Study design
In order to evaluate the effects of Ginsenoside-Rg1 on HUVECs under hyperoxia, various concentrations of Ginsenoside-Rg1 were added to endothelial cells cultured under normoxia and hyperoxia in the experiment period. Cell proliferation and protein production will be measured. In clinical condition, Fick’s first law is used to describe the exchange of oxygen in the lung capillaries, where oxygen passes from the alveoli through the alveolar membrane and entering the blood stream. Fick’s law can also be applied to cell culture in a similar manner if the oxygen concentration at the air-culture medium interface is considered similar to the oxygen concentration of gas at the surface of the alveolar membrane, and oxygen right above the cell monolayer is paralleled to the oxygen concentration in the blood stream [11]. Thus the culture atmosphere was set at 60% O2, which is similar to the oxygen percentage administrated to preterm infants with respiratory distress syndrome.
Materials
Cell culture: Primary human umbilical vein endothelial cells (HUVEC, CRL-1730TM) were used in our study. The cells were purchased from ATCC (Manassas, VA). Culture medium M199 and endothelial cell growth supplement (ECGS) were purchased from Sigma. Fetal bovine serum (FBS) was from Gibco.
Ginsenoside-Rg1: Experimental reagent Ginsenoside-Rg1 is a reference compound, with purity around 97.7%, purchased from the Division of Chinese Material Medica and Natural Products, National Institute for the Control of Pharmaceutical and Biological Products, Ministry of Public Health, China. A stock solution of Ginsenoside-Rg1 50 mM was prepared in sterile double distilled H2O and stored at -80℃.
Antibodies: β-actin was used as a loading control. Anti-β-actin antibody was obtained from Millipore (Temecula, CA). Antibodies for VEGF, GR, GPx (Glutathione Peroxidase), Bax, Cyt c (cytochrome c), PARP (Poly ADP-ribose polymerase), caspase 3 were purchased from Cell Signaling Technology, Beverly, MA, USA. Cell proliferation was measured by MTT colorimetric assay (CytoSelect™ MTT Cell Proliferation Assay, CELL BIOLABS, INC.).
Methods
Cell culture
HUVECs were cultured in medium M199 with 10% FBS, 25μg/ml ECGS, and 1% penicillin/streptomycin in an incubator at 37℃. After seeding, the cells were stabilized in incubator under room air over night before experiment. In the experiments, cells were divided into normoxia and hyperoxia groups. In normoxia group, cells remained in incubator supplied with 95% room air (approximately 20% O2) and 5% CO2. In hyperoxia group, cells were transferred to a chamber supplied with mixed air containing 60% O2, 35% N2, and 5% CO2. Culture medium was saturated with mixed air previously before experiments.
The duration for experiments were 24, 48, and 72 hours after cells were exposed to normoxia or hyperoxia. In hyperoxia group, NexBiOxy Hypoxia/Hyperoxia System (NexBiOxy, Taiwan) was used to monitor and maintain the oxygen concentration in the culture chamber.
Ginsenoside-Rg1 administration
According to references, 0, 75, 150, 300nM of Ginsenoside-Rg1 were used in our experiments [6]. Various concentrations of Ginsenoside-Rg1 were prepared and added to the medium just before experiment. Medium was changed every day to keep Ginsenoside-Rg1 within half-life period.
Western blot
HUVECs were washed in phosphate-buffered saline and then extracted with lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton, 0.1% SDS, and 0.5% sodium deoxycholate) and protease inhibitors. Lysates were centrifuged at 12,000 rpm for 5 min, and the resulting supernatant was collected. The extracted protein was quantized by protein assay. Aliquots (25 μg) of cellular lysate was separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane. After blocking with 5% BSA, blots were incubated with primary antibodies and then corresponding secondary antibodies. An enhanced chemiluminescence kit (Amersham, Piscataway, NJ) was used for immunodetection. The protein bands were quantified using the Image J software (NIH, Bethesda, MD).
Statistical analysis
Each experiment was repeated for at least three times. Data were expressed as means ± S.D. Statistical comparisons were carried out by one-way ANOVA for multiple groups or t test for two groups. Significance was accepted at the p≦0.05 level and was marked with * in the figures.