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R&D Systems nmol l tumor necrosis factor
Nmol L Tumor Necrosis Factor, supplied by R&D Systems, used in various techniques. Bioz Stars score: 97/100, based on 1694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 97 stars, based on 1694 article reviews

nmol l tumor necrosis factor - by Bioz Stars, 2026-09

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In vivo immunomodulatory effects of different modified surfaces in a DM model. (A) Schematic representation of the animal modeling and experimental treatment workflow. (B, C) hematoxylin and eosin staining of the peri-implant tissues in the femurs of DM rats 1 week after implantation, accompanied by quantitative analysis of the fibrous capsule thickness (scale bar = 100 μm, n = 5). (D–G) Immunofluorescence staining evaluating the polarization state of macrophages surrounding the implants (green: macrophage marker cluster of differentiation (CD) 68; red: M1 marker CD86 and M2 marker CD206; blue: nuclei), along with corresponding quantitative analysis of the fluorescence signals (scale bar = 100 μm, n = 5). (H–K) Immunohistochemical staining assessing the expression of the pro-inflammatory marker tumor necrosis factor-α and the anti-inflammatory marker interleukin-10 in the peri-implant area, with quantitative results of the positive staining areas (scale bar = 100 μm, n = 5). Data are expressed as the mean ± standard deviation, with statistical analysis performed using one-way ANOVA and Tukey's post-hoc test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate statistical significance.

Journal: Bioactive Materials

Article Title: Integrated apoptotic extracellular vesicle-recruitment peptide coating reprograms the diabetic bone microenvironment and orchestrates enhanced implant osseointegration

doi: 10.1016/j.bioactmat.2026.05.059

Figure Lengend Snippet: In vivo immunomodulatory effects of different modified surfaces in a DM model. (A) Schematic representation of the animal modeling and experimental treatment workflow. (B, C) hematoxylin and eosin staining of the peri-implant tissues in the femurs of DM rats 1 week after implantation, accompanied by quantitative analysis of the fibrous capsule thickness (scale bar = 100 μm, n = 5). (D–G) Immunofluorescence staining evaluating the polarization state of macrophages surrounding the implants (green: macrophage marker cluster of differentiation (CD) 68; red: M1 marker CD86 and M2 marker CD206; blue: nuclei), along with corresponding quantitative analysis of the fluorescence signals (scale bar = 100 μm, n = 5). (H–K) Immunohistochemical staining assessing the expression of the pro-inflammatory marker tumor necrosis factor-α and the anti-inflammatory marker interleukin-10 in the peri-implant area, with quantitative results of the positive staining areas (scale bar = 100 μm, n = 5). Data are expressed as the mean ± standard deviation, with statistical analysis performed using one-way ANOVA and Tukey's post-hoc test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 indicate statistical significance.

Article Snippet: For IHC analysis, sections underwent heat-induced antigen retrieval and blocking prior to incubation with antibodies against TNF-α (GB11188, Servicebio, China), IL-10 (GB11534, Servicebio, China), and VEGF (GB15165, Servicebio, China) to identify differences in local inflammatory and angiogenic profiles.

Techniques: In Vivo, Modification, Staining, Immunofluorescence, Marker, Fluorescence, Immunohistochemical staining, Expressing, Standard Deviation

SEI inhibits DSS‐induced colonic inflammation and suppresses M1 polarization of macrophages. (A) qRT‐PCR analysis of inflammatory cytokines ( IL‐1β , IL‐6 , TNF‐α and IL‐23 ) ( n = 6). (B) ELISA for inflammatory cytokine production in colonic tissue, including IL‐1β, IL‐6, TNF‐α and IL‐23 ( n = 6). (C) The protein expression of iNOS and CD86 in colonic tissue was determined by WB ( n = 6). (D) Representative immunofluorescence images of colonic tissue stained for F4/80 (red) and CD86 (green) with DAPI (blue) for nuclear counterstaining. Scale bar = 50 µm. (E) Relative fluorescence intensity of CD86 + F4/80 + cells was quantified ( n = 4). (F) Flow cytometry gating strategy. (G) Representative flow cytometry dot plot. SEI reduced the levels of CD86 + M1‐type macrophages in the colonic lamina propria and increased the levels of F4/80 + CD206 + M2‐type macrophages. Bars are color‐coded to represent experimental groups: light blue = H 2 O‐treated normal control; red = DSS‐induced acute UC model; pale blue = 5‐ASA‐treated positive control (co‐administered with DSS); dark blue = low‐dose SEI (12.5 mg/kg) + DSS; purple = medium‐dose SEI (25 mg/kg) + DSS; green = high‐dose SEI (50 mg/kg) + DSS. Values were expressed as mean ± SD. # p < 0.05, ### p < 0.001 versus H 2 O group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS group.

Journal: Advanced Science

Article Title: Senkyunolide I Inhibits mtDNA‐cGAS‐STING Signaling in Macrophages via Targeting VDAC1 Oligomerization to Attenuate Ulcerative Colitis

doi: 10.1002/advs.77045

Figure Lengend Snippet: SEI inhibits DSS‐induced colonic inflammation and suppresses M1 polarization of macrophages. (A) qRT‐PCR analysis of inflammatory cytokines ( IL‐1β , IL‐6 , TNF‐α and IL‐23 ) ( n = 6). (B) ELISA for inflammatory cytokine production in colonic tissue, including IL‐1β, IL‐6, TNF‐α and IL‐23 ( n = 6). (C) The protein expression of iNOS and CD86 in colonic tissue was determined by WB ( n = 6). (D) Representative immunofluorescence images of colonic tissue stained for F4/80 (red) and CD86 (green) with DAPI (blue) for nuclear counterstaining. Scale bar = 50 µm. (E) Relative fluorescence intensity of CD86 + F4/80 + cells was quantified ( n = 4). (F) Flow cytometry gating strategy. (G) Representative flow cytometry dot plot. SEI reduced the levels of CD86 + M1‐type macrophages in the colonic lamina propria and increased the levels of F4/80 + CD206 + M2‐type macrophages. Bars are color‐coded to represent experimental groups: light blue = H 2 O‐treated normal control; red = DSS‐induced acute UC model; pale blue = 5‐ASA‐treated positive control (co‐administered with DSS); dark blue = low‐dose SEI (12.5 mg/kg) + DSS; purple = medium‐dose SEI (25 mg/kg) + DSS; green = high‐dose SEI (50 mg/kg) + DSS. Values were expressed as mean ± SD. # p < 0.05, ### p < 0.001 versus H 2 O group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus DSS group.

Article Snippet: Senkyunolide I (HY‐N0745), Erastin (HY‐15763), H‐151 (HY‐112693), TNF‐α (HY‐P7058 or HY‐P7090), IFN‐γ (HY‐P7025 or HY‐P7071), Lipopolysaccharides (LPS, HY‐D1056), Adenosine 5'‐triphosphate (ATP, HY‐B2176) were purchased from MedChem Express.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining, Fluorescence, Flow Cytometry, Control, Positive Control

ABPP proteomic analysis confirms VDAC1 as target protein of SEI. (A) Chemical structures of SEI and SEI probe (SEI‐P). (B) Cell viability of iBMDM cells treated with SEI or SEI‐P. (C) Release of inflammatory cytokine IL‐1β and (D) cell viability in TNF‐α plus IFN‐γ‐induced iBMDM cells. (E) Cellular imaging of SEI‐P with different exposure times in iBMDM cells. (F) Dose‐dependent labeling of proteins by SEI‐P in iBMDM cells. (G) Competition between SEI and SEI‐P for protein binding in situ (red star: 35 kD). (H) Chemical proteomics analysis workflow for identifying potential targets of SEI, created using Figdraw. (I) Volcano plot of proteins identified in the ABPP method. The graph displayed the log 2 FC of the competition group (100 µ m SEI + 50 µ m SEI‐P) versus SEI‐P (50 µ m ) ( x ‐axis) against the −log 10 ( p‐ value) ( y ‐axis). Among these, points with p < 0.05 and log 2 FC < −1 (blue) were selected as target protein candidates. Values were expressed as mean ± SD ( n = 3).

Journal: Advanced Science

Article Title: Senkyunolide I Inhibits mtDNA‐cGAS‐STING Signaling in Macrophages via Targeting VDAC1 Oligomerization to Attenuate Ulcerative Colitis

doi: 10.1002/advs.77045

Figure Lengend Snippet: ABPP proteomic analysis confirms VDAC1 as target protein of SEI. (A) Chemical structures of SEI and SEI probe (SEI‐P). (B) Cell viability of iBMDM cells treated with SEI or SEI‐P. (C) Release of inflammatory cytokine IL‐1β and (D) cell viability in TNF‐α plus IFN‐γ‐induced iBMDM cells. (E) Cellular imaging of SEI‐P with different exposure times in iBMDM cells. (F) Dose‐dependent labeling of proteins by SEI‐P in iBMDM cells. (G) Competition between SEI and SEI‐P for protein binding in situ (red star: 35 kD). (H) Chemical proteomics analysis workflow for identifying potential targets of SEI, created using Figdraw. (I) Volcano plot of proteins identified in the ABPP method. The graph displayed the log 2 FC of the competition group (100 µ m SEI + 50 µ m SEI‐P) versus SEI‐P (50 µ m ) ( x ‐axis) against the −log 10 ( p‐ value) ( y ‐axis). Among these, points with p < 0.05 and log 2 FC < −1 (blue) were selected as target protein candidates. Values were expressed as mean ± SD ( n = 3).

Techniques: Imaging, Labeling, Protein Binding, In Situ

SEI directly interacts with VDAC1 to inhibit its oligomerization. (A) The interaction between SEI and VDAC1 was detected using molecular docking. (B) Lysates from iBMDM cells were incubated with or without SEI (100 µ m ) for 24 h. Different concentrations of pronase E were added for 20 min, and VDAC1 content was analyzed using WB analysis. (C) Lysates from iBMDM cells were incubated with SEI at the indicated concentrations for 24 h, with a final concentration of 0.01% pronase E added for 20 min. The level of VDAC1 was assessed through WB analysis. (D) iBMDM cells were incubated with SEI (100 µ m ) for 24 h. These samples were then analyzed using CETSA. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01 versus SEI group. (E) MST demonstrating a direct interaction between SEI and EGFP‐tagged VDAC1 in lysates from EGFP‐VDAC1 expressing HEK293T cells. (F) Immunoblotting analysis of VDAC1 cross‐linking in iBMDM cells, untreated or stimulated with TNF‐α plus IFN‐γ, with or without addition of SEI (100 µ m ). (G) The VDAC1‐OE plasmid was transfected into iBMDM cells, and cGAMP production was subsequently measured by ELISA. (H) iBMDM cells were transfected with VDAC1‐OE, and then the phosphorylation levels of STING and IRF3 were measured by WB. (I) The mRNA expression levels of CCL5 , CXCL10 , and ISG15 in iBMDM cells transfected with VDAC1‐OE were measured by qRT‐PCR. (J) qRT‐PCR analysis of cytoplasmic mtDNA ( mt‐Nd1 , D‐loop and mt‐Cytb ). Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Advanced Science

Article Title: Senkyunolide I Inhibits mtDNA‐cGAS‐STING Signaling in Macrophages via Targeting VDAC1 Oligomerization to Attenuate Ulcerative Colitis

doi: 10.1002/advs.77045

Figure Lengend Snippet: SEI directly interacts with VDAC1 to inhibit its oligomerization. (A) The interaction between SEI and VDAC1 was detected using molecular docking. (B) Lysates from iBMDM cells were incubated with or without SEI (100 µ m ) for 24 h. Different concentrations of pronase E were added for 20 min, and VDAC1 content was analyzed using WB analysis. (C) Lysates from iBMDM cells were incubated with SEI at the indicated concentrations for 24 h, with a final concentration of 0.01% pronase E added for 20 min. The level of VDAC1 was assessed through WB analysis. (D) iBMDM cells were incubated with SEI (100 µ m ) for 24 h. These samples were then analyzed using CETSA. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01 versus SEI group. (E) MST demonstrating a direct interaction between SEI and EGFP‐tagged VDAC1 in lysates from EGFP‐VDAC1 expressing HEK293T cells. (F) Immunoblotting analysis of VDAC1 cross‐linking in iBMDM cells, untreated or stimulated with TNF‐α plus IFN‐γ, with or without addition of SEI (100 µ m ). (G) The VDAC1‐OE plasmid was transfected into iBMDM cells, and cGAMP production was subsequently measured by ELISA. (H) iBMDM cells were transfected with VDAC1‐OE, and then the phosphorylation levels of STING and IRF3 were measured by WB. (I) The mRNA expression levels of CCL5 , CXCL10 , and ISG15 in iBMDM cells transfected with VDAC1‐OE were measured by qRT‐PCR. (J) qRT‐PCR analysis of cytoplasmic mtDNA ( mt‐Nd1 , D‐loop and mt‐Cytb ). Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Incubation, Concentration Assay, Expressing, Western Blot, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Quantitative RT-PCR

SEI directly binds to K12 on VDAC1. (A) The RMSD of the VDAC1 backbone was simulated for a range of 100 ns. (B) The RMSF values of all amino acid residues were simulated. (C) Radius of gyration of the apo (blue) and protein when bound to the ligand (red) for the 100 ns simulation. (D) DCCM analysis matrix of VDAC1 protein; the region in red indicates residue pairs in horizontal and vertical coordinates have positive correlation in movement patterns, while the region in blue indicates negative correlation. (E) DCCM analysis matrix of SEI‐VDAC1 complex. (F–H) Movement correlation of each residue with a negative correlation coefficient ranged from −0.6 to −0.8 of VDAC1 protein (F), ranged from −0.4 to −0.6 of VDAC1 protein (G), and ranged from −0.4 to −0.6 of SEI‐VDAC1 complex (H). (I) The total binding free energy was calculated, and a series of contribution components were analyzed. Data are presented as energy changes in units of kJ/mol. (J) Ten residues of the VDAC1‐SEI complex were selected and analyzed. Data were presented as energy changes in a unit of kJ/mol with different contributors indicated by colors. (K) Free energy landscape. (L) Sequence conservation analysis of VDAC1 protein using ESPript 3.0. (M) The iBMDM cells were transfected with K174, K12, and G172 mutation plasmids and then treated with DMSO or SEI (100 µ m ) for 1 h. The interaction between SEI and VDAC1 was detected using the CETSA assay. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl group. (N) VDAC1 −/− iBMDM cells were transfected with Flag‐VDAC1(WT), Flag‐VDAC1(K174A), Flag‐VDAC1(K12A), or Flag‐VDAC1(G172A). Immunoblotting analysis of VDAC1 cross‐linking in iBMDM cells stimulated with TNF‐α plus IFN‐γ, with or without addition of SEI (100 µ m ). (O) VDAC1 −/− iBMDM cells were transfected with an empty vector, Flag‐VDAC1‐WT, Flag‐VDAC1(K174A), Flag‐VDAC1(K12A), or Flag‐VDAC1(G172A). WB analyses of the quantity of p‐TBK1 and p‐IRF3 after stimulation with TNF‐α plus IFN‐γ and treatment with SEI (100 µ m ) or left untreated (control) for 24 h. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Advanced Science

Article Title: Senkyunolide I Inhibits mtDNA‐cGAS‐STING Signaling in Macrophages via Targeting VDAC1 Oligomerization to Attenuate Ulcerative Colitis

doi: 10.1002/advs.77045

Figure Lengend Snippet: SEI directly binds to K12 on VDAC1. (A) The RMSD of the VDAC1 backbone was simulated for a range of 100 ns. (B) The RMSF values of all amino acid residues were simulated. (C) Radius of gyration of the apo (blue) and protein when bound to the ligand (red) for the 100 ns simulation. (D) DCCM analysis matrix of VDAC1 protein; the region in red indicates residue pairs in horizontal and vertical coordinates have positive correlation in movement patterns, while the region in blue indicates negative correlation. (E) DCCM analysis matrix of SEI‐VDAC1 complex. (F–H) Movement correlation of each residue with a negative correlation coefficient ranged from −0.6 to −0.8 of VDAC1 protein (F), ranged from −0.4 to −0.6 of VDAC1 protein (G), and ranged from −0.4 to −0.6 of SEI‐VDAC1 complex (H). (I) The total binding free energy was calculated, and a series of contribution components were analyzed. Data are presented as energy changes in units of kJ/mol. (J) Ten residues of the VDAC1‐SEI complex were selected and analyzed. Data were presented as energy changes in a unit of kJ/mol with different contributors indicated by colors. (K) Free energy landscape. (L) Sequence conservation analysis of VDAC1 protein using ESPript 3.0. (M) The iBMDM cells were transfected with K174, K12, and G172 mutation plasmids and then treated with DMSO or SEI (100 µ m ) for 1 h. The interaction between SEI and VDAC1 was detected using the CETSA assay. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl group. (N) VDAC1 −/− iBMDM cells were transfected with Flag‐VDAC1(WT), Flag‐VDAC1(K174A), Flag‐VDAC1(K12A), or Flag‐VDAC1(G172A). Immunoblotting analysis of VDAC1 cross‐linking in iBMDM cells stimulated with TNF‐α plus IFN‐γ, with or without addition of SEI (100 µ m ). (O) VDAC1 −/− iBMDM cells were transfected with an empty vector, Flag‐VDAC1‐WT, Flag‐VDAC1(K174A), Flag‐VDAC1(K12A), or Flag‐VDAC1(G172A). WB analyses of the quantity of p‐TBK1 and p‐IRF3 after stimulation with TNF‐α plus IFN‐γ and treatment with SEI (100 µ m ) or left untreated (control) for 24 h. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Residue, Binding Assay, Sequencing, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Control

The VDAC1 K12 site is required for the protective roles of SEI in UC mice. (A) Daily assessments of body weight change and (B) DAI were conducted ( n = 6). (C) Gross morphology images of the colon were captured on day 9 after DSS treatment, and (D) colon length was measured ( n = 6). (E) The spleen index of mice after DSS treatment. (F) Colonic sections from mice were subjected to H&E staining ( U ‐shaped curve: U ‐shaped crypt; arrow: goblet cell; circle: inflammatory cells), and (G) a semiquantitative histological score was assessed ( n = 6). (H) Representative fluorescent images of MUC2, ZO1, Claudin1 and Occludin in the colonic tissues ( n = 4). (I) Representative fluorescent images of TFF3 in the colonic tissues ( n = 4). (J) IF staining for Ki67 and E‐cadherin in colon tissues ( n = 4). (K) ELISA for inflammatory cytokine production in colonic tissues, including IL‐1β, IL‐6, TNF‐α, and IL‐23 ( n = 6). (L) IF staining for F4/80 (red) and CD86 (green) in colon tissues ( n = 4). Scale bar = 50 µm. Values were expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Journal: Advanced Science

Article Title: Senkyunolide I Inhibits mtDNA‐cGAS‐STING Signaling in Macrophages via Targeting VDAC1 Oligomerization to Attenuate Ulcerative Colitis

doi: 10.1002/advs.77045

Figure Lengend Snippet: The VDAC1 K12 site is required for the protective roles of SEI in UC mice. (A) Daily assessments of body weight change and (B) DAI were conducted ( n = 6). (C) Gross morphology images of the colon were captured on day 9 after DSS treatment, and (D) colon length was measured ( n = 6). (E) The spleen index of mice after DSS treatment. (F) Colonic sections from mice were subjected to H&E staining ( U ‐shaped curve: U ‐shaped crypt; arrow: goblet cell; circle: inflammatory cells), and (G) a semiquantitative histological score was assessed ( n = 6). (H) Representative fluorescent images of MUC2, ZO1, Claudin1 and Occludin in the colonic tissues ( n = 4). (I) Representative fluorescent images of TFF3 in the colonic tissues ( n = 4). (J) IF staining for Ki67 and E‐cadherin in colon tissues ( n = 4). (K) ELISA for inflammatory cytokine production in colonic tissues, including IL‐1β, IL‐6, TNF‐α, and IL‐23 ( n = 6). (L) IF staining for F4/80 (red) and CD86 (green) in colon tissues ( n = 4). Scale bar = 50 µm. Values were expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

The cellular uptake and anti-inflammatory effect of HPSL in vitro . (A) Flow cytometry analysis and (B) semi-quantitative analysis of cellular uptake of PSL and blank NPs by M1 macrophages. n = 3. (C) Representative Giemsa staining images of LPS and high glucose-stimulated RAW 264.7 cells with different formulations, scale bar = 50 μm. (D) Immunofluorescence staining and semi-quantitative analysis of CD68 (red) and iNOS (green) in RAW 264.7 cells from different treatment groups, scale bar = 50 μm. n = 6. (E) Immunofluorescence staining and semi-quantitative analysis of CD68 (green) and Arg-1 (red) in RAW 264.7 cells from different treatment groups, scale bar = 50 μm. n = 6. Western blotting analysis and corresponding semi-quantitative analysis of (F) STING/ p -STING, (G) TBK1/ p -TBK1, (H) IRF3/ p -IRF3, (I) NF-κB, (J) TNF-α, and (K) IL-6, Lane 1: Normal group, Lane 2: Model group, Lane 3: PSL group, Lane 4: Free H151 group, Lane 5: HPSL group. n = 3. All data are shown as mean ± SEM.

Journal: Bioactive Materials

Article Title: Glucose/ROS-responsive and redox-gated adaptive hydrogel dressing for accelerating diabetic wound repair via synergistic cGAS/STING pathway inhibition and oxidative stress alleviation

doi: 10.1016/j.bioactmat.2026.03.025

Figure Lengend Snippet: The cellular uptake and anti-inflammatory effect of HPSL in vitro . (A) Flow cytometry analysis and (B) semi-quantitative analysis of cellular uptake of PSL and blank NPs by M1 macrophages. n = 3. (C) Representative Giemsa staining images of LPS and high glucose-stimulated RAW 264.7 cells with different formulations, scale bar = 50 μm. (D) Immunofluorescence staining and semi-quantitative analysis of CD68 (red) and iNOS (green) in RAW 264.7 cells from different treatment groups, scale bar = 50 μm. n = 6. (E) Immunofluorescence staining and semi-quantitative analysis of CD68 (green) and Arg-1 (red) in RAW 264.7 cells from different treatment groups, scale bar = 50 μm. n = 6. Western blotting analysis and corresponding semi-quantitative analysis of (F) STING/ p -STING, (G) TBK1/ p -TBK1, (H) IRF3/ p -IRF3, (I) NF-κB, (J) TNF-α, and (K) IL-6, Lane 1: Normal group, Lane 2: Model group, Lane 3: PSL group, Lane 4: Free H151 group, Lane 5: HPSL group. n = 3. All data are shown as mean ± SEM.

Article Snippet: VEGF-A and TNF-α-specific antibodies were purchased from Wanleibio (Shenyang, China).

Techniques: In Vitro, Flow Cytometry, Staining, Immunofluorescence, Western Blot

Characterization of Res-PD-L1@nmEVs . (A) Schematic illustration of the Res-PD-L1@nmEVs synthesis procedure. (B-D) Representative transmission electron microscopy (TEM) images, dynamic light scattering (DLS) size distributions, and zeta potential measurements of nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs. (E) PD-L1 expression in PD-L1-overexpressing MSCs (OE-PD-L1) and negative control (NC) MSCs, and CD11b expression in HL60 cells before and after DMSO stimulation, as determined by Western blot. (F) Expression levels of neutrophil membrane markers (CD11b, CXCR2, RAGE, TLR2) and the exosomal marker CD63 in the four EV types. (G) Fluorescence co-localization images of DiO-labeled nEVs (green) and DiL-labeled PD-L1@mEVs (red) after fusion, demonstrating hybrid vesicle formation. (H) Size stability of Res-PD-L1@nmEVs stored at 4 °C and 37 °C for 7 days. (I-K) Binding and neutralization capacity of Res-PD-L1@nmEVs against inflammatory cytokines (TNF-α, IL-6, IL-1β) in vitro. ∗ vs. 0ug/ml; # vs. 100 μg/ml, p < 0.05, n = 5.

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Characterization of Res-PD-L1@nmEVs . (A) Schematic illustration of the Res-PD-L1@nmEVs synthesis procedure. (B-D) Representative transmission electron microscopy (TEM) images, dynamic light scattering (DLS) size distributions, and zeta potential measurements of nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs. (E) PD-L1 expression in PD-L1-overexpressing MSCs (OE-PD-L1) and negative control (NC) MSCs, and CD11b expression in HL60 cells before and after DMSO stimulation, as determined by Western blot. (F) Expression levels of neutrophil membrane markers (CD11b, CXCR2, RAGE, TLR2) and the exosomal marker CD63 in the four EV types. (G) Fluorescence co-localization images of DiO-labeled nEVs (green) and DiL-labeled PD-L1@mEVs (red) after fusion, demonstrating hybrid vesicle formation. (H) Size stability of Res-PD-L1@nmEVs stored at 4 °C and 37 °C for 7 days. (I-K) Binding and neutralization capacity of Res-PD-L1@nmEVs against inflammatory cytokines (TNF-α, IL-6, IL-1β) in vitro. ∗ vs. 0ug/ml; # vs. 100 μg/ml, p < 0.05, n = 5.

Article Snippet: These cells were then stimulated with 40 ng/mL recombinant TNF-α (C008, Novoprotein, China) for 6 h to induce an N1-type neutrophil phenotype.

Techniques: Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Expressing, Negative Control, Western Blot, Membrane, Marker, Fluorescence, Labeling, Binding Assay, Neutralization, In Vitro

Res-PD-L1@nmEVs Attenuate Inflammation and Oxidative Damage in Lung Epithelial Cells In Vitro . (A-B) Flow cytometric analysis and quantification (B) of DiO-labeled Res-PD-L1@nmEVs uptake by BEAS-2B cells under H/R conditions after pretreatment with different endocytic inhibitors (chlorpromazine, chloroquine, and filipin) or incubation at 4 °C. (C) mRNA expression levels of IL-6, TNF-α, and IL-1β in BEAS-2B cells with or without H/R injury following pretreatment with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs. (D-E) Representative fluorescence images (D) and quantitative analysis (E) of cell proliferation assessed by BrdU incorporation (red; nuclei stained with DAPI, blue). Scale bar: 50 μm. (F-G) Apoptosis rates detected by flow cytometry (F) and flow cytometric analysis of Annexin V-positive BEAS-2B cells under the indicated conditions (G). (H–K) Fluorescence microscopy images and quantitative analysis of intracellular nitric oxide (NO, green) (H-I) and reactive oxygen species (ROS, red) (J-K). Scale bar: 100 μm. (L) Flow cytometry analysis of intracellular ROS levels. (M − O) Levels of malondialdehyde (MDA) (M), superoxide dismutase 2 (SOD2) activity (N), and glutathione (GSH) content (O) in cells. (P-Q) Cell migration ability evaluated by wound healing assay under different treatments. ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Journal: Bioactive Materials

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Attenuate Inflammation and Oxidative Damage in Lung Epithelial Cells In Vitro . (A-B) Flow cytometric analysis and quantification (B) of DiO-labeled Res-PD-L1@nmEVs uptake by BEAS-2B cells under H/R conditions after pretreatment with different endocytic inhibitors (chlorpromazine, chloroquine, and filipin) or incubation at 4 °C. (C) mRNA expression levels of IL-6, TNF-α, and IL-1β in BEAS-2B cells with or without H/R injury following pretreatment with Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs. (D-E) Representative fluorescence images (D) and quantitative analysis (E) of cell proliferation assessed by BrdU incorporation (red; nuclei stained with DAPI, blue). Scale bar: 50 μm. (F-G) Apoptosis rates detected by flow cytometry (F) and flow cytometric analysis of Annexin V-positive BEAS-2B cells under the indicated conditions (G). (H–K) Fluorescence microscopy images and quantitative analysis of intracellular nitric oxide (NO, green) (H-I) and reactive oxygen species (ROS, red) (J-K). Scale bar: 100 μm. (L) Flow cytometry analysis of intracellular ROS levels. (M − O) Levels of malondialdehyde (MDA) (M), superoxide dismutase 2 (SOD2) activity (N), and glutathione (GSH) content (O) in cells. (P-Q) Cell migration ability evaluated by wound healing assay under different treatments. ∗ vs. Control; # vs. H/R; & vs. H/R + PD-L1@nmEVs, p < 0.05.

Article Snippet: These cells were then stimulated with 40 ng/mL recombinant TNF-α (C008, Novoprotein, China) for 6 h to induce an N1-type neutrophil phenotype.

Techniques: In Vitro, Labeling, Incubation, Expressing, Fluorescence, BrdU Incorporation Assay, Staining, Flow Cytometry, Microscopy, Activity Assay, Migration, Wound Healing Assay, Control

Res-PD-L1@nmEVs Suppresses Neutrophil Activation HL60 cells were differentiated into neutrophil-like cells using DMSO and subsequently stimulated with TNF-α to induce activation under conditions simulating IRI. The effects of Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs on neutrophil activation were evaluated. (A) Cell surface PD-1 expression analyzed by flow cytometry. (B) Representative immunofluorescence images of CD206 expression (red). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (C) Flow cytometric analysis of cell surface CD206 expression. (D) Flow cytometric analysis of cell surface CD95 expression. (E-G) Levels of myeloperoxidase (MPO) (E), neutrophil elastase (NE) (F), and MMP-9 (G) in neutrophil culture supernatants, measured by ELISA. (H-J) BEAS-2B cells were co-cultured with neutrophils in the presence or absence of TNF-α stimulation. Apoptosis levels (I) and migration capacity (J) of BEAS-2B cells were assessed under different treatment conditions. ∗ vs. Control; # vs. TNF-a; & vs. TNF-a+PD-L1@nmEVs, p < 0.05.

Journal: Bioactive Materials

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation HL60 cells were differentiated into neutrophil-like cells using DMSO and subsequently stimulated with TNF-α to induce activation under conditions simulating IRI. The effects of Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs on neutrophil activation were evaluated. (A) Cell surface PD-1 expression analyzed by flow cytometry. (B) Representative immunofluorescence images of CD206 expression (red). Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (C) Flow cytometric analysis of cell surface CD206 expression. (D) Flow cytometric analysis of cell surface CD95 expression. (E-G) Levels of myeloperoxidase (MPO) (E), neutrophil elastase (NE) (F), and MMP-9 (G) in neutrophil culture supernatants, measured by ELISA. (H-J) BEAS-2B cells were co-cultured with neutrophils in the presence or absence of TNF-α stimulation. Apoptosis levels (I) and migration capacity (J) of BEAS-2B cells were assessed under different treatment conditions. ∗ vs. Control; # vs. TNF-a; & vs. TNF-a+PD-L1@nmEVs, p < 0.05.

Article Snippet: These cells were then stimulated with 40 ng/mL recombinant TNF-α (C008, Novoprotein, China) for 6 h to induce an N1-type neutrophil phenotype.

Techniques: Activation Assay, Expressing, Flow Cytometry, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture, Migration, Control

Res-PD-L1@nmEVs Effectively Attenuates MRSA-Induced Pneumonia (A-B) Rats with MRSA-induced pneumonia received three bronchial nebulization treatments over one week with different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs). (A) Representative H&E-stained lung sections and (B) corresponding lung injury scores are shown (n = 5). (C) TUNEL staining of lung tissues to assess apoptosis. (D) Representative micro-CT images of anesthetized rats. (E-G) Flow cytometric analysis of immune cell proportions in lung single-cell suspensions: CD8 + T cells (E), neutrophils (F), and classical monocytes (G). (H-J) Plasma levels of inflammatory cytokines IL-6 (H), IL-1β (I), and TNF-α (J) (n = 5). (K) Immunofluorescence staining of tight junction proteins Occludin (green) and ZO-1 (red) in lung tissues (nuclei stained with DAPI). Scale bar: 50 μm. (L-N) Pulmonary function parameters: lung compliance (L), airway resistance (M), and oxygenation index (N) (n = 4). ∗ vs. Sham; # vs. MRSA; & vs. MRSA + PD-L1@nmEVs, p < 0.05.

Journal: Bioactive Materials

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Effectively Attenuates MRSA-Induced Pneumonia (A-B) Rats with MRSA-induced pneumonia received three bronchial nebulization treatments over one week with different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs). (A) Representative H&E-stained lung sections and (B) corresponding lung injury scores are shown (n = 5). (C) TUNEL staining of lung tissues to assess apoptosis. (D) Representative micro-CT images of anesthetized rats. (E-G) Flow cytometric analysis of immune cell proportions in lung single-cell suspensions: CD8 + T cells (E), neutrophils (F), and classical monocytes (G). (H-J) Plasma levels of inflammatory cytokines IL-6 (H), IL-1β (I), and TNF-α (J) (n = 5). (K) Immunofluorescence staining of tight junction proteins Occludin (green) and ZO-1 (red) in lung tissues (nuclei stained with DAPI). Scale bar: 50 μm. (L-N) Pulmonary function parameters: lung compliance (L), airway resistance (M), and oxygenation index (N) (n = 4). ∗ vs. Sham; # vs. MRSA; & vs. MRSA + PD-L1@nmEVs, p < 0.05.

Article Snippet: These cells were then stimulated with 40 ng/mL recombinant TNF-α (C008, Novoprotein, China) for 6 h to induce an N1-type neutrophil phenotype.

Techniques: Staining, TUNEL Assay, Micro-CT, Single Cell, Clinical Proteomics, Immunofluorescence

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