Mesalazine

As microdialysis perfusate A argon-deoxygenated (5 min) and urea-enriched (final concentration 20 mmol/L) phosphate-buffered saline (PBS, pH 7.4) was used. 5-ASA, also known as mesalazine (Sigma-Aldrich), was added to this solution (final concentration 1 mmol/L) as an anti-inflammatory substance to produce microdialysis perfusate B. Both solutions were prepared on day 1 and used throughout the whole experiment. Five-day stability of these solutions without precipitate formation was verified spectrophotometrically at room temperature and exposure to daylight.

Standards, certified reference material, for calibration and validation of biomimetic columns were: Acetylsalicylic acid, caffeine, cefalexin, chloramphenicol, cimetidine, codeine hydrochloride dihydrate, furosemide, ibuprofen, mesalazine, metronidazole, nifedipine, paracetamol, propranolol hydrochloride, salicylic acid, sulfadiazine, sulfamethoxazole, thiamine hydrochloride and warfarin purchased from Sigma-Aldrich (St. Louis, MO, USA). Azathioprine; folic acid; 6-mercaptopurine and 6-thioguanine, United States Pharmacopeia reference standard and 6-methylthioguanine (≥95%) have also been obtained from Sigma-Aldrich. Acetonitrile, ethanol, iso-propanol, methanol, formic acid, all HPLC grade; sodium hydroxide; ACS reagent (97.0%) pellets; sodium nitrate (≥99.0); phosphate-buffered saline tablets (0.01 M, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4 at 25 °C); potassium phosphate monobasic and potassium phosphate dibasic (HPLC grade) were provided by Sigma-Aldrich. Ultra-pure water was obtained using a MilliQ UF-Plus system (Millipore, Darmstadt, Germany); resistivity MΩcm⁻¹ >18 at 25 °C and TOC <5 ppb. The excipients included hydroxypropyl methylcellulose Methocel K100M Premium CR (Colorcon, Harleysville, PA, USA), magnesium stearate (Acros Organics, Princeton, NJ, USA), lactose monohydrate, stearic acid, wheat, rice and corn starch (Kemig, Zagreb, Croatia).

Dextran sulfate sodium was purchased from MP Biomedicals (Aurora, OH, United States). Compound C and AICAR were purchased from MedChemExpress (MCE, United States). Antibody for phosphorylated AMPK (Thr172) was purchased from Cell Signaling Technology (Beverly, MA, United States), antibodies for AMPKα1/2, ZO-1, occludin, claudin-1, Actin, Tubulin were purchased from Santa Cruz (Santa Cruz, CA, United States), IgG for rabbit and mouse were purchased from (Beyotime Biotechnology, China). Human recombinant interferon-γ (IFN-γ) was purchased from R&D Systems (Minneapolis, MN, United States). Lipopolysaccharide (LPS), metformin, mesalazine and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States). Alexa Fluor 488-conjugated anti-rabbit IgG was purchased from Life Technology (Carlsbad, CA, United States)

TGFβ is one of the most potent inductors of virtually all types of fibrosis (Rockey et al. 2015 ). Unless stated otherwise, 24 h after seeding, the cells were stimulated with TGFβ (10 ng/mL in medium) (100-21C, Peprotech, USA) for 72 h, followed by either 72 h of medium (solvent control) or 10 mmol/L mesalazine (A3537, Sigma-Aldrich, USA) solved in medium (Fig. 1). TGFβ treatment followed by solvent control is stated as “TGFβ” in the results, whereas TGFβ treatment followed by mesalazine is stated as “TGFβ + Mesa.” Medium and drugs (TGFβ and mesalazine) were changed daily.Fig. 1

Schematic illustration of the TGFβ stress model and mesalazine treatment protocol

mesalazine solution was prepared by dissolving powdered mesalazine (>99.9% purity) (Sigma Aldrich, St. Louis, MO, USA) in sterilised Mili Q water and adjusted to pH to 7 using 1 mM sodium hydroxide (NaOH) solution. The mesalazine solution was then diluted into either PneumaCult™-Ex Plus Basal Medium (STEMCELL Technologies, Tullamarine, VIC, Australia), PneumaCult™-Ex Plus 50X Supplement (STEMCELL Technologies, Tullamarine, VIC, Australia), and penicillin-streptomycin (Thermo Scientific, Walthman, MA, USA) (designated as Ex Plus complete media)] or PneumaCult™-ALI Basal Medium (STEMCELL Technologies, Tullamarine, VIC, Australia); PneumaCult™-ALI 10X Supplement; penicillin-streptomycin/amphotericin B (Thermo Scientific, Waltham, MA, USA); And PneumaCult™-ALI Maintenance Supplement (STEMCELL, Vancouver, BC, Canada) (ALI complete media)] to achieve the final concentrations of 0.5 mM, 1 mM, 10 mM, 20 mM and 50 mM. The mesalazine solution was covered with foil paper in all experiments unless specified.

After seeding, cells were cultured in control medium for 72 h, followed by either 72 h of medium (solvent control) or 10 mmol/L mesalazine (A3537, Sigma Aldrich, USA) solved in medium (Fig. 1). Medium and reagents were changed daily.Fig. 1

Schematic illustration of the invitro mesalazine treatment protocol