative image of CD86 and CD206 IHC staining expression in rat liver sections, scale bar = 100 μm. Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01).

Article Snippet: The primary antibodies used for IHC staining were rabbit anti-p47phox antibody (1:100, Abbkine, ABP52116 , USA), rabbit anti-iNOS1 antibody (1:50, Abclonal, A14031, China), rabbit anti-Arginase1 antibody (1:100, CST, 93668S, USA), rabbit anti-CD68 antibody (1:100, Absin, abs120102, China), rabbit anti-CD206 antibody (1:200, CST, #24595S, USA).

Techniques: Injection, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Expressing

Journal: Materials Today Bio

Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102697

Figure Lengend Snippet: miR-19b-3p suppressed p47phox mediated ROS production via targeting p47phox 3′UTR. (A) KEGG enrichment analysis of rat liver RNA-Seq from ALF (n = 3) and miR-19-EXO group (n = 4). (B) miRNA-mRNA network analysis of the top six ADSC-EXO exosomal miRNA (hsa-miR-221-3p, hsa-miR-29a-3p, hsa-miR-24-3p, hsa-miR-19b-3p, hsa-miR-193b-3p, hsa-miR-214-3p). (C) Dual-luciferase reporter system was constructed with psi-Check2 dual-luciferase plasmid cloned with p47phox mRNA 3′UTR, containing with miR-19b-3p predicted targeting sequence (p47phox WT) or mutated targeting sequence (p47phox MUT). The mutant nucleotides were highlighted. (D) The p47phox WT/MUT were co-transfected with miR-19b-3p Mimic NC/Mimic to 293T cells. After 48 h for transfection, the luciferase activity was determined. Data shown as 3 independent experiments with triplicate measurements. (E) Schematic represented the p47phox status and interaction network in gp91phox complex during NADPH oxidase activation or inactivation. (F–G) Western blot analysis for determining the protein expression of key protein in NADPH oxidase, including p47phox, gp91phox, RAC1 in RMa-bm cells transfected with miR-19b-3p Mimic or Inhibitor. The level of p47phox relative to GAPDH was statistically analyzed in (G). (H–I) Protein level of p47phox in rat liver were determined using western blots analysis (n = 3). The level of p47phox relative to GAPDH was statistically analyzed in (I). (J) ROS level in rat liver was measured using DCFH-DA probe. Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01).

Article Snippet: The primary antibodies used for IHC staining were rabbit anti-p47phox antibody (1:100, Abbkine, ABP52116 , USA), rabbit anti-iNOS1 antibody (1:50, Abclonal, A14031, China), rabbit anti-Arginase1 antibody (1:100, CST, 93668S, USA), rabbit anti-CD68 antibody (1:100, Absin, abs120102, China), rabbit anti-CD206 antibody (1:200, CST, #24595S, USA).

Techniques: RNA Sequencing, Luciferase, Construct, Plasmid Preparation, Clone Assay, Sequencing, Mutagenesis, Transfection, Activity Assay, Activation Assay, Western Blot, Expressing

Journal: Materials Today Bio

Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102697

Figure Lengend Snippet: miR-19-EXO dampened M1 macrophages polarization via inhibiting NF-κB canonical activation. (A) The procedure of THP1-based macrophage polarization assay. THP1 monocytes were treated with 100 ng/mL PMA for 48 h (for M0 induction), then treatment treated with 20 ng/mL LPS and 20 ng/mL IFN-γ for M1 subtype polarization, or 20 ng/mL IL4 and 20 ng/mL IL-13 or M2 subtype polarization, respectively. Representative morphology of M0, M1, M2 were recorded (scale bar = 100 μm). (B) and (D) RT-PCR analysis of M1 marker TNF-α , Il-6 and M2 marker Il-10 , CD163 in WT-EXO/miR-19-EXO treated M1 or M2 cells. (C) and (E) The macrophage polarization status in WT-EXO/miR-19-EXO treated M1/M2 cells was estimated by CD86 + and CD206 + cells ration using flow cytometry. (F) Thp1-M1 cells treated with miR-19-EXO or miR-19KD-EXO (100 μg/mL) for 24 h, then protein level of p47phox, NF-κB P65 and p-P65 were examined by immunoblotting. Thp1-M0 as control. (G) Thp1-M1 cell administrated with miR-19-EXO (100 μg/mL) for 24 h or NC were treated with CHX (100 μg/mL) for 0, 30, 60, 120, 240 min. Immunoblotting for iKBα expression levels in whole-cell extraction. (H) Thp1-M1 cell administrated with miR-19-EXO for 24 h or NC were treated with MG132 (10 mmol/L) for an extra 12h, then immunoblotting for iKBα protein levels in each group. (I) Thp1-M1 cell administrated with miR-19-EXO for 24 h or NC were immunoprecipitated with IgG control or iKBα antibody and then immunoblotted for Ubiquitin and iKBα. (J) Thp1-M1 cells treated with miR-19-EXO for 24 h were subjected to nuclear cytoplasmic separation assay, NF-κB P65 and IKKα level and translocation were examined by immunoblotting. LaminB1 was used as an internal control of nuclear protein, GAPDH as cytoplasmic protein control. (K) Schematic diagram for the mechanisms of IKK/NF-κB pathway activation suppressed by miR-19-EXO. (L)Thp1-M1 cells treated with miR-19-EXO or Thp1-M1 shp47phox cells or Thp1-M1 shp47phox cells treated with miR-19-EXO (100 μg/mL) for 24 h were detected the protein level of p47phox, NF-κB P65 and p-P65 by immunoblotting. Thp1-M1 as control (Ctrl). (M)Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01).

Article Snippet: The primary antibodies used for IHC staining were rabbit anti-p47phox antibody (1:100, Abbkine, ABP52116 , USA), rabbit anti-iNOS1 antibody (1:50, Abclonal, A14031, China), rabbit anti-Arginase1 antibody (1:100, CST, 93668S, USA), rabbit anti-CD68 antibody (1:100, Absin, abs120102, China), rabbit anti-CD206 antibody (1:200, CST, #24595S, USA).

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Flow Cytometry, Western Blot, Control, Expressing, Extraction, Immunoprecipitation, Ubiquitin Proteomics, Translocation Assay

Journal: Materials Today Bio

Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102697

Figure Lengend Snippet: The ROS-scavenging drug NAC exerted finite ameliorated effect in ALF rats relative to miR-19-EXO. (A) Pattern diagram of ALF model and NAC treatment strategy. SD rats were divided to Sham group or subjected to D-GalN/LPS induced ALF. For ALF + NAC group, NAC treatment (200 mg/kg) were administrated 3 h after intraperitoneal injection of D-GalN/LPS in ALF rat model. (B) 24h-survival rate of Sham group,ALF group and ALF treated NAC (20 mg/kg) group were recorded using Kaplan–Meier survival analysis (n = 8). (C) H&E staining of liver sections, scale bar = 50 μm.(D) Hepatic function indicators ALT, AST, ALB, LDH, TBIL and ALP level in rat serum were measured using Full-automatic chemistry analyzer (n = 8). (E) p47phox and NF-κB P65 and p-P65 protein level change in rat liver were determined using western blot. (F) ROS level in liver tissues were examined using DCFH-DA probe method. (G) Pro-inflammation associated genes of Tnfα , Il-6 , Il-1β , IFN-γ , IL1α , Icam , Mcp1 , Cxcl1 and (H) anti-inflammation associated genes Il-10 and Il1ra mRNA level were detected using RT-PCR in rat liver tissues (I) Representative image of CD86 and CD206 IHC staining expression in rat liver sections, scale bar = 100 μm. Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01).

Article Snippet: Following antibodies were used in our study: anti-Tsg101 antibody (A2216, ABclonal, China), anti-Alix antibody (92880S, CST, USA), anti-CD63 antibody (sc5275, Santa Cruz, USA), anti-Calnexin antibody (ABclonal, A4846, China), anti-LaminB antibody (Absin, abs131244, China), anti-iKBα antibody (Proteintech, 10268-1-AP60004-1-Ig, China), anti-NF-κB P65 antibody (Santa Cruz,sc-8008, China), anti-phospho-NFκb-p65 antibody (CST, 3033SS, USA), anti-p47phox antibody (Abbkine, ABP52116 , USA), anti-NOX2/gp91phox antibody (Abclonal, A1636, China), anti-Ubiquitin antibody (Invitrogen, 13–1600, USA),rabbit anti-GAPDH antibody (CST, 2118S,USA), mouse anti-GAPDH antibody (Proteintech, 60004-1-Ig, China), anti-rabbit IgG HRP-linked antibody (CST, 7074S, USA), and anti-mouse IgG HRP-linked antibody (CST, 7076S, USA).

Techniques: Injection, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Expressing

Journal: Materials Today Bio

Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102697

Figure Lengend Snippet: miR-19-EXO dampened M1 macrophages polarization via inhibiting NF-κB canonical activation. (A) The procedure of THP1-based macrophage polarization assay. THP1 monocytes were treated with 100 ng/mL PMA for 48 h (for M0 induction), then treatment treated with 20 ng/mL LPS and 20 ng/mL IFN-γ for M1 subtype polarization, or 20 ng/mL IL4 and 20 ng/mL IL-13 or M2 subtype polarization, respectively. Representative morphology of M0, M1, M2 were recorded (scale bar = 100 μm). (B) and (D) RT-PCR analysis of M1 marker TNF-α , Il-6 and M2 marker Il-10 , CD163 in WT-EXO/miR-19-EXO treated M1 or M2 cells. (C) and (E) The macrophage polarization status in WT-EXO/miR-19-EXO treated M1/M2 cells was estimated by CD86 + and CD206 + cells ration using flow cytometry. (F) Thp1-M1 cells treated with miR-19-EXO or miR-19KD-EXO (100 μg/mL) for 24 h, then protein level of p47phox, NF-κB P65 and p-P65 were examined by immunoblotting. Thp1-M0 as control. (G) Thp1-M1 cell administrated with miR-19-EXO (100 μg/mL) for 24 h or NC were treated with CHX (100 μg/mL) for 0, 30, 60, 120, 240 min. Immunoblotting for iKBα expression levels in whole-cell extraction. (H) Thp1-M1 cell administrated with miR-19-EXO for 24 h or NC were treated with MG132 (10 mmol/L) for an extra 12h, then immunoblotting for iKBα protein levels in each group. (I) Thp1-M1 cell administrated with miR-19-EXO for 24 h or NC were immunoprecipitated with IgG control or iKBα antibody and then immunoblotted for Ubiquitin and iKBα. (J) Thp1-M1 cells treated with miR-19-EXO for 24 h were subjected to nuclear cytoplasmic separation assay, NF-κB P65 and IKKα level and translocation were examined by immunoblotting. LaminB1 was used as an internal control of nuclear protein, GAPDH as cytoplasmic protein control. (K) Schematic diagram for the mechanisms of IKK/NF-κB pathway activation suppressed by miR-19-EXO. (L)Thp1-M1 cells treated with miR-19-EXO or Thp1-M1 shp47phox cells or Thp1-M1 shp47phox cells treated with miR-19-EXO (100 μg/mL) for 24 h were detected the protein level of p47phox, NF-κB P65 and p-P65 by immunoblotting. Thp1-M1 as control (Ctrl). (M)Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01).

Article Snippet: Following antibodies were used in our study: anti-Tsg101 antibody (A2216, ABclonal, China), anti-Alix antibody (92880S, CST, USA), anti-CD63 antibody (sc5275, Santa Cruz, USA), anti-Calnexin antibody (ABclonal, A4846, China), anti-LaminB antibody (Absin, abs131244, China), anti-iKBα antibody (Proteintech, 10268-1-AP60004-1-Ig, China), anti-NF-κB P65 antibody (Santa Cruz,sc-8008, China), anti-phospho-NFκb-p65 antibody (CST, 3033SS, USA), anti-p47phox antibody (Abbkine, ABP52116 , USA), anti-NOX2/gp91phox antibody (Abclonal, A1636, China), anti-Ubiquitin antibody (Invitrogen, 13–1600, USA),rabbit anti-GAPDH antibody (CST, 2118S,USA), mouse anti-GAPDH antibody (Proteintech, 60004-1-Ig, China), anti-rabbit IgG HRP-linked antibody (CST, 7074S, USA), and anti-mouse IgG HRP-linked antibody (CST, 7076S, USA).

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Flow Cytometry, Western Blot, Control, Expressing, Extraction, Immunoprecipitation, Ubiquitin Proteomics, Translocation Assay