Diosmetin and diosmin were purchased from ChemFaces (Wuhan, China), their purity was over 98%. 2,4-Dinitrochlorobenzene (DNCB) was obtained from Sigma-Aldrich Co (St. Louis, MO, USA). DMEM (Dulbecco’s Modified Eagle’s Media) and FBS (Fetal Bovine Serum) were obtained from HyClone Laboratories, Inc (Logan, UT, USA). Penicillin-streptomycin solution and Fast SYBR® Green Master Mix were purchased from Life Technologies (Waltham, MA, USA). An RNeasy mini kit and a ImProm-II Reverse Transcription System kit were purchased from Qiagen (Valencia, CA, USA) and Promega (Madison, WI, USA), respectively.
2 4 dinitrochlorobenzene dncb
Manufactured by Merck Group
Sourced in United States
2,4-dinitrochlorobenzene (DNCB) is a chemical compound commonly used as a laboratory reagent. It is a yellowish crystalline solid with the molecular formula C6H3ClN2O2. DNCB is primarily utilized as a reference substance and an intermediate in the synthesis of other organic compounds.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Cytiva (2 mentions)
Cinnamic aldehyde, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Improm 2 reverse transcription system kit, by Qiagen (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Cytiva (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Diosmin, by ChemFaces (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
P mk2, by Cell Signaling Technology (1 mentions)
P mkk3 6, by Cell Signaling Technology (1 mentions)
Sb203580, by Merck Group (1 mentions)
Mkk3 6, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
P jnk, by Santa Cruz Biotechnology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
15 protocols using 2 4 dinitrochlorobenzene dncb
1
Diosmetin and Diosmin Characterization
2
Roxatidine Modulates Inflammatory Signaling
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For this study, roxatidine (≥98% purity, Fig. 1A ), primary antibodies for p65, p–IκBα, IκBα, p-ERK, p-p38, p-JNK, ERK, p38, JNK, PARP, α-tubulin, caspase-1, MKK3/6, MAPK-activated protein kinase 2 (MK2), and β-actin and peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). p-IKK, IKK, p-Akt, Akt, p-MKK3/6, and p-MK2 primary antibodies were obtained from Cell Signaling Technology (Danvers, MA) and M5/114.15.2 (MHC class II) antibody was purchased from eBioscience (eBioscience, Affimatrix Phorbol 12-myristate 13-acetate (PMA), calciumionophore A23187 (Calcymycin; C29H37N3O6), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tertazolium bromide (MTT), fluorescein isothiocyanate (FITC) isomer-I, 2,4-dinitrochlorobenzene (DNCB) and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Iscove’s modified Dulbecco’s medium (IMDM) fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY). The enzyme immunoassay (EIA) kits for TNF-α, IL-6, and IL-1β were obtained from R&D Systems (Minneapolis, MN, USA). SYBR Premix Ex Taq was purchased from Takara (Shuzo, Shiga, Japan). TNF-α, IL-6, IL-1β, and GAPDH oligonucleotide primers were purchased from Bioneer (Daejeon, Chungbuk, South Korea). SB203580 was purchased from Sigma Chemical Co. (St. Louis, MO).
3
Synthesis and Characterization of Acetophenone Azine
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Acetophenone Azine; Chemical name: 1‐phenyl‐ethanone(1‐phenylethylidene)hydrazine (CAS no. 729‐43‐1; batch number: 10138845 l) were purchased from Alfa Aesar and the purity of the compound was 97%; Cinnamic aldehyde (CA), (CAS no.14371‐10‐9) and dimethylsulfoxide (DMSO) were purchased from Sigma‐Aldrich. 2,4‐Dinitrochlorobenzene (DNCB) (CAS no.97‐00‐7) and Nickel Sulphate (NiSO4) (CAS no.10101‐97‐0) were purchased from Sigma‐Aldrich and Merck, respectively. The purity of the compounds was reported equal to or higher than 99% by the respective suppliers.
Acetone:Olive oil 4:1 (vol/vol) mixture, N,N‐dimethylformamide (DMF), methyl ethyl ketone, propylene glycol, dimethyl sulfoxide and 1% aqueous Pluronic PE9200 were used as standard OECD vehicles.
α‐Hexylcinnamaldehyde (HCA) (CAS no.101‐86‐0; batch number: MKCD2910) was purchased from Sigma‐Aldrich Co.
Acetone:Olive oil 4:1 (vol/vol) mixture, N,N‐dimethylformamide (DMF), methyl ethyl ketone, propylene glycol, dimethyl sulfoxide and 1% aqueous Pluronic PE9200 were used as standard OECD vehicles.
α‐Hexylcinnamaldehyde (HCA) (CAS no.101‐86‐0; batch number: MKCD2910) was purchased from Sigma‐Aldrich Co.
4
Murine Macrophage Cytokine Profiling
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Minimum essential medium-α (MEM-α), fetal bovine serum (FBS), and penicillin–streptomycin (P/S) were purchased from Life Technologies (Carlsbad, CA, USA). Recombinant mouse macrophage colony-stimulating factor (M-CSF) and enzyme-linked immunosorbent assay (ELISA) kits for interleukin-1β (IL-1β) were obtained from R&D Systems (Minneapolis, MN, USA). An ELISA kit for immunoglobulin E (IgE), interleukin-6 (IL-6), interleukin-18 (IL-18), and thymic stromal lymphopoietin (TSLP) was supplied by Invitrogen (Carlsbad, CA, USA). Lipopolysaccharide from Escherichia coli O26:B6 (LPS), adenosine 5′-triphosphate disodium salt hydrate (ATP) and 2,4-dinitrochloro benzene (DNCB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Radioimmunoprecipitation assay buffer (RIPA buffer) and skim milk powder were supplied by ELPIS Biotech (Deajeon, Republic of Korea) and BD Difco (Sparks, MD, USA), respectively. All other materials and reagents used in the study were of the highest quality available.
5
Dexamethasone Modulates Inflammatory Responses
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Dexamethasone (DEX; C22H29FO5; molecular weight, 392.46 g/mol; cat. no. D4902), a representative GC, lipopolysaccharide (LPS), scopoletin and 2,4-dinitrochlorobenzene (DNCB) were obtained from Sigma-Aldrich (Merck KGaA). Institute of Cancer Research (ICR) mice were supplied by the Animal Center of NARA Biotech. Phosphorylated (p)-NF-κB (cat. no. 3033) and p-GR antibodies (cat. no. 4161S) were purchased from Cell Signaling Technology, Inc. Bone morphogenetic protein 2 (BMP-2, cat. no. ab14933) and Runt-related transcription factor 2 (Runx2, cat. no. ab76956) antibodies were purchased from Abcam. Actin (cat. no. sc-8432) and Lamin B antibody (cat. no. sc-374015) was obtained from Santa Cruz Biotechnology, Inc., calbindin-D28k antibody (cat. no. PA1-931) was obtained from Thermo Fisher Scientific, Inc., and HRP-conjugated IgG antibody (cat. no. 115-035-062) was obtained from Jackson ImmunoResearch Laboratories, Inc. FBS, penicillin/streptomycin and PBS were provided by Invitrogen; Thermo Fisher Scientific, Inc. CellTiter 96® AQueous Non-Radioactive Cell Proliferation assay was obtained from Promega Corporation. Reverse transcription PCR (RT-PCR) primers were purchased from GenoTech Corp.
6
Inflammatory Signaling Pathway Modulation
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Arctigenin, hederagenin, and baicalein were purchased from Chemfaces (Hubei, China). 2,4-Dinitrochlorobenzene (DNCB), corticotropin-releasing hormone CRH, substance P (SP), 1-thioglycerol, U0126, SB202190, and SP600125 were obtained from Sigma-Aldrich (St. Louis, MO, USA). High-glucose Dulbecco's modified Eagle's medium (DMEM) was obtained from Welgene Inc. (Gyeongsangbuk, Korea). Iscove's modified Dulbecco's medium (IMDM) was purchased from Merck Millipore (Darmstadt, Germany). Fetal bovine serum (FBS) and antibiotics were obtained from Invitrogen Inc. (Carlsbad, CA, USA). Human IL-6, ICAM-1, VEGF, and mouse IL-13, TNF-α, IFN-γ, IL-12, VEGF, and IgE ELISA kits were purchased from Koma Biotech Inc. (Seoul). Human TARC/CCL17, MDC/CCL22, and mouse IL-4 ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies for phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated p38 (p-p38) were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP-conjugated anti-β-actin was obtained from Sigma-Aldrich (St. Louis, MO, USA).
7
DNCB-induced Atopic Dermatitis Model
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Eight-week-old female BALB/c mice were purchased from the Guangdong Animal Centre (Guangzhou, China) and housed under specific pathogen-free conditions at 22°C ± 2°C, 45% ± 10% relative humidity, and a 12 h light/dark cycle. Furthermore, 2,4-dinitrochlorobenzene (DNCB) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetone and olive oil were obtained from Aladdin (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits were purchased from BD Biosciences (San Jose, CA, USA). The TRIzol reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
8
DNCB-Induced Atopic Dermatitis in Mice
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2,4-Dinitrochlorobenzene (DNCB; 1%) (Sigma-Aldrich, Seoul, Republic of Korea) in propylene glycol:EtOH (7:3 v/v) was used to induce AD-like symptoms. First, mice in the DNCB control group and the DNCB/isolated compound groups were sensitized by applying 1% DNCB in propylene glycol:EtOH (7:3 v/v) to dorsal skin once daily for a week (experimental day 1 (ED1) to ED7). DNCB controls were then challenged by administering 100 μL of 0.1% DNCB from ED8 every 3 days for 2 weeks. Animals were painted with 100 μL solution of 0.5% chamaejamine or 1% pimecrolimus, a positive control, in propylene glycol:EtOH (7:3 v/v) and with DNCB, as described above, twice a day from ED8 for 2 weeks. In these groups, chamaejamine or pimecrolimus were applied 4 h after DNCB application. Mice in the untreated control group were treated with propylene glycol:EtOH (7:3 v/v) daily using the method described above.
9
Keratinocyte Activation and Inflammatory Regulation
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Human keratinocyte HaCaT cells were obtained from the Cell Lines Service (Eppelheim, Germany). Human primary neonatal keratinocyte HEKn cells were purchased from ThermoFisher Scientific Korea (Seoul, Korea). HaCaT cells were maintained in DMEM medium (1.8 mM CaCl2) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and HEKn cells were cultured in EpiLife medium supplemented with Human Keratinocyte Growth Supplement (HKGS) (ThermoFisher Scientific Korea). Agerarin was isolated as described previously [15 (link)]. 2,4-Dinitrochlorobenzene (DNCB) was obtained from Sigma–Aldrich (St. Louis, MO, USA). TNFα and IFNγ were obtained from Prospec-Tany Technogene Ltd. (Ness-Ziona, Israel). The hematoxylin and eosin (H&E) stain kit and toluidine blue dye were obtained from Sigma–Aldrich. The Firefly Luciferase Assay System was from Promega (Madison, WI, USA). Antibodies against phospho-JAK1 (Y1034/1035), JAK1, phospho-JAK2 (Y1007/1008), JAK2, phospho-STAT3 (Y705), and STAT3 were purchased from Cell Signaling Technology (Beverly, MA, USA).Antibodies against FLG and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-FLG antibody for immunofluorescent staining was from BioLegend (San Diego, CA, USA).
10
Murine Model of Atopic Dermatitis
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Bee venom phospholipase A2 (bvPLA2, Apis mellifera) and dexamethasone (DEXA) were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). The American house dust mite in the form of freeze-dried crude Dermatophagoides farinae extract (DFE) (Greer Laboratories, Lenoir, NC, USA) was used as an antigen and 2,4-dinitrochlorobenzene (DNCB) (Sigma-Aldrich) was used as a sensitizer to induce AD in the mice. bvPLA2 was dissolved in phosphate-buffered saline (PBS) and DFE was dissolved in PBS containing 0.5% Tween 20. DNCB was dissolved in acetone and olive oil (AOO) at a ratio of 3:1. AOO or AOO containing 0.5% Tween 20 was used as a vehicle.
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