Glutathione (gsh)

The lipid peroxidation end-product MDA (Catalog #K739, BioVision, USA) and the antioxidative enzymes SOD (Catalog#K335, BioVison), CAT (Catalog #K773, BioVision), and GSH (Catalog #K261, BioVision) were detected using ELISA kits according to the manufacturer’s instructions.

Levels of SOD (Thermo Fisher Scientific, Waltham, MA, USA), CAT and GPx (Abcam, Cambridge, UK), GSH (BioVision, San Francisco, CA, USA) and MDA (Cayman, Hamburg, Germany) were determined by ELISA kits following the manufacturer’s instructions.

Serum concentration of IgE was determined using the mouse IgE ELISA kit (Bethyl Laboratories, Montgomery, TX, United States) and the OVA-specific IgE ELISA kit (Cayman Chemical, Ann Arbor, MI, United States). The levels of prostaglandin E₂ (PGE₂) and cytokines IL-4, IL-5, IL-13, IL-6, tumor necrosis factor (TNF)-α, tumor growth factor (TGF)-β1, and vascular endothelial growth factor (VEGF) were examined using their respective ELISA kits (R&D System, Minneapolis, MN, United States). Further tests included histamine (Enzo Life Sciences, Ann Arbor, MI, United States), mast cell tryptase (Cusabio Biotech., Wuhan, Hubei, China), and leukotrienes E4 (LTE4; MyBioSource, San Diego, CA, United States) in the BALF were also quantified using their respective ELISA kits. The absorbance was read at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, United States). The contents of MDA (Sigma, St. Louis, Mo. United States) and GSH (BioVision, Milpitas, CA, United States), and activity of SOD (BioVision, Milpitas, CA, United States) in lung tissues were measured using the ELISA kits.

Colorimetric kits and enzyme immunoassay kits were used. Colorimetric kits: thiobarbituric acid reactive substances, total antioxidant capacity (Cell Biolabs Inc., Bionova Científica S.L., Madrid, Spain), and glutathione (Abcam, Cambridge, UK). Enzyme immunoassay kits: 3-nitrotyrosine, 8-iso-prostaglandin F2α (8-isoprostane), 8-hydroxy-2-deoxyguanosine (Cell Biolabs Inc., Bionova Científica S.L., Madrid, Spain), 11-dehydro-thromboxane B2, and 6-keto-prostaglandin F1α (Cayman Chemical Co., Ann Arbor, MI, USA). Sigma Chemical Corp. (St. Louis, MO, USA) provided the rest of the necessary reagents.
3’,4’-dihydroxyphenylglycol (DHPG), with a purity of 95%, was supplied by Fernández-Bolaños J (Instituto de la Grasa, CSIC, Spain). It was obtained from a by-product of olive oil following a two-phase extraction process in oil mills and a subsequent purification process, according to the patent of the same researcher (WO2010070168A1).

Piperine standard was acquired from Sigma Aldrich (Bangalore, India), Piperine API was procured from Yucca enterprises (Mumbai, India). Phospholipon 90G was procured from Lipoid (Ludwiashafen, Germany), sodium cholate was procured from DC Fine Chemicals (Mumbai, India), polyethylene glycol-400, methanol, chloroform span 60, and cholesterol were acquired from SD fine chemicals (Mumbai, India). Oxidative stress markers (superoxide dismutase, catalase, glutathione, malondialdehyde) kits were acquired from Abcam, Cambridge (UK). Pentylenetetrazole was procured from Sigma Aldrich (Bangalore, India). All HPLC solvents were acquired from SD Fine chemicals (Mumbai, India).

Streptozotocin (STZ) and curcumin were purchased from Sigma-Aldrich Inc., St. Louis, MO, USA. The antioxidant enzyme kits including catalase, glutathione, and superoxide dismutase were acquired from Abcam, Cambridge, UK. The inflammatory markers kits for TNF-α, IL-6 and IL-1β were also obtained from Abcam, UK. Antioxidant enzyme and myeloperoxidase kits were purchased from Abcam, UK. For fibrosis evaluation, trichrome stain and a Sirius red kit were purchased from the same company. IL-6 and TNF-α monoclonal antibodies and the Specific HRP/DAB Detection IHC kit were acquired from Abcam, United Kingdom. All supportive chemicals used in this study were of analytical grade and purchased from a local vendor.