Rho kinase inhibitor y 27632

Human primary CoEpiCs and CjEpiCs were obtained from donor corneas (CorneaGen, Seattle Eye Bank, Seattle, WA, USA). The cells were isolated and cultured following a previously described method [16 (link)] with slight modifications. The corneal and conjunctival epithelium were peeled off after overnight treatment with dispase type II (Godo Shusei, Tokyo, Japan) at a concentration of 1000 PU/mL at 4°C. The epithelial cells were then dissociated into single cells by incubation with TrypLE™ Express (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at 37°C and seeded into a single well of a 6-well plate. The CoEpiCs and CjEpiCs were cultured at 37°C, 95% humidity, and 5% CO₂ in complete medium [17 (link)], consisting of Dulbecco’s modified Eagle’s medium and Ham’s F-12 media (DMEM/F12, 1:1 mixture) (Thermo Fisher Scientific), B-27™ supplement (2%) (Thermo Fisher Scientific), Rho-kinase inhibitor Y-27632 (10 μM) (Selleck Chemicals, Houston, TX, USA), keratinocyte growth factor (10 ng/ml) (Thermo Fisher Scientific), and penicillin-streptomycin (50 IU/ml) (Nacalai Tesque, Kyoto, Japan). The cells were used for subsequent experiments after one passage.

Geniposide was extracted and purified from G. jasminoides Ellis (purity > 98%), and provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Fetal bovine serum (FBS) was obtained from HyClone (Logan, United States). Rabbit monoclonal antibodies against p38, p-p38, NF-κB p65 and NF-κB p65 were supplied from Cell Signaling Technology (Beverly, MA, United States). Anti-RhoA antibody was purchased from Abcam (Cambridge, MA, United States). β-actin and HRP-conjugated goat anti-rabbit antibodies were provided by ZSGB-BIO (Beijing, China). Rho-kinase inhibitor (Y-27632) was obtained from Selleck (Houston, TX, United States). Rat IL-4, IL-1β, IL-17 and TGF-β ELISA kits were purchased from Elabscience Biotechnology (Wuhan, China). Freund’s Complete Adjuvant (FCA) and LPS were supplied from Sigma Chemical Company (St. Louis, MO, United States). Other chemicals that have been used in this work were of research grade.

We utilized primary CEnCs isolated from human donor corneas (CorneaGen, Seattle Eye Bank, WA, USA). The hCEnCs were cultured following established protocols with slight modifications [32 (link), 41 (link)]. Briefly, the Descemet’s membranes with the CEnCs were stripped from donor corneas and digested at 37°C using 1 mg/mL collagenase A (Roche Applied Science, Penzberg, Germany) for 12 h. Subsequently, the hCEnCs obtained from a single donor cornea were collected following two washes with OptiMEM-I (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then seeded in one well of a 6-well plate that had been pre-coated with laminin E8 fragments (iMatrix-511; Nippi, Incorporated, Tokyo, Japan) [42 (link)].
hCEnCs were cultured at 37°C with 95% humidity and 5% CO2 in complete hCEnC medium, which consisted of OptiMEM-I (Thermo Fisher Scientific), 8% fetal bovine serum (FBS), 20 μg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 200 mg/L calcium chloride, 0.08% chondroitin sulfate (Sigma-Aldrich), Rho-kinase inhibitor Y-27632 (10 μM) (Selleck Chemicals, Houston, TX), and penicillin-streptomycin (50 IU/ml) (Nacalai Tesque, Kyoto, Japan). Cultured hCEnCs were passaged after harvest with 10xTrypLE Select (Thermo Fisher Scientific, Inc.) treatment at 37°C for 12 min when they reached confluence and used for subsequent experiments. hCEnCs at passage 2 were used for all experiments.