2 4 6 trinitrobenzenesulfonic acid tnbs

(+)-Pentazocine, haloperidol, and buffer components (TRIS-HCl, inhibitors) were purchased from Sigma-Aldrich Kft. (Budapest, Hungary). The radioligand [³H]-(+)-pentazocine (s.a. 1.98 TBq/mmol) was prepared in the Laboratory of Chemical Biology (BRC, Szeged, Hungary). Tritium labeling was carried out in a self-designed vacuum manifold [68 (link)] and radioactivity was measured with a Packard Tri-Carb 2100 TR liquid scintillation analyzer (Packard, Perkin Elmer, Waltham, MA, USA) using Insta Gel scintillation cocktail of PerkinElmer. Drugs were dissolved at 1 mM in dimethyl sulfoxide (DMSO) and stored at −20 °C, and then diluted in the binding buffer. 2,4,6-trinitrobenzenesulfonic acid (TNBS) was purchased from Sigma-Aldrich (Budapest, Hungary), and was dissolved in 50% ethanol and distilled water mixture. Fluvoxamine (Fluvoxamine maleate, Sigma-Aldrich, Budapest, Hungary) was dissolved in 3% dimethyl sulfoxide (DMSO). BD1063, purchased from Tocris (Bio-Techne R&D Systems Kft., Budapest, Hungary), was dissolved in physiological saline (0.9%). Sulfasalazine (SASP) was used as a positive control and purchased from Sigma-Aldrich (Budapest, Hungary). The anesthetic agent Thiopental (Tiobarbital Braun, 0.5 g, B. Braun Medical SA, Barcelona, Spain) was dissolved in saline (0.9%).

Chitosan oligosaccharide (CSO) with average molecular weight of 18.0 kDa was prepared by enzymatic degradation of chitosan (CS) (95% acetylate, M_w = 450 kDa; Yuhuan Marine Biochemistry Co., Ltd, Zhejiang, China) as described in a previous work (Zhao et al., 2012 (link); Yi et al., 2015 ). Angiopep-2 peptide (Ap, TFFYGGSRGKRNNFKTEEY) was synthesized by Chinese Peptide Company (Hangzhou, China). NH₂-PEG-NH₂ (PEG₂₀₀₀) and 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) were purchased from Sigma-Aldrich (Diegem, Belgium). Pyrene was purchased from Aldrich Chemical Co (Milwaukee, WI). Di-tert-butyl dicarbonate ((Boc)₂O), 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Shanghai Medpep Co, Ltd (Pudong, Shanghai, China). N,N′-Disuccinimidyl carbonate (DSC) was obtained from Bio Basic Inc., (Toronto, Canada). Doxorubicin hydrochloride (DOX·HCl) was purchased from Zhejiang Hisun Pharmaceutical Co, Ltd, (Taizhou, Zhejiang, China). 1, 1′-Dioctadecyl-3, 3, 3′, 3′-tetramethylindotricarbocyanine iodide (DiR) was purchased from Life Technologies (Carlsbad, CA). D-Luciferin was supplied by Shanghai Sciencelight Biology Science and Technology Co., Ltd (Shanghai, China). All of the other chemicals were of analytical or chromatographic grade.

SNT granules, composed of Fu Zi, Gan Jiang, and Gan Cao based on Chinese Pharmacopoeia (2015 edition) in a ratio of 3:2:3, were purchased from the Outpatient Department of Traditional Chinese Medicine of the Chinese Academy of Traditional Chinese Medicine (the chemical structures of the pharmacoactive compounds are shown in the Figure S1. Sulfasalazine (SASP) enteric-coated tablets were obtained from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd. (batch no. 09170502; $3.92; Shanghai, China). The 2,4,6-trinitrobenzenesulfonic acid (TNBS) was obtained from Sigma ($397.7; St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-6, TNF-α, prostaglandin E₂ (PGE₂), CRP, and collagen alpha-1(XII) chain (COL12A1) were obtained from Cusabio Biotech Co. Ltd. (Wuhan, China), and a nitric oxide (NO) test kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).