Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: cfDNA promotes the polarization of macrophages towards the M1 phenotype by activating the TLR9-MyD88 and cGAS-STING pathways (A- D) CpG DNA treatment increased the protein levels of IL-1α, TNF-α, and IL-6. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h and then subjected to western blotting (A). IL-1α (B), TNF-α (C), and IL-6 (D) levels were normalized to the β-actin level in each sample, and the normalized values were used for statistical analysis, n = 4. (E-F) CpG DNA treatment significantly enhanced CD86 expression in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (E) and quantitative fluorescence graph (F) are shown, n = 6. Scale bar: 50 μm. (G-J) CpG DNA treatment significantly upregulated the expression of key proteins associated with the TLR9-MyD88 and cGAS-STING pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h and then subjected to western blotting, n = 8/4(G, I). STING, cGAS, TLR9, and MyD88 levels were normalized to the β-actin level in each sample. p-NF-κB, p-IκB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, IκB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis, n = 8/4(H, J). For (B-D, F, H, J), error bars represent mean ± standard deviation. P values were calculated using one-way analysis of variance (ANOVA) (B-D, F, H, J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Western Blot, Expressing, Fluorescence, Microscopy, Standard Deviation

Journal: Materials Today Bio

Article Title: Toll-like receptor 9-overexpressing red blood cell biomimetic microbubbles adsorb cell-free DNA to relieve steatotic liver ischemia-reperfusion injury

doi: 10.1016/j.mtbio.2026.103558

Figure Lengend Snippet: RBC-MBs promotes the polarization of macrophages towards the M2 phenotype by inhibiting the TLR9-MyD88 and cGAS-STING pathways (A-F) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of pro-inflammatory cytokines (A-B), key proteins in the TLR9-MyD88 (C-D) and cGAS-STING (E-F) pathways. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, and then subjected to western blot analysis (A, C, E). IL-1α, TNF-α, IL-6, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample. p-NF-κB, p-TBK1, and p-IRF3 levels were normalized to their respective total protein levels (NF-κB, TBK1, and IRF3) in each sample. These normalized values were then used for statistical analysis (B, D, F), n = 6. (G) RBC-MB treatment significantly attenuated CpG DNA-induced upregulation of CD86 in macrophages. RAW264.7 cells were treated with 1 μg mL -1 CpG DNA for 24 h, followed by incubation with 0.5 mL RBC-MBs (concentration: 1 × 10 8 particles mL -1 ) for 24 h, then immunostained with an anti-CD86 antibody (green) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Representative images (left panel) and quantitative fluorescence graph (right panel) are shown. Scale bar: 50 μm, n = 6. (H-J) RBC-MB treatment significantly inhibited hepatic IRI-induced polarization of macrophages toward the M1 phenotype (H) and activation of the TLR9-MyD88 and cGAS-STING pathways (I-J). MASLD rats underwent 45 min of hepatic ischemia followed by reperfusion. At 0, 6, 12, 18, and 24 h post-reperfusion, the rats were treated with either Con-MBs or RBC-MBs. Primary macrophages were isolated from rat livers, then co-stained with CD68 and CD163 followed by flow cytometric analysis (H), n = 3/4, or subjected to western blotting (I-J). CD206, iNOS, TLR9, MyD88, STING, and cGAS levels were normalized to the β-actin level in each sample, and these normalized values were used for statistical analysis (J), n = 3. For (B, D, F, G right panel , H right panel , J), error bars represent mean ± standard deviation. P values were calculated using the unpaired Student's t-test (B, D, F, G right panel ) or one-way analysis of variance (ANOVA) (H right panel , J). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies used included: rabbit polyclonal antibodies against TLR9 (A14642, Abclonal), NF-κB (A2547, Abclonal), MyD88 (A0980, Abclonal), cGAS (A8335, Abclonal), IL-1α (A2170, Abclonal), TNF-α (A0277, Abclonal), IL-6 (A0286, Abclonal), CD206 (A8301, Abclonal), CD86 (A16805, Abclonal), CD47 (A1838, Abclonal), and ATP1A (A0643, Abclonal); rabbit monoclonal antibodies against STING (13647, CST, Beverly, MA, USA), p-NF-κB (3033, CST), p-TBK1 (5483, CST), TBK1 (ab40676, Abcam), p-IκB (2859, CST), IκB (4812, CST), p-IRF3 (4947, CST), and β-actin (AC026, Abclonal).

Techniques: Incubation, Concentration Assay, Western Blot, Fluorescence, Microscopy, Activation Assay, Isolation, Staining, Standard Deviation

Journal: Journal of Pharmacopuncture

Article Title: Effects of Acupuncture at ST36 and SP6 on Activity Levels and Pain Thresholds Through Interleukin-1, Interleukin-6, Cortisol, and Matrix Metalloproteinase-13 Modulation in an Osteoarthritis Rat Model

doi: 10.3831/KPI.2026.29.3.286

Figure Lengend Snippet: Serum levels of interleukin (IL)-1 (A) and IL-6 (B) in an untreated osteoarthritis rat model, healthy control rats, and three experimental groups of osteoarthritis model rats following acupuncture treatment. Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance followed by Duncan’s multiple range test. Different letters indicate significant differences between groups (p < 0.05). K−, negative control; K+, positive control; K + A, acupuncture to the ST36 + SP6 points; K + B, acupuncture to the ST36 point only; acupuncture to the K + C, SP6 point only.

Article Snippet: These were the Rat IL-1 ELISA Kit (cat. no. E0119Ra) and the Rat IL-6 ELISA Kit (cat. no. E0135Ra), both from BT-LAB (Shanghai, China).

Techniques: Control, Standard Deviation, Analysis, Negative Control, Positive Control

Journal: Journal of Pharmacopuncture

Article Title: Effects of Acupuncture at ST36 and SP6 on Activity Levels and Pain Thresholds Through Interleukin-1, Interleukin-6, Cortisol, and Matrix Metalloproteinase-13 Modulation in an Osteoarthritis Rat Model

doi: 10.3831/KPI.2026.29.3.286

Figure Lengend Snippet: Pearson correlations between inflammatory and hormonal biomarkers and behavioral outcomes in a monosodium iodoacetate-induced osteoarthritis rat model

Article Snippet: These were the Rat IL-1 ELISA Kit (cat. no. E0119Ra) and the Rat IL-6 ELISA Kit (cat. no. E0135Ra), both from BT-LAB (Shanghai, China).

Techniques: Biomarker Discovery, Activity Assay

Journal: Aging Cell

Article Title: uPAR ‐Targeting T Cell Engager Exerts Senolytic Effects in Mice and Non‐Human Primates With Serum Aminotransferase Activity as a Safety Monitor

doi: 10.1111/acel.70742

Figure Lengend Snippet: hGFD‐CD3 mediates the clearance of senescent cells by T cells in vitro. (A) A schematic diagram of hGFD‐CD3 and the negative control used in this study. (B) Semiquantitative analysis of the cell surface binding of hGFD‐CD3 to young uPAR‐HEL1 cells and uPAR+ HEL1 senescent cells via flow cytometry. (C) Semiquantitative analysis of the cell surface binding of hGFD‐CD3 and hCD3 to human CD3+ T cells via flow cytometry. (D) Dose–response study of hGFD‐CD3 mediated T cell cytotoxicity against senescent cells (E: T = 1: 1). Each data point represents the mean of a biological replicate ( n = 2). (E) Cytokine release studies using ELISA from cell supernatants in experiments where 100 ng/mL hGFD‐CD3 was applied. (F) T cell activation was assessed by measuring CD25 and CD69 levels on CD3+ T cells by flow cytometry. All experiments were performed in biological triplicate. Data are presented as mean ± SD of triplicate samples and analyzed by two‐tailed unpaired Student's t ‐test with Welch's correction (e, f). ** p ≤ 0.05; ** p ≤ 0.01; ns, not significant.

Article Snippet: The cytokine IL‐6 was measured with an ELISA kit (Proteintech, KE10007).

Techniques: In Vitro, Negative Control, Analysis, Binding Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activation Assay, Two Tailed Test

Journal: Aging Cell

Article Title: uPAR ‐Targeting T Cell Engager Exerts Senolytic Effects in Mice and Non‐Human Primates With Serum Aminotransferase Activity as a Safety Monitor

doi: 10.1111/acel.70742

Figure Lengend Snippet: mGFD‐CD3 safely and effectively mediates T‐cell clearance of senescent cells in naturally aged mice. (A) The half‐lives of mGFD‐CD3 and mCD3 negative control in naturally aged mice were analyzed by ELISA. Each data point represents the mean of a biological replicate ( n = 2). (B) The mRNA expression levels for mouse uPAR, P16 INK4a , and P21 were detected by qPCR analysis from the indicated tissues from mice treated with mCD3 or mGFD‐CD3 on day 21 ( n = 5 mice per group). (C) Representative images of SA‐β‐Gal staining of liver and lung tissue samples collected from naturally aged mice treated with mCD3 or mGFD‐CD3 ( left ), with quantification of average staining intensities (ImageJ) ( right ) ( n = 5). Scale bar, 100 μm. (D) Images of subcutaneous adipose tissue (SAT) after SA‐β‐Gal staining ( left ) and quantification of the average staining intensities (ImageJ) ( right ). SAT was collected from mice treated with mCD3 or mGFD‐CD3 on day 21 ( n = 5 individual samples per condition). Results from mouse experiments are representative of two independent experiments. (E) Analysis of Senmayo enrichment dataset of liver tissue samples from naturally aged mice treated with mGFD‐CD3 or mCD3 as negative control ( n = 3 mice per condition). (F) Gene expression volcano plot from the analysis of liver tissue samples collected from mice treated with mCD3 or mGFD‐CD3 ( n = 3 per condition). (G) Hierarchical clustering heatmap of the senescence‐associated secretory phenotype (SASP) profiles of liver samples ( n = 3 per condition). (H) Representative images of BiTEs (green), CD3 (red), and SA‐β‐Gal staining (blue green) for liver and lung samples collected from mice treated with BiTEs 24 h post‐injection. (I) Flow cytometry results of CD3 + T cells analyzed for CD25 expression after administration of mCD3 or mGFD‐CD3 ( n = 3 mice per condition). Data is presented as mean ± SD and analyzed by one‐way ANOVA followed by Tukey's multiple comparisons test (C, D, I). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; ns, not significant.

Article Snippet: The cytokine IL‐6 was measured with an ELISA kit (Proteintech, KE10007).

Techniques: Negative Control, Enzyme-linked Immunosorbent Assay, Expressing, Analysis, Staining, Tissue, Gene Expression, Injection, Flow Cytometry

Journal: Aging Cell

Article Title: uPAR ‐Targeting T Cell Engager Exerts Senolytic Effects in Mice and Non‐Human Primates With Serum Aminotransferase Activity as a Safety Monitor

doi: 10.1111/acel.70742

Figure Lengend Snippet: Elimination of senescent cells with mGFD‐CD3 ameliorated age‐related phenotypes in mice. (A) ELISA analysis for TNF‐α and IL‐1β in blood samples collected from mice treated with mGFD‐CD3 or mCD3 at 3 Months. ( n = 5 for young group; n = 3 for mCD3 group; n = 3 for mGFD‐CD3 group). (B) ( left ) H&E staining of IAT samples collected from mice described in (a). Quantification of adipocyte areas ( right ). Each data point represents an individual mouse. Scale bar, 50 μm. (C) ( left ) Masson staining of liver and lung tissues collected from the mice described in (a). Black arrows indicate positive staining. Scale bar, 50 μm. ( right ) Quantification of the fibrotic areas of liver or lung tissues using Image J. (D) PAS staining of kidney samples from mice described in (a). Scale bar, 50 μm (original) and 15 μm (magnified). Quantification was achieved using Image J. (E) TRAP and Masson staining of femur samples from mice described in (a). Black arrows indicate positive staining. Scale bar, 50 μm. Quantification was achieved with Image J. Data are presented as mean ± SD and analyzed by one‐way ANOVA followed by Tukey's multiple comparisons test (A–E). * p ≤ 0.05.

Article Snippet: The cytokine IL‐6 was measured with an ELISA kit (Proteintech, KE10007).

Techniques: Enzyme-linked Immunosorbent Assay, Analysis, Blood/Plasma/Serum Samples, Staining

Journal: Aging Cell

Article Title: uPAR ‐Targeting T Cell Engager Exerts Senolytic Effects in Mice and Non‐Human Primates With Serum Aminotransferase Activity as a Safety Monitor

doi: 10.1111/acel.70742

Figure Lengend Snippet: Assessing safety attributes of hGFD‐CD3 treatment in aged nonhuman primates. (A) Semiquantitative analysis of cell surface binding of hGFD‐CD3 to macaque T cells via flow cytometry. (B) Monitoring of liver function after administration of each different dose within a 72‐h period. (C) Schematic diagram of the experiment to investigate clearance of senescent cells from macaques, where hGFD‐CD3 was administered at a dose of 20 μg/kg every three days. (D) Liver function was evaluated throughout the injection phase. (E) Changes in cytokine levels in serum samples from three macaques before, as well as 24‐ and 72 h after the first injection of hGFD‐CD3, as determined via ELISA. Data are presented as mean ± SD of triplicate samples analyzed by two‐tailed unpaired Student's t ‐test with Welch's correction. (F) Hematological parameters were assessed throughout the injection phase. (G) Six accessible vital sign parameters were continuously monitored during the injection period. Data is presented as mean ± SD of triplicates and analyzed by two‐tailed unpaired Student's t test with Welch's correction (E). *** p ≤ 0.001.

Article Snippet: The cytokine IL‐6 was measured with an ELISA kit (Proteintech, KE10007).

Techniques: Analysis, Binding Assay, Flow Cytometry, Injection, Blood/Plasma/Serum Samples, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Aging Cell

Article Title: uPAR ‐Targeting T Cell Engager Exerts Senolytic Effects in Mice and Non‐Human Primates With Serum Aminotransferase Activity as a Safety Monitor

doi: 10.1111/acel.70742

Figure Lengend Snippet: hGFD‐CD3 clears senescent cells in aged nonhuman primates. (A) SA‐β‐Gal staining of subcutaneous adipose tissue and quantification before injection, as well as at Days 42 and 90 after the first injection. (B) Fold‐changes in mRNA expression levels of uPAR in subcutaneous adipose tissue, muscle, and skin before injection and at Days 42 and 90 after the first injection. (C) Fold‐changes in mRNA expression levels of the senescence markers P16 INK4a and P21 in subcutaneous adipose tissue, muscle, and skin before injection and at Days 42 and 90 after the first injection. (D) The expression levels of representative genes from T cell‐, NK‐, Monocyte‐, Macrophage‐, DC‐, B cell‐ and Plasma‐cell types in liver, fat, and skin tissue collected from three macaques before injection, and at Days 42 and 90 after the first injection of hGFD‐CD3, and the data of pre‐administration were normalized and compared to those of the data of day 42. ( n = 3). (E) Changes in cytokine levels in serum samples from three macaques before injection, and at Days 42 and 90 after the first injection of hGFD‐CD3, as determined via ELISA. Data is presented as mean ± SD of triplicates and analyzed by paired two‐tailed Student's t ‐test. ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: The cytokine IL‐6 was measured with an ELISA kit (Proteintech, KE10007).

Techniques: Staining, Tissue, Injection, Expressing, Clinical Proteomics, Blood/Plasma/Serum Samples, Enzyme-linked Immunosorbent Assay, Two Tailed Test