Sesamin

Chemicals, i.e., gallic acid, ascorbic acid, acetic acid, sodium thiosulphate, sodium hydroxide, potassium iodide, starch, ethanol 95%, methanol, hexane, butanol, iso-octane, panisidine, Folin-Ciocalteu’s phenol reagent, butylated hydroxytoluene (BHT), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), sesamol, sesamin, and sesamolin, were supplied from Sigma-Aldrich Company (St. Louis, MO, USA) and Merck Company (Darmstadt, Germany). All chemicals were of HPLC grade. Refined olive oil (ROO) without antioxidants, season 2020, was freshly supplied from Khoshala Olive Oil Company (Obour city, Cairo, Egypt). The samples were directly transported to the oil technology lab, Biochemistry Department, Benha University, and placed in a dark condition at ambient temperature. Dark sesame seed coats (SSCs) by-products were obtained from El-Bawady Company (Alexandria, Egypt). The powdered SSCs was packed and stored in a dry and dark environment until preparation.

The human colon mucosal epithelial cell line NCM-460 was purchased from American Type Culture Collection (ATCC, Manassas, VA). Sesamin and LPS were purchased from Sigma-Aldrich (St. Louis, MO). The HMGB1 antagonist ammonium glycyrrhizinate (AG, HY-N0184B) was purchased from MedChemExpress (MCE, Monmouth Junction, NJ). Sesamin was initially dissolved in dimethyl sulphoxide (DMSO) and stored at −20 °C and was then thawed to be diluted in the cell culture medium to the required concentrations. NCM-460 cells were maintained in RPMI 1640 (Gibco, Gran Island, NY) with 15% foetal bovine serum (FBS) at 37 °C with 5% CO₂. To establish an in vitro sepsis model, NCM-460 cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and cultured overnight. The cells were then incubated alone or with LPS (1 μg/mL), LPS + Sesamin (5 μM)/AG (50 µg/mL), or LPS + Sesamin + AG as described above for 48 h and then collected for subsequent RT-qPCR and Western blotting analyses.

The pGL4 luciferase vector harboring human BMP2 promotor sequence was constructed by GenePharma (Shanghai, China). The Renilla luciferase vector was obtained from Cell Signaling Technology (Albany, NY, USA). Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) reagent was used for transfection according to the manufacturers’ instruction. The human bone marrow mesenchymal stem cells (BMSCs) were isolated and kept in our laboratory as previously described [20 (link)]. In general, the human BMSCs were seeded into a 12-well plate and grown until 70–80% confluence. For transfection, 1 μg plasmid DNA with 1 μL Lipofectamine^TM 3000 were mixed in 500 μL Opti-MEM^TM (Thermo scientific, Waltham, MA, USA), and added into the culture medium. After 12-h incubation, cells were treated with 0.5 μM sesamin (59867, Sigma-Aldrich, St. Louis, MO, USA), 100 nM estradiol (E8875, Sigma-Aldrich, St. Louis, MO, USA), 0.2 nM amcenestrant (Sigma, St. Louis, MO, USA) or the combinations for an additional 24 h. For dual-luciferase assay, Dual-Luciferase^® Reporter Assay Kit (Promega, Madison, WI, USA) was applied as mentioned before [20 (link)]. The luciferase activity was measured by PerkinElmer VictorTM X2 2030 multilateral reader (PerkinElmer, Waltham, MA, USA). The firefly luciferase activity was normalized to renilla luciferase activity.

Sesamin is a pure substance bought from Sigma-Aldrich (St. Louis, MO, USA), the purity of which is over 98%. By using dimethyl sulfoxide (DMSO), Sesamin stock was prepared at a concentration of 100 mM; respectively diluted to 0, 10, 20, and 40 mM; and stored at −20 °C. The content of DMSO in all medicines should be less than 0.2%. While processing when medicine was added, medicine at the appropriate concentration was mixed and added into the cell culture dish. Cells were put into an incubator at 37 °C and 5% CO₂ following the required conditions and time for the experiment.