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: Synthetic RBC-MBs with high TLR9 expression (A-B) TLR9 protein levels on erythrocyte membranes were significantly decreased in both patients with MASLD (A), n = 9/7, and MASLD rats (B), n = 8/6. Erythrocytes were collected from patients with or without MASLD (A) and from rats fed a normal diet or a high-fat diet (B). Erythrocyte membranes were isolated and subjected to western blotting ( upper panels ). TLR9 levels were normalized to ATP1A levels in each sample, and the normalized values were used for statistical analysis ( lower panels ). (C) Lipopolysaccharide (LPS) treatment significantly increased TLR9 protein levels on erythrocyte membranes. Rats were intravenously injected with 12 mg kg -1 LPS for 6 h. Erythrocytes were collected, and the membranes were isolated and analyzed by western blotting ( upper panel ). TLR9 levels were normalized to ATP1A levels in each sample, and the normalized values were used for statistical analysis ( lower panel ), n = 6. (D) Schematic diagram illustrating the synthesis of RBC-MBs. (E) The ultrasound microbubbles appeared as a milky suspension. (F-G) Size distribution of Con-MBs (F) and RBC-MBs (G) in phosphate-buffered saline. (H-I) Average hydrodynamic diameters (H) and surface charges (ζ-potential) (I) of Con-MBs and RBC-MBs, as determined by dynamic light scattering, n = 6. (J) RBC-MBs were round, uniform, and well-dispersed. Erythrocyte membranes were labeled with DiL (red), and phospholipids were labeled with DiO (green). Scale bar: 30 μm. (K) RBC-MBs contained TLR9, CD47, and ATP1A proteins. Red blood cells, erythrocyte membranes, and RBC-MBs were subjected to western blotting. (L) RBC-MBs effectively adsorbed cfDNA in vitro . BRL-3A cells were treated with 250 μM sodium palmitate and 500 μM sodium oleate for 24 h, followed by hypoxia for 9 h and reoxygenation for 2 h. Subsequently, the cells were co-incubated with DiO-labeled (green) Con-MBs or RBC-MBs for 20 min, then immunostained with an anti-DNA antibody (red) and DAPI (blue, nuclei), and imaged by fluorescence microscopy. Scale bar: 20 μm. For (A-C, H-I), error bars represent mean ± standard deviation. P values were calculated using the unpaired Student's t-test (A, B, C, H, I). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.(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: Expressing, Isolation, Western Blot, Injection, Suspension, Saline, Labeling, In Vitro, Incubation, 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: 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: Matrix metalloproteinase-13 expression in the articular cartilage of an untreated osteoarthritis rat model, healthy control rats, and three experimental groups of osteoarthritis model rats following acupuncture treatment. (A) Representative immunohistochemical staining. The brown stain indicates positive MMP-13 expression (scale bar = 60 μm). (B) Quantitative analysis of the MMP-13-positive area (mean ± standard deviation). 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; MMP-13, matrix metalloproteinase-13.

Article Snippet: The tissue sections were deparaffinized, rehydrated, and incubated overnight at 4°C with rabbit polyclonal anti-MMP-13 antibody (bs-10581R, Bioss Antibodies, MA, USA).

Techniques: Expressing, Control, Immunohistochemical staining, Staining, Analysis, Standard Deviation, 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: The tissue sections were deparaffinized, rehydrated, and incubated overnight at 4°C with rabbit polyclonal anti-MMP-13 antibody (bs-10581R, Bioss Antibodies, MA, USA).

Techniques: Biomarker Discovery, Activity Assay