Growth kit

DXMa was purchased from LA COOPER (Melun, France). Hydroxypropyl-γ-cyclodextrin (HPγCD, W8HP, DS = 0.6, and Mw = 1576 Da) was a kind gift from ASHLAND (Schaffhausen, Switzerland) and hydroxypropyl-β-cyclodextrin (HPβCD, KLEPTOSE DS = 0.63 and Mw = 1391 Da) was obtained from ROQUETTE (Lestrem, France). CELLUVISC^® (sodium carboxymethylcellulose) and VISMED^® (sodium hyaluronate) are marketed gels used for the treatment of dry eye syndrome. DEXAFREE^® (DXM sodium phosphate 1% solution eye drops), MAXIDEX^® (DXM 0.1% suspension eye drops) and BSS^® (Alcon Laboratories, Rueil-Malmaison, France) are human authorized ocular medicines. Normal human primary corneal epithelial cells (ATCC PCS 700-010), medium (ATCC PCS-700-030), growth kit (ATCC PCS-700-040), PBS (ATCC 30-2200), trypsin EDTA (ATCC PCS-999-003 and 005), and antibiotics (gentamicin, streptomycin, and amphotericin BATC PCS-999-002) were obtained from LGC standard - ATCC^® (Molsheim, France). Thioglycollate with resazurine medium and Tryptic soy broth were obtained from BIOMERIEUX (Craponne, France). ALAMARBLUE^® was purchased from BIO-RAD (Marnes-la-Coquette, France) and DMSO from SIGMA-ALDRICH (Lyon, France). Purified water was prepared by DIRECT-Q^®3UV water purifier (MILLIPORE, Molsheim, France). All other solvents and chemicals were of HPLC and analytical grade, respectively.

In this study, HCECs were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were resuspended in corneal epithelial cell basal medium (ATCC) supplemented with a growth kit (ATCC). The cells were plated in 75 cm² tissue flasks, and then maintained at 37 °C in a 5% CO₂ and a 95% humidified atmosphere. Culture medium was changed every 3 d and the cells were passed using 0.05% Trypsin-EDTA (Gibco BRL, CA, USA). A passage number of ≤5 was used in this study.

Primary human vaginal epithelial cells (ATCC480-010, VA) were purchased and cultured in basal medium (ATCC 480-030) with a growth kit (ATCC 480-040). After fresh flowers having low temperature ripening at 10 °C for 48 h, the leaves of A. helianthus were dried with infrared ray for 2 h and grinded into microparticles (400 mesh). The powder was extracted with vacuum at 0.08 MPa, 70 °C for 2 h. After adjusting the treatment dosage of the A. helianthus water extract (800 µg/mL, cultured for 72 h), supported by Life Together (Gangwon-Do, Korea), the extract was added to the HVECs for 72 h at 37 °C, under 5% CO₂. The supernatant of the treated cells, conditioned medium was collected. The activated HVECs were treated with hydrogen peroxide, 50 µM/mL (Daejung, Daejeon, Seoul, Korea), and fungal protease, 50 µg/mL (Sigma, MO, USA) for 2 h. The conditioned medium (100 µl/mL) was added to THP-1 cells (KCLB 40202, Seoul, Korea), which were cultured in RPMI 1640. Progenitor DCs (Lonza, MD, USA) were cultured with lymphocyte growth medium (CC-3211, Lonza), and after activation by conditioned medium for 24 h, the cells were exposed to the two stresses for 2 h.