Azilsartan

Cell viability assay was achieved using MTT reagent [3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyltetrazolium bromide]. MCF-7, MDA-MB-231 or MCF 10A cells (10⁴ cells per well) were seeded in triplicate in 96-well plates and allowed to grow in fresh DMEM medium for 24 h. Then, medium was changed with fresh DMEM containing different concentrations (7.81, 15.61, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL) of azilsartan (Sigma-Aldrich, Inc., St Louis, MO, USA). After 48 h, 10 μL of MTT (5 μg/mL) was added per well and incubated in the dark for 3 h at 37 °C. To dissolve the Formazan crystals that were formed, 100 μL of DMSO was used, and absorbance was measured using an ELISA reader at 570 nM [66 (link)]. The IC₅₀ of azilsartan was calculated for each cell line using Graph Pad Prism-9 software for macOS (GraphPad version 9.4.1 (458), La Jolla, CA, USA).

All the experiments were designed in accordance with the “World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects”. The study was approved by the Ethical Committee of Ganzhou People’s Hospital (GZH-2017021). HUVECs were purchased from the Type Culture Collection of the Chinese Academy of Sciences (TCCCAS, Shanghai, China) and cultured with an EGM-2 Bullet Kit (Lonza, Switzerland) containing 0.1% gentamicin sulfate (GA-1000). HUVECs were routinely maintained in 2% low serum complete growth media EGM2 for less than seven passages, and all cultures were maintained in a humidified 37° C atmosphere. Azilsartan (98% purity) was purchased from Sigma-Aldrich, MO, USA (#SML0432), and Ox-LDL from Thermo Fisher Scientific, MA, USA (#L34357). For treatment experiments, HUVECs were plated on 35 or 60-mm dishes at a density of 2x10⁴ cells/cm² area overnight, HUVECs were treated with Ox-LDL (100 μg/ml) [18 (link), 19 (link)] in the presence or absence of Azilsartan (3, 6 μM) [17 (link)] for 24 hours.

Male C57BL6/J mice that were eight to ten weeks old were acquired from CLEA Japan, Inc. (Tokyo, Japan) and maintained at an animal facility. The Science Animal Care and Use Committee of Tohoku University’s guidelines were followed in full for all animal care and experimental procedures. Each experimental group consisted of four randomly assigned mice. Azilsartan was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in DMSO before being kept at 4°C. LPS from Escherichia coli were bought from Sigma-Aldrich (St. Louis, MO). As previously mentioned, recombinant murine TNF-α (38 (link)) and M-CSF (CMG14-12 cell line) (39 (link)) were produced.