Journal: Scientific reports

Article Title: Myricetin ameliorates ox-LDL-induced HUVECs apoptosis and inflammation via lncRNA GAS5 upregulating the expression of miR-29a-3p.

doi: 10.1038/s41598-021-98916-7

Figure Lengend Snippet: Figure 7. Myr inactivated the TLR4/NF-κB signalling pathway in ox-LDL-treated HUVEC by down- regulating GAS5 or upregulating miR-29a-3p. Western blot analysis of p-p65, p65T, p-IκBα, IκBα and TLR4. *P < 0.05 compared with Control; #P < 0.05 compared with ox-LDL + Myr + NC. &P < 0.05 compared with ox-LDL + Myr + mimic control.

Article Snippet: Protein samples isolated from HUVEC cells (25 μg)) were resolved using SDS-PAGE and then moved to polyvinylidene fluoride (PVDF) membrane (Millipore, USA) with being probed with GAPDH (#2118, 1:5000, Cell Signaling), Bax (#2772S, 1:1000, Cell Signaling), Bcl-2 (#15071S, 1:1000, Cell Signaling), caspase3 (#9662S, 1:1000, Cell Signaling), CD31 (ab9498, 1:1000, Abcam), SM22a (ab14106, 1:1000, Abcam), p-p65 (10745-1-AP, 1:1000, Proteintech), p65 (66535-1-Ig, 1:1000, Proteintech), p-IkBa (10268-1-AP, 1:1000, Proteintech), IkBa (66418-1-Ig, 1:1000, Proteintech) and TLR4 (66350-1-Ig, 1:1000, Proteintech) antibody followed by appropriate secondary antibody.

Techniques: Western Blot, Control

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 2. Upregulation of HMGB1, TLR4, pIKBα and GFAP in astrocytes and brain microvessel endothelial cells in MCAO rats. (A, B and D) Compared with the sham group, the expression of HMGB1, TLR4, pIKBα and GFAP was potentiated in the cerebral cortex tissues of MCAO rats. The scale bars represent 60 and 30 μm in (A) and (B) respectively. (C and E) Consistently, the HMGB1 (red) in astrocytes in MCAO group translocated from nucleus (Blue color represents DAPI) to the cytoplasm (Green color represents GFAP). The expression of TLR4 (yellow) and pIKBα (yellow) in brain microvessel endothelial cells (Green color represents VIII factor) in MCAO group was potentiated respectively. The expression of GFAP (green) in astrocytes (Blue color represents DAPI) in MCAO group was also increased. The scale bars represent 10 μm. (D and E) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 8. HMGB1 contributed to upregulation of P-gp via TLR4/NF-kB pathway. (A) The relationship among HMGB1 and changes of TLR4, TIRAP, NF-kB and P-gp in rBMECs after OGD was investigated using related positive agents. (B) Changes of TLR4 (green) and TIRAP (red) after OGD and treatment with positive agents in rBMECs. (C) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group; #P < 0.05 vs. OGD group. The scale bars represent 10 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques:

Journal: Life science alliance

Article Title: Mast cell extracellular trap formation underlies vascular and neural injury and hyperalgesia in sickle cell disease.

doi: 10.26508/lsa.202402788

Figure Lengend Snippet: Figure 1. Sickle microenvironment drives mast cell extracellular trap (MCET) release. (A) Dorsal skin of female control HbAA mice treated with the vehicle and sickle HbSS mice treated with the vehicle or GSK484 (sc, 20 mg/kg/day) was imaged with a laser scanning confocal microscope in 100-μm immunostained (see the Materials and Methods section for details) sections. Z-stacks of 1.0 μm were sequentially acquired at 60x magnification. Association of mast cells with vasculature and nerve bundles is shown: nerve bundles (cyan, anti-neurofilament H- 200) surrounded by degranulating mast cells (green, anti-c-Kit) loaded with tryptase granules (red, anti- tryptase). Mast cells are extending green nanotubes interspersing and disrupting the nerve bundles, and tryptase granules are intertwined around the nerve bundles, suggestive of MCET formation in HbSS mice. (B) Mast cells, indicated with c-Kit and tryptase, associate with CD31+ (magenta) endothelial cells in HbSS skin. White arrows indicate mast cell interactions with nf or bv. (C) Incitement of a sickle microenvironment with TNF-α (1 ng/ml), hemin (40 μM), or TNF-α/hemin caused MCET formation in mast cells cultured from skin of HbSS female mice, shown with the formation of rigid DNA fibers (stained with cell-permeable DAPI and SYTO 13 and cell-impermeable SYTOX Orange). (D) HbSS mast cells treated with TNF-α/hemin showed citrullinated histones (red) and DNA-binding fluorescent probes, DAPI (cyan), SYTO 13 (green), and SYTOX Orange (red), indicating an extranuclear DNA that overlaps with histone citrullination. (E) MCETs were elongated in HbSS and HbAA mast cells with TNF-α, hemin, or combined treatment, and to a greater extent in HbSS mast cells compared with TNF-α or hemin alone. The data are analyzed with regular two-way ANOVA with Tukey’s post hoc multiple comparisons test. Data shown are the mean ± SEM of N = 17–24 cells. (F) TNF-α/hemin-induced MCET formation was abrogated by pretreatment with scrambled siRNA (10 μM), the TLR4 inhibitor TAK242 (1 μM), siRNA silencing of FcεR1 and TLR4 (10 μM each), and PAD4 inhibition with GSK484 (10 μM). Each image represents multiple images from five different 5.0-mo-old female mice. Abbreviations: bv, blood vessel; MCET, mast cell extracellular trap; nf, nerve fiber; PAD4, peptidylarginine deiminase 4; TLR4, Toll-like receptor 4; TNF-α, tumor necrosis factor alpha; trp, tryptase.

Article Snippet: Mast cells were also transfected with FcεR1 siRNA (10 μM, #sc-45268; Santa Cruz), TLR4 siRNA (10 μM, #sc-40261; Santa Cruz), or scramble siRNA negative control (10 μM, # 4390843; Thermo Fisher Scientific) using Lipofectamine RNAiMAX Transfection Reagent (#13778100; Thermo Fisher Scientific) per the manufacturer’s recommendation.

Techniques: Control, Microscopy, Cell Culture, Staining, Binding Assay, Inhibition

Journal: Inflammation Research

Article Title: Wnt5a, TLR2 and TLR4 are elevated in advanced human atherosclerotic lesions

doi: 10.1007/s00011-013-0697-x

Figure Lengend Snippet: Quantitative RT-PCR revealed higher expression of TLR4 and TLR2 in diseased regions of the atherosclerotic arteries relative to less advanced regions. RNA isolated ( n = 14) from the areas defined in Fig. was reverse transcribed, and subjected to quantitative RT-PCR for analyzing the transcription levels of TLR4 (a) and TLR2 (b) in all four regions using HPRT1 as an endogenous control. Individual comparison of 14 samples for TLR4 (c) and TLR2 (d) transcripts in less advanced arterial tissue (region 1) compared to the three different advanced regions, i.e. regions 2, 3 and 4, was also performed. Results are expressed as mean ± SEM. $ P < 0.001, * P < 0.002, € P < 0.003, Ω P < 0.004 compared to less advanced regions of the arteries, as determined by nonparametric Wilcoxon test

Article Snippet: Taqman Gene Expression Assays GEx (Hs99999909_m1 HPRT1, Hs00180103_m1 Wnt5a, Hs00152939_m1 TLR4, and Hs00610101_m1 TLR2) and Taqman Gene Expression Master Mix (Applied Biosystems) were used according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Expressing, Isolation, Reverse Transcription, Control, Comparison

Journal: Frontiers in Nutrition

Article Title: A Cocoa Diet Can Partially Attenuate the Alterations in Microbiota and Mucosal Immunity Induced by a Single Session of Intensive Exercise in Rats

doi: 10.3389/fnut.2022.861533

Figure Lengend Snippet: Changes in small intestine gene expression: Toll-like receptor (TLR) 2 (A) , TLR4 (B) , TLR5 (C) , TLR7 (D) , free fatty acid receptor (FFAR) 2 (E) and FFAR3 (F) , polymeric immunoglobulin receptor (pIgR) (G) , retinoic acid receptor (RAR) α (H) , forkhead box P3 (FoxP3) (I) ; occludin (Ocldn) (J) , claudin-4 (Cldn4) (K) , zonula occludens (ZO) 1 (L) and ZO2 (M) gene expression. Results expressed as fold change with respect to the REF/C group. The control (C) groups are represented by smooth bars and the runner (R) groups by striped bars. REF, reference diet; CF, 5% cocoa fiber-enriched diet; C10, 10% cocoa-enriched diet. Data are expressed as mean ± SEM ( n = 8). Statistical differences (Kruskal-Wallis followed by Dunnet’s test): * p < 0.05 vs. the C group in the same diet; # p < 0.05 vs. the same exercise condition in the REF diet; λ p < 0.05 vs. the same exercise condition in the CF diet.

Article Snippet: Afterward, the Real Time (RT)-PCR was carried out using the ABI Prism 7900 HT quantitative RT-PCR system (AB) and the following specific PCR TaqMan primers (AB): pIgR (Rn00562362_m1, Inventoried, I), RARα (Rn00580551_m1, I), FoxP3 (Rn01525092_m1, I), TLR2 (Rn02133647_s1, I), TLR4 (Rn00569848_m1, I), TLR5 (Rn04219239_s1, I), TLR7 (Rn01771083_m1, I), FFAR2 (Rn02345824_s1, I), FFAR3 (Rn01457614_g1, I), Ocldn (Rn00580064_m1, I), Cldn4 (Rn01196224_s1, I), ZO1 (Rn02116071_s1, I) and ZO2 (Rn01501483_m1, I).

Techniques: Gene Expression, Control