Journal: Frontiers in Nutrition

Article Title: Altered intestinal barrier contributes to cognitive impairment in old mice with constipation after sevoflurane anesthesia

doi: 10.3389/fnut.2023.1117028

Figure Lengend Snippet: Sevoflurane exposure increased the serum concentration of inflammatory cytokines and activated the TLR4/NF-κB signaling pathway of the PFC in aged STC mice. (A–H) Statistical analysis of the concentrations of IL-2 (A, F 3,16 = 4.16, p = 0.02), IL-4 (B, F 3,16 = 1.00, p = 0.42), IL-1 (C, F 3,16 = 5.77, p < 0.01), IL-5 (D, F 3,16 = 5.48, p < 0.01), IL-10 (E, F 3,16 = 5.42, p = 0.01), INF-γ (F, F 3,16 = 6.28, p < 0.01), TNF-α (G, F 3,16 = 7.58, p < 0.01), and GM-CSF (H, F 3,16 = 3.96, p = 0.03) at 4 h after sevoflurane anesthesia. (I) Representative western blot bands for TLR4 and GAPDH proteins. (J) Representative western blot bands for active-NF-κB, NF-κB, and GAPDH proteins. (K) Quantitative analysis of the expression of TLR4 at 24 h after sevoflurane anesthesia ( n = 6, F 3,16 = 13.37, p < 0.01). (L) Quantitative analysis of the relative expression of active-NF-κB/NF-κB at 24 h after sevoflurane anesthesia ( n = 6, F 3,16 = 7.04, p < 0.01). Data were presented as mean ± SD. One-way analysis of variance (ANOVA) with Tukey’s post hoc comparison. * p < 0.05; ** p < 0.01. CTRL, control; Sev, sevoflurane exposure; STC, slow transit constipation.

Article Snippet: After blocking for 1 h in 5% milk in PBS or tris-buffered saline and 5% bovine serum albumin, membranes were incubated with mouse anti-Toll-like receptor 4 (TLR4) (1:250, Cat.No.sc-293,072, Santa Cruz Biotechnology, Santa-Cruz, United States), rabbit anti-nuclear factor-κB (NF-κB) (1:1000, Cat.No.06–418, Millipore, United States), rabbit anti-active-NF-κB (1:1000, Cat.No.MAB3026, Millipore, United States), rabbit anti-GAPDH (1:1000, Cat.No.5174, Cell Signaling Technology, United States) overnight at 4°C.

Techniques: Concentration Assay, Western Blot, Expressing, Comparison, Control

Journal: Frontiers in Pharmacology

Article Title: Periplaneta americana extract alleviates steatohepatitis in a mouse model by modulating HMGB1-mediated inflammatory response

doi: 10.3389/fphar.2022.995523

Figure Lengend Snippet: GLC blocks M1 polarization by decreasing HMGB1 released by hepatocytes. (A) Immunofluorescence staining of HMGB I in mouse liver tissues. Scale bars, 50 μm. (B) Statistics of cytoplasmic HMGB1 positive cells of IF staining sections (C) Dataset (GEO database- GSE28619) of patients with alcoholic hepatitis were analyzed. Expression levels of M1 related markers (CD86, CXCL9, CXCL1 0 and TLR4) in HMGB1-High group and HMGB1-Low group. (D) Schematic flow chart of co-culture system. NCTC1469 cells induced by EtOH with or without treatment of GLC serum. Supemetant was collected for ELISA and culturing macrophages. The treatment of NCTC1469 cells was displayed in table (E) Supernatant HMGB1 levels of NCTC1469 cells induced by EtOH. ** P < 0.01 vs. the NCTC1469 cells with EtOH treatment. (F) Representative iNOS immunofluorescence staining and statistics of macrophages treated with condition medium (CM) of NCTC1469 cells. Scale bars, 50 μm. (G) Quantitative PCR analysis of J774A.1 cell cocultured with NCTCI469 cells. The data were obtained from three dependent experiments per group. * P < 0.05 vs. G#1, *** P < 0.001 vs. G#1.

Article Snippet: The membranes were washed, blocked and incubated with specific primary anti-rabbit antibodies against CYP2E1 (Proteintech, #19937-1-AP), GAPDH (Cell signaling Technology, #2118S), pNF-kB p65 (Wanleibio, #WL02169), NF-kB p65 (Wanleibio, #WL01273b), TLR4 (PTMBIO, #PTM-5192).

Techniques: Immunofluorescence, Staining, Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Periplaneta americana extract alleviates steatohepatitis in a mouse model by modulating HMGB1-mediated inflammatory response

doi: 10.3389/fphar.2022.995523

Figure Lengend Snippet: GLC ameliorates progression of steatohepatitis via HMGB1-mediated TLR4/NF-KB pathway (A) Western blot analysis of indicated proteins expression. (B) The relative intensity of indicated proteins expression. The data were obtained from three dependent experiments per group. (C) Process of GLC inhibiting HMGB1-mediated macrophage activation by TLR4/NF-KB in the progression of steatohepatitis. Alcoholics and free fatty acids cause oxidative stress injury and lipid accumulation of hepatocytes. The dying/dead hepatocytes release DAMPs - HMGB1 to the extracellular. HMGB1 combined with TLR4 of macrophages to activate the inflammatory response through the NF-KB pathway, which aggravated the hepatocyte injury. GLC exerts hepatoprotective properties by reducing lipid accumulation and oxidative stress. It decreases the HMGB1 in extracellular and blocks TLR4/NF-KB mediated inflammatory cytokines expression of macrophages.

Article Snippet: The membranes were washed, blocked and incubated with specific primary anti-rabbit antibodies against CYP2E1 (Proteintech, #19937-1-AP), GAPDH (Cell signaling Technology, #2118S), pNF-kB p65 (Wanleibio, #WL02169), NF-kB p65 (Wanleibio, #WL01273b), TLR4 (PTMBIO, #PTM-5192).

Techniques: Western Blot, Expressing, Activation Assay

Journal: Iranian Journal of Public Health

Article Title: Exosomal ITGB2 Mediates Immune Evasion in Triple-Negative Breast Cancer by Suppressing Dendritic Cell Activation via TLR4

doi: 10.18502/ijph.v54i6.18903

Figure Lengend Snippet: Modulation of TLR4 Expression in DCs by Exosomal ITGB2 from TNBC Cells. (A–B) Flow cytometry analysis of dendritic cells (DCs) induced from TNBC patient PBMCs, showing increased proportions of CD80 and CD83 positive cells post-induction, indicating successful DC culture. (C–D) Effects of exosomes from MD-AMB-231 cells with varied ITGB2 expression levels on TLR4 expression in DCs. Exosomes from MD-AMB-231 cells decreased TLR4 expression, while exosomes from ITGB2-knockdown cells restored it, and exosomes from ITGB2-overexpressing cells further reduced TLR4 expression. (E–F) The effect of exosomal ITGB2 on DC maturation (CD80, CD83) in the presence of LPS was evaluated by FCM

Article Snippet: After SDS-PAGE separation and PVDF membrane transfer, membranes were incubated overnight at 4°C with primary antibodies against ITGB2 (#72607), TLR4 (#14358), and β-actin (#3700), which all were purchased from Cell Signaling Technology.

Techniques: Expressing, Flow Cytometry, Knockdown

Journal: Iranian Journal of Public Health

Article Title: Exosomal ITGB2 Mediates Immune Evasion in Triple-Negative Breast Cancer by Suppressing Dendritic Cell Activation via TLR4

doi: 10.18502/ijph.v54i6.18903

Figure Lengend Snippet: Modulation of Cytokine Secretion in DCs by Exosomal ITGB2 from TNBC Cells. (A–E) Levels of cytokines IL-12, IL-4, TNF-α, TGF-β, and IFN-β in DCs treated with exosomes from MD-AMB-231 cells, showing downregulation in MD-AMB-231 and OE- ITGB2 exosome-treated groups and restoration in sh- ITGB2 exosome-treated group. (F) Schematic representation of the mechanism by which TNBC-derived exosomal ITGB2 regulates TLR4 and inhibits dendritic cell maturation and immune response

Article Snippet: After SDS-PAGE separation and PVDF membrane transfer, membranes were incubated overnight at 4°C with primary antibodies against ITGB2 (#72607), TLR4 (#14358), and β-actin (#3700), which all were purchased from Cell Signaling Technology.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: Inhibition of Sphingosine Kinase-2 Suppresses Inflammation and Attenuates Graft Injury after Liver Transplantation in Rats

doi: 10.1371/journal.pone.0041834

Figure Lengend Snippet: Livers were harvested at 18 h after transplantation (TX) or sham-operation (Sham). Levels of TLR4, NF-κB p65 subunit, phosphorylated NF-κB p65 subunit (p-NF-κB p65), intracellular adhesion molecule-1 (ICAM-1) and actin were determined by immunoblotting. Representative blot images are shown in A . Images were quantified by densitometry ( B–D ). ABC, ABC294640; a, p<0.05 vs sham; b, p<0.05 vs the TX group (n = 4 per group).

Article Snippet: Livers were harvested at 18 h after transplantation or sham operation, and immunoblotting was performed as described elsewhere using specific antibodies against cleaved caspase-3 (Cell Signaling Technology, Danvers, MA), toll-like receptor-4 (TLR4, Abcam, Cambridge, MA), NF-κB p65, phosphorylated NF-κB p65 (Ser 536), ED2 (Santa Cruz Biotechnology, Santa Cruz, CA), intracellular adhesion molecule-1 (ICAM-1; BD Biosciences Pharmingen, San Diego, CA), myeloperoxidase (MPO; DAKO Corp., Carpinteria, CA), CD4 (Origene Technologies, Rockville, MD), interferon-γ (IFNγ, Biolegend, San Diego, CA) at concentrations of 1∶500–1∶1000 , or actin (Cell Signaling Technology; 1∶3000) at 4°C over night.

Techniques: Transplantation Assay, Western Blot

Journal: Translational Neuroscience

Article Title: Euxanthone inhibits traumatic spinal cord injury via anti-oxidative stress and suppression of p38 and PI3K/Akt signaling pathway in a rat model

doi: 10.1515/tnsci-2021-0012

Figure Lengend Snippet: Effect of Euxanthone on TLR4/NF-κB protein expression in t-SCI-induced rats. (a) Quantification of TLR4 protein expression. (b) Representative Western blot images of the effects of Euxanthone on TLR4/NF-κB protein expression. (c) Quantification of NF-κB protein expression. GAPDH served as an internal control. The data are represented as the mean value ± standard error of mean. # p < 0.01 relative to sham group, * p < 0.05 relative to t-SCI model group, and $ p < 0.05 relative to t-SCI model group and Eux30 study group. t-SCI, traumatic spinal cord injury; Eux30 and Eux60, Euxanthone 30 and 60 mg/kg, respectively, in t-SCI-induced rats; TLR4, toll-like receptor 4; NF-κB, nuclear factor; p, phosphorylated.

Article Snippet: The membranes were then incubated for 12 h at 4°C with primary antibodies purchased from Bio-rad Laboratories, Shanghai, China, namely – Receptor activator of nuclear factor-κB ligand (RANKL) (MCA5971), toll-like receptor 4(TLR4) (AHP1822), Nuclear factor (NF)-κB (VPA00015), phosphorylated (p)-NF-κB (AHP1342), PI3K (AHP2224), Akt (VMA00253) and GAPDH (HCA272), p38 (AHP2410), and p-p38 (#9211, Cell Signaling Technology, Shanghai, China).

Techniques: Expressing, Western Blot