Journal: Heliyon

Article Title: Pharmacodynamics of Sishen decoction in relieving rheumatoid arthritis: Chemical composition, regulatory pathway and online prediction simulation

doi: 10.1016/j.heliyon.2024.e37257

Figure Lengend Snippet: SSD treatment ameliorated RA rats. Representative images of hind paws from different groups and changes in foot circumference. Hematoxylin eosin staining and immunohistochemical staining (CD31, CD39, CD73, CCR6 and IL1R1) of knee joint synovial slices. * P < 0.05, ** P < 0.01 vs Model group.

Article Snippet: Additionally, antibodies for IL1R1, CD39, CD73, and CCR6 were obtained from Bosterbio in Wuhan, China.

Techniques: Staining, Immunohistochemical staining

Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: (A) 2303 natural compound libraries were tested for their ability to inhibit the interaction between recombinant human IL-1β and IL1 Receptor 1. Microtiter 96-well plates were coated with hIL-1β (100 ng/well) overnight and the blocking buffer without any single compound was used as a positive control. Diluted single compounds (20 μM) were added to each well and incubated for 2 h. After washing, recombinant human IL-1R1 (125 ng/ml) was added and incubated for 2 h. Subsequently, HRP-conjugated Anti-Human IgG Fc (1:2000) was added and incubated for 1 h. An OD450 was obtained following the TMB reaction. Data indicate mean ± SD (n = 3). (B) Dose-dependency test of selected natural compound for blocking the interaction between recombinant human IL-1β and IL-1R1. Every step was identical to primary screening except for concentrations of selected natural compound (10, 40, or 160 μM) and washing buffer (PBS containing 0.05% Tween-20 and 0.01% Triton X-100). * p <0.05 compared to the control group of IL-1β and IL1 Receptor 1 interaction without any natural compounds. (C) IL-1β-dependent HEK-Blue IL-1β cells (5×10 4 cell/well) were seeded onto a 96-well plate and treated with pre-incubation (20 min) of human IL-1β (10 ng/ml) with various concentrations of TA (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 μM). After a 24 h incubation, SEAP activity was assessed using QUANTI-Blue™ and the optical ensity (OD) at 620 nm. Cell viability was measured by analyzing the OD at 450 nm using D-Plus CCK. The IC 50 value of tannic acid was determined using the GraphPadPrism 10 software. Data indicate mean ± SD (n = 3). (D) Surface plasmon resonance (SPR) assay was used to analyze the direct binding of tannic acid to human IL-1β. Human IL-1β protein (50 μg/ml) was immobilized on a CM5 sensor chip and various concentrations of TA (1.56, 3.125, 6.25, 12.5, 25, 37.5, 50, 62.5, 75, 87.5, or 100 μΜ) were injected into the flow system with a flow rate 20 μl/min for 300 s and allowed to dissociate for 600 s. The K D values of the tannic acid against human IL-1β were obtained using the T200 BIA evaluation software.

Article Snippet: ELISA-based binding assays were performed for IL-1β-blocking candidates to block the interaction between IL-1β and IL-1R1 using 2303 natural compound libraries (TargetMol L-6000, Wellesley Hills, MA, USA; Selleckhem L-1400, Matsonford Road Radnor, PA, USA).

Techniques: Recombinant, Blocking Assay, Positive Control, Incubation, Control, Activity Assay, Software, SPR Assay, Binding Assay, Injection

Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: Human articular chondrocytes from OA patients were seeded onto 12-well plates (2×10 5 cells/well) and serum-starved cells were co-treated with various concentrations of TA (0.5, 1, or 2 μM) or IL-1R1 (1 μg/ml) and IL-1β (10 ng/ml) for 48 h. (A) The mRNA expression levels of iNOS , COX-2 , IL-6 , and TNF were measured using qRT-PCR. The relative quantity of each gene expression was normalized to the relative quantity of human GADPH. (B) Griess reaction was used to measure the NO levels in the culture supernatants and PGE2, IL-6, and TNF levels in the culture supernatants were evaluated using ELISA. # p < 0.05 compared with medium only control group and * p <0.05 compared with IL-1β-treated group.

Article Snippet: ELISA-based binding assays were performed for IL-1β-blocking candidates to block the interaction between IL-1β and IL-1R1 using 2303 natural compound libraries (TargetMol L-6000, Wellesley Hills, MA, USA; Selleckhem L-1400, Matsonford Road Radnor, PA, USA).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Enzyme-linked Immunosorbent Assay, Control

Journal: PLOS ONE

Article Title: Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

doi: 10.1371/journal.pone.0281834

Figure Lengend Snippet: Human articular chondrocytes from OA patients were seeded onto 6-well plates (3×10 5 cells/well) and serum-starved cells were co-treated with various concentrations of TA (2 μM), IL-1R1 (1 μg/ml), or anti-IL-1 neutralizing antibody (1 μg/ml) and IL-1β (10 ng/ml) for 48 h. mRNA and protein expressions of MMPs, ADAMTSs, collagen type II, and aggrecan were detected by qRT-PCR (A) and Western blot analysis (B). The relative quantity of each gene expression was normalized to the relative quantity of human GADPH and β-actin was used as a loading control. # p < 0.05 compared with medium only control group and * p <0.05 compared with IL-1β-treated group.

Article Snippet: ELISA-based binding assays were performed for IL-1β-blocking candidates to block the interaction between IL-1β and IL-1R1 using 2303 natural compound libraries (TargetMol L-6000, Wellesley Hills, MA, USA; Selleckhem L-1400, Matsonford Road Radnor, PA, USA).

Techniques: Quantitative RT-PCR, Western Blot, Gene Expression, Control

Journal: NPJ Breast Cancer

Article Title: IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer

doi: 10.1038/s41523-021-00305-w

Figure Lengend Snippet: a Quantification (photons/sec (p/s)) of primary tumour growth in IL1R1 fl/fl ( n = 8 primary tumours from n = 4 mice) and IL1R1 −/− ( n = 8 primary tumours from n = 4 mice) mice up to 12 days of post-orthotopic injection of E0771-luc2-V5-GFP cells and b correspondent micrographs. c Quantification (p/s) of primary tumour growth in IL-1B fl/fl ( n = 7) and IL-1B −/− ( n = 6) mice up to 26 days of post-orthotopic injection of E0771-luc2- GFP cells and d correspondent micrographs. IL-1B fl/fl : n = 13 primary tumours from n = 7 mice (day 7 and 14); n = 12 primary tumours from n = 6 mice (day 20 and 26). IL-1B −/− : n = 12 primary tumours from n = 6 mice (day 7); n = 10 primary tumours from n = 6 mice (day 14, 20, and 26). 2.3-fold increase in primary tumour growth in IL-1B −/− mice (7.6 × 10 8 p/s) compared to IL-1B fl/fl mice (3.2 × 10 8 p/s) ( P = 0.01). Data are mean +/− SEM, Two-way ANOVA with Sidak’s post-hoc test.

Article Snippet: The following plasmids were used for overexpression studies: IL-1B (MR226719L4, Origene), IL1R1 (MR227508L4, Origene), GFP (17448 pLenti CMV GFP Puro (658-5), Addgene), Luciferase (21474 pLenti CMV V5-LUC Blast (w567-1), Addgene).

Techniques: Injection

Journal: NPJ Breast Cancer

Article Title: IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer

doi: 10.1038/s41523-021-00305-w

Figure Lengend Snippet: a , b Images and quantification of primary tumour development in IL-1B fl/fl ( n = 8 primary tumours from n = 4 mice) and IL-1B −/− ( n = 10 primary tumours from n = 5 mice) mice after intra-ductal administration of E0771 Luc2 V5 IL-1B-GFP cells. Data are mean +/− SEM, Two-way ANOVA with Sidak’s post-hoc test. c , d Quantification of F4/80 + macrophages ( c ) and CD163 + macrophages ( d ) in tumour core and periphery in IL-1B fl/fl and IL-1B −/− mice. Data are mean ± SEM, Two-way ANOVA with Sidak’s post hoc test. e , f Quantification of CD34 + blood vessels and MPO + neutrophils in IL-1B fl/fl and IL-1B −/− mice. Data are shown as mean +/− SEM, Two-tailed unpaired t -test. g , h Images and quantification of primary tumour development in IL1R1 fl/fl ( n = 18 primary tumours from n = 9 mice) and IL1R1 −/− ( n = 14 primary tumours from n = 7 mice) mice after intra-ductal administration of E0771 Luc2 V5 IL-1B-GFP. Normalised data are shown as mean +/− SEM, Two-tailed unpaired t -test.

Article Snippet: The following plasmids were used for overexpression studies: IL-1B (MR226719L4, Origene), IL1R1 (MR227508L4, Origene), GFP (17448 pLenti CMV GFP Puro (658-5), Addgene), Luciferase (21474 pLenti CMV V5-LUC Blast (w567-1), Addgene).

Techniques: Two Tailed Test

Journal: NPJ Breast Cancer

Article Title: IL-1B drives opposing responses in primary tumours and bone metastases; harnessing combination therapies to improve outcome in breast cancer

doi: 10.1038/s41523-021-00305-w

Figure Lengend Snippet: a , b Tumour proliferation after injection of E0771 luc2 V5 IL1R1-GFP cells in IL1R1 fl/fl ( n = 8 primary tumours from n = 4 mice) and IL1R1 −/− ( n = 6 primary tumours from n = 3 mice) mice. Data are mean +/− SEM, Two-way ANOVA with Sidak’s multiple comparisons test. c In vitro relative tumour growth of E0771 luc2 V5 GFP, IL-1B GFP or IL1R1 GFP cells upon stimulation with 40 pg/ml mouse recombinant IL-1B. Data are mean (+/− SEM), Two-way ANOVA with Sidak’s multiple comparison test ( n = 6 technical repeats from two biological replicates). d Transwell cell migration of IL-1B overexpressing ( P = 0.0009) and IL1R1 overexpressing ( P < 0.0001) E0771 luc2 V5 cancer cells compared to control GFP-expressing cells (no treatment with exogenous IL-1B). Comparison of cell migration in vitro between E0771 luc2 V5 IL-1B GFP and IL1R1 GFP tumour cells ( P = 0.0018). Data are mean (+/− SEM) cell number from 4 fields of view derived from three biological experiments ( n = 12 technical repeats). Two-tailed unpaired t -test with Welch’s correction. e Representative images of haematoxylin-stained control, IL-1B and IL-1R1 overexpressing cells migrated through the membrane. Scale bar = 200 µm.

Article Snippet: The following plasmids were used for overexpression studies: IL-1B (MR226719L4, Origene), IL1R1 (MR227508L4, Origene), GFP (17448 pLenti CMV GFP Puro (658-5), Addgene), Luciferase (21474 pLenti CMV V5-LUC Blast (w567-1), Addgene).

Techniques: Injection, In Vitro, Recombinant, Migration, Expressing, Derivative Assay, Two Tailed Test, Staining

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: Specificity of the anti-IL-1R1 antibody and distribution of IL-1R1 immunoreactivity in the spinal dorsal horn. a Adsorption of anti-IL-1R1 antibody to recombinant IL-1R1 peptide completely abolished the immunostaining. b Western blot analysis reinforces the specificity of the anti-IL-1R1 antibody. The single immunoreactive band indicates that the antibody detects a protein with a molecular mass of ~80 kDa that corresponds to the molecular weight of IL-1R1. c , d Micrographs showing immunoreactivity for IL-1R1 in control ( c ) and CFA-injected rats ( d ) 3 days after CFA-injection. Bars 100 μm

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Adsorption, Recombinant, Immunostaining, Western Blot, Molecular Weight, Injection

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: CFA-evoked inflammation of the hindpaw initiates an overproduction of IL-1R1 protein in the spinal dorsal horn of rats. a Representative immune-blots showing immunoreactive bands for IL-1R1 and β-tubulin (loading control) in Western blots of tissue samples obtained from the L3–L5 lumbar segments of the spinal dorsal horn of the control and CFA-injected animals at post-injection day 3. b Histogram showing the optical densities of IL-1R1 immunostained bands (see on insert a ) calculated in proportion to the optical densities of β-tubulin (loading control) immunostained bands (see on insert a ). IL-1R1 protein level was found to be significantly higher than the control value ( p = 0.029). Data are shown as mean ± SEM

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Western Blot, Injection

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: Mechanical withdrawal threshold and thermal withdrawal latency of wild type and IL-1R1 knockout mice during the course of CFA-induced inflammation of the hind paw. a The histogram shows the mechanical withdrawal threshold (MWT) of wild type (BL6) and IL-1R1 knockout (IL-1R1 KO) mice receiving different treatments in the three experimental groups: group (1) complete Freund-adjuvant (CFA) injection (BL6 CFA, IL-1R1 KO CFA); group (2) physiological saline injection (BL6 sham, IL-1R1 KO sham); and group (3) without any treatment (BL6 control, IL-1R1 KO control). Measurements were made only on the right hind paw which received the physiological saline or CFA injections. b The histogram shows the thermal withdrawal latency (TWL) of wild type (BL6) and IL-1R1 knockout (IL-1R1 KO) mice receiving different treatments in the three experimental groups: group (1) complet Freund-adjuvant (CFA) injection (BL6 CFA, IL-1R1 KO CFA); group (2) physiological salt solution injection (BL6 sham, IL-1R1 KO sham); and group (3) without any treatment (BL6 control, IL-1R1 KO control). Measurements were made only on the right hind paw which received the physiological saline or CFA injections. Data are shown as mean ± SEM

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Knock-Out, Injection

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: Localization of IL-1R1 on neurons and glial cells in the superficial spinal dorsal horn of rats. Micrographs of single 1-μmthick laser scanning confocal optical sections illustrating the co-localization between immunolabeling for IL-1R1 ( red ; a – d , e , h , k , n , q ) and immunoreactivity for markers that are specific for axon terminals of peptidergic (CGRP, green ; a ) and non-peptidergic (IB4 binding, green ; b ) primary afferents, axon terminals of excitatory (VGLUT2, green ; c ) and inhibitory (VGAT, green ; d ) intrinsic neurons, astrocytes (GFAP, green ; o ) and microglial cells (CD11b, green ; r ), and postsynaptic membranes of excitarory (PSD95, green ; i ) and inhibitory (gephyrin, green ; l ) synapses in the superficial spinal dorsal horn. Mixed colors ( yellow ; marked by white arrowheads ) on the superimposed images ( j , m , p , s ) indicate double-labeled structures . The absence of yellow color on a – d indicates a lack of IL-1R1 expression on axon terminals of various origin. IL-1R1 immunoreactive spots appear in two different localization on the micrographs showing immunostaining also for KCC2 ( green , f , g ): (1) They can be aligned along the lines defined by the KCC2 immunostaining (cell membrane localization; white arrowheads on f and g ). (2) They can also be located in areas surrounded by the KCC2-immunostained cell membranes (cytoplasmic localization, yellow arrowhead on g . Bars 2 μm ( a – d ), 5 μm ( n – s ), and 10 μm ( e – m )

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Immunolabeling, Binding Assay, Labeling, Expressing, Immunostaining

Journal: Journal of Neuroinflammation

Article Title: Interleukin-1 receptor type 1 is overexpressed in neurons but not in glial cells within the rat superficial spinal dorsal horn in complete Freund adjuvant-induced inflammatory pain

doi: 10.1186/s12974-017-0902-x

Figure Lengend Snippet: Histogram showing the CFA-evoked inflammation induced changes in the degree of co-localization between immunoreactivity for IL-1R1 and selected neuronal and glial markers in the superficial spinal dorsal horn of rats. Columns indicate the percentages of profiles immunoreactive for IL-1R1 that were found to be labeled also for the selected markers, and the ones that were aligned along KCC2 immunoreactive membranes (localization on the somatodendritic membrane of neurons) or were located within areas surrounded by KCC2 immunoreactive membranes (localization within the cytoplasm of the somatodendritic compartment of neurons). White columns show data obtained from control animals, whereas black columns represent values found in CFA-injected animals 3 days after CFA injection into the right hind paw. Asterisk indicate that CFA-evoked inflammation significantly increased the number of spots immunoreactive for IL-1R1 on the somato-denditic membrane of neurons ( p = 0.000001). Data are shown as mean ± SEM

Article Snippet: As a part of the immunohistochemical protocol, we tested the specificity of the primary antibody on tissue sections by treating the diluted anti-IL-1R1 with recombinant rat IL-1R1 protein (Sino Biological Inc, Beijing, China, catalog no.: 80028R08H50) for the purpose of antibody adsorption.

Techniques: Labeling, Injection