Journal: Neural Regeneration Research

Article Title: Porcine decellularized nerve matrix hydrogel attenuates neuroinflammation after peripheral nerve injury by inhibiting the TLR4/MyD88/NF-κB axis

doi: 10.4103/NRR.NRR-D-24-00302

Figure Lengend Snippet: TLR4/MyD88/NF-κB pathway plays an indispensable role in regulating pDNM-gel-mediated macrophage polarization and anti-inflammatory reactions in vitro . (A) WB detection of TLR4, MyD88, p-NF-κB, NF-κB, p-IκBα, and IκBα in RAW264.7 cells treated with LPS (100 ng/mL) + pDNM-gel (0.5%) for 24 hours, followed by the addition of CRX-527 (500 µM), TAK242 (100 nM), CL075 (2 µM), or T6167923 (100 µM) for another 24 hours. (B–E) Quantitative analysis of the expression levels of these four proteins from A. (F, G) Co-staining of CD68 (red) with iNOS (green) or Arg1 (green) in the LPS + pDNM-gel, LPS + pDNM-gel + CRX-527, LPS + pDNM-gel + TAK242, LPS + pDNM-gel + CL075, and LPS + pDNM-gel + T6167923 treatment groups. Nuclei were stained with DAPI (blue). Scale bars: 10 μm. (H, I) Quantification of the fluorescence intensity of iNOS and Arg1 from F and G. (J, K) Flow cytometry detection of M1-type and M2-type macrophages in each group. (L, M) Quantitative analysis of the percentages of double-positive CD68 + CD86 + and CD68 + CD206 + cells from J and K. (N, O) qRT-PCR analysis of the mRNA expression of pro-inflammatory signature genes TNF-α , IL-1 β, and IL-6 , and anti-inflammatory signature genes IL-4 , IL-10 , and TGF- β, in each group. (P, Q) ELISAs of inflammatory cytokine levels in RAW264.7 cell culture supernatants in each group. Data are expressed as mean ± SEM from three independent assays in vitro ; * P < 0.05, ** P < 0.01, *** P < 0.001. Arg1: Arginase 1; CD206: cluster of differentiation protein 206; CD68: cluster of differentiation protein 68; CD86: cluster of differentiation protein 86; DAPI: 4′,6-Diamidino-2-phenylindole; ELISA: Enzyme-linked immunosorbent assay; IL-10: interleukin-10; IL-1β: interleukin-1β; IL-4: interleukin-4; IL-6: interleukin-6; iNOS: inducible nitric oxide synthase; IκBα: nuclear factor kappa B inhibitory protein alpha; LPS: lipopolysaccharides; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor-κB; n.s,: not significant; pDNM-gel: porcine decellularized nerve matrix hydrogel; p-IκBα: phosphorylated nuclear factor kappa B inhibitory protein alpha; p-NF-κB: phosphorylated nuclear factor-κB; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; TGF-β: transforming growth factor-beta; TLR4: Toll-like receptor 4; TNF-α: tumor necrosis factor-alpha.

Article Snippet: Then, the medium was added with one of the following agonists/inhibitors and treated the cells for a further 24 hours: 500 μM CRX-527 (InvivoGen, Toulouse, France, Cat# tlrl-crx527), 100 nM TAK242 (MedChemExpress, Monmouth Junction, NJ, USA, Cat# HY-11109), 2 μM CL075 (MedChemExpress, Cat# HY-117066), or 100 μM T6167923 (MedChemExpress, Cat# HY-19744; Additional Figure 3 ).

Techniques: In Vitro, Expressing, Staining, Fluorescence, Flow Cytometry, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Porcine decellularized nerve matrix hydrogel attenuates neuroinflammation after peripheral nerve injury by inhibiting the TLR4/MyD88/NF-κB axis

doi: 10.4103/NRR.NRR-D-24-00302

Figure Lengend Snippet: Promotional effect of pDNM-gel on nerve regeneration is abolished using TLR4/MyD88/NF-κB agonists in vivo . (A) Immunofluorescence staining for neurofilament-H (green) and MBP (red) in sciatic nerves from rats treated with pDNM-gel, pDNM-gel + CRX-527, or pDNM-gel + CL075 at 21 dpi ( n = 5/group). Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (B) Representative TEM images of regenerated sciatic nerve cross-sections from the three groups at 21 dpi. Scale bars: 10 μm. (C, D) Calculated area (%) of positive staining of neurofilament-H and MBP per 100 μm 2 in the immunofluorescence images for the three groups ( n = 5/group). (E, F) Statistical analysis of the number and thickness of myelin sheaths in the three groups using ImageJ software ( n = 3/group). Data are expressed as mean ± SEM; ** P < 0.01, *** P < 0.001. DAPI: 4′,6-Diamidino-2-phenylindole; dpi: days post-injury; MBP: myelin basic protein; MyD88: myeloid differentiation factor 88; NF-κB: nuclear factor-κB; pDNM-gel: porcine decellularized nerve matrix hydrogel; PNI: peripheral nerve injury; SEM: standard error of the mean; TEM: transmission electron microscopy; TLR4: Toll-like receptor 4.

Article Snippet: Then, the medium was added with one of the following agonists/inhibitors and treated the cells for a further 24 hours: 500 μM CRX-527 (InvivoGen, Toulouse, France, Cat# tlrl-crx527), 100 nM TAK242 (MedChemExpress, Monmouth Junction, NJ, USA, Cat# HY-11109), 2 μM CL075 (MedChemExpress, Cat# HY-117066), or 100 μM T6167923 (MedChemExpress, Cat# HY-19744; Additional Figure 3 ).

Techniques: In Vivo, Immunofluorescence, Staining, Software, Transmission Assay, Electron Microscopy

Journal: Frontiers in Immunology

Article Title: De novo COVID-19-associated insulin resistance drives dysregulated neutrophil extracellular trap formation (NETosis) four months after infection

doi: 10.3389/fimmu.2026.1787799

Figure Lengend Snippet: Evaluation of NETosis in neutrophils of the study cohort by flow cytometry. (A) Flow cytometry plot strategy for the identification of PMN populations, neutrophils (using CD-16/anti-Singlec8 staining), quantification of TLR7/8 and identification of NETosis (using Live/Dead Dye and Sytox Blue staining). (B) Scatter plots showing the quantification of the percentage of baseline NETosis in neutrophils from the three patient groups of the study cohort. Ordinary one-way ANOVA *p <0.05. (C) Paired data scatter plots comparing the percentage of baseline NETosis and when stimulated with IL-6 (20ng/mL) and TNFα (2ng/mL). 2-way ANOVA **p <0.01. Scatter plots showing the quantification of the expression of (D) TLR7 and (E) TLR8 according to median fluorescence intensity (MFI). Ordinary one-way ANOVA. (F) Scatter plots of paired data comparing the percentage of baseline NETosis against stimuli on TLR receptors such as ImiQ (2μg/mL), (G) R848 (2μg/mL), (H) CL075 (1μg/mL), (I) ssRNA40 (1μg/mL) and (J) ssRNA41 (1μg/mL). For panels (F–J) , 2-way ANOVA *p <0.05, **p <0.01 ***p <0.005.

Article Snippet: For NETosis, 1 x 10 6 cells were incubated for 30 minutes at 37 °C and 5% CO2 under basal conditions or using IL-6 (20ng/mL) and TNFα (2ng/mL), or TLR7/8 agonists (CL075 (1μg/mL), ImiQ (2μg/mL), and R848 (2μg/mL), ssRNA40 (1μg/mL) and ssRNA 41(1μg/mL) (Catalog No.tlrl-kit3hw3, InvivoGen).

Techniques: Flow Cytometry, Staining, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Pregnancy loss due to early developmental defects in lupus mice expressing human TLR8

doi: 10.64898/2026.02.07.701591

Figure Lengend Snippet: HuTLR8 induces fetal resorption and spontaneous pregnancy loss in Sle1 mice. A) C57Bl/6.huTLR8tg mice were crossed with Yaa males and then with Sle1 females . Sle1.huTLR8tg female mice express one (Sle1.huTLR8 +/- ) or two (Sle1.huTLR8 +/+ ) copies of the single human TLR8 (huTLR8) transgenic locus. B. Outcomes of observed pregnancies. Adverse outcome was defined as maternal distress or death at delivery with evidence of an impacted pup or birth of dead or runted pups. p values calculated using Chi square analysis. C. I. An impacted pup in a Sle1.huTLR8 +/+ female II. An impacted pup together with an unaffected littermate; III. Representative uteri from Sle1 (top) and Sle1.huTLR8 +/+ (bottom) mice; IV. Growth retarded pup with an associated small placenta (*); V. A normal sized fetus and a resorbed fetus from the same pregnancy. D. Fetal weights (in gram) are depicted for full term Sle1 (n=5) and C57BL/6.huTLR8tg control live pregnancies and Sle1.huTLR8 +/- (n=16) and Sle1.huTLR8 +/+ (n=18) pregnancies with dystocia. Impacted/resorbed/dead fetuses are depicted in red. Maternal genotype and number of pregnancies are shown below the x axis. E, F. Total number of pups (E) and number of male (blue bars) and female (pink bars) pups (F) per live litter. Maternal genotype and number of pregnancies are shown below the x axis. Each symbol represents an individual pregnancy; bars represent the mean + SD. D-F: p values calculated using Kruskal Wallis ANOVA with Dunn’s correction for multiple analyses. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Article Snippet: Bone marrow neutrophils were isolated from 3– and 6-month-old homozygous female Sle1.huTLR8tg, Sle1 and C57BL/6 controls using EasySep mouse neutrophil enrichment kit (STEMCELL) and were treated with PMA 50nM for 4 hours at 37°C with or without pre-treatment with PMA or TLR8 agonist CL075 (Invivogen) 10mg/ml for 20 minutes.

Techniques: Transgenic Assay, Control