Artemisinin

Chemicals and reagents were from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Artemisinin was from Cayman Chemical (Ann Arbor, MI, USA); deoxyArtemisinin from Toronto Research Chemicals (North York, ON, Canada). Lipopolysaccharide was Escherichia coli serotype O111:B4 and dissolved in sterile water at 1 mg/mL before storage at −20 °C. LEGEND MAX^TM Enzyme-linked immunosorbent assay (ELISA) kits for rat TNF-α and IL-6 were from BioLegend (San Diego, CA, USA). ELISA kits for rat IL-10 were from Thermo Fisher Scientific (Waltham, MA, USA). P450-Glo CYP2B6 and CYP3A4 kits were from Promega (Madison, WI, USA). Human liver microsomes (HLMs) from a 200-donor pool of male and female donors were from Sekisui XenoTech (Kansas City, KS, USA).

Drug treatments were performed as follows. A. annua tea was diluted in a growth media to 50 µM ART; A. afra tea was diluted with water to 1/2, 1/4, or 1/8 strength of the A. annua tea on a dry-weight basis as needed; artemisinin (ART; Cayman Chemical, Ann Arbor, MI, USA, 11816), artesunate (AS; Cayman Chemical 11817), artemether (AM; gift from Prof. J. Plaizier-Vercammen (Brussels, Belgium)), dihydroartemisinin (DHA; Cayman Chemical 19846), and deoxyartemisinin (dART; Toronto Research Chemicals, Toronto, Ontario, Canada, D232150) were dissolved in DMSO as 1000× stock solutions, filter sterilized, and added to a final concentration of 50 μM. DMSO and water were the solvent controls for the artemisinic compounds and teas, respectively. Fibroblasts (passages 10–14) were seeded in 6-well plates in 2 mL medium at a density of 6250 cells/cm². After overnight attachment, media were aspirated and replaced with test drugs in growth media at the indicated concentrations and incubated at 37 °C and 5% CO₂ in high humidity. After 4 d of drug treatment, cells were harvested, washed with 1× Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific 14190136), snap frozen in liquid nitrogen, and stored at −80 °C until the time of RNA extraction for analysis.