Griess reagent

Nobiletin (purity ≥ 98%), collagenase-II, and dimethyl sulfoxide (DMSO) were obtained from Solarbio (Beijing, China). Recombinant mouse IL-1β was purchased from Novoprotein (China). Primary antibodies directed against A disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS5), collagen II, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Abcam (Cambridge, MA, USA), while primary antibodies against iNOS, MMP-3, MMP-13, cyclooxygenase-2 (COX-2), p-IkBα, IkBα, p-p65, and p65 were obtained from ProteinTech (Wuhan, China). Cell-Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM)/F12, 0.25% trypsin ethylenediaminetetraacetic acid (trypsin–EDTA), and bovine serum albumin (BSA) were purchased from Healthcare Life Sciences (Hyclone; Logan, UT, USA). Griess reagent and Bicinchoninic acid radioimmunoprecipitation assay lysis buffer were purchased from Solarbio (Beijing, China). The NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC) was obtained from Abcam (Cambridge, UK). Goat anti-mouse horseradish peroxidase conjugates and goat anti-rabbit were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Griess reagent and PGE2 ELISA kits were obtained from Bio-Swamp Life Science (Shanghai, China).

The production of NO in the culture supernatant was quantified by evaluating the nitrite content according to the standard Griess reaction (33 (link), 34 (link)). RAW264.7 cells were inoculated at a density of 1×10⁵ cells/mL in a 96-well plate and incubated with 5% CO₂ at 37°C. Cells were pretreated with dexamethasone (MCE, New Jersey, USA) or specific concentrations of HM0539 (25, 50, 100 ng/mL) for 24 h and then further stimulated with or without LPS (1 µg/mL) for 24 h. The cell culture medium (100 μL) in each case was collected and incubated with 100 μL of Griess reagent (Solarbio, Beijing, China) at room temperature for 15 min. At the end point of the treatment, the absorbance was measured at a wavelength of 550 nm in a microplate reader. NO (μM) levels were calculated on the basis of the sodium nitrite standard (35 (link)).

DiR, coumarin-6 (C6), and thrombin from bovine plasma (37KD) were obtained from Meilun Biotech Co. Ltd., (Dalian, China). BPA, and Sodium nitrite (NaNO₂) were purchased from Macklin Biochemical Co., Ltd (Shanghai, China). Griess Reagent was obtained from was purchased from Beijing Solarbio Science & Technology Co., ltd. 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG_2K) was purchased from A.V.T. Pharmaceutical Co., Ltd (Shanghai, China). DSPE-PEG_2K-CREKA was synthesized by Premierbiochem Co., Ltd (Suzhou, China). sCD40L Assay Kit was purchased from Beijing Solarbio Science & Technology Co., ltd. Cell culture dishes and plates were bought from NEST Biotechnology Co., Ltd (Wuxi, China). All solvents and reagents used were analytical or HPLC standard.