Ursolic acid inhibits lymphocyte proliferation in vitro.

<p>(A) For cell proliferation analysis CFSE labelled lymphocytes were treated with UA (0.25–5 µM, 4 h) and stimulated with the Con A (10 µg/ml) at 37°C for 72 h. Twenty thousand cells were acquired in a flowcytometer. Vehicle treated cells served as control. Percent daughter cells were calculated from decrease in mean fluorescence intensity. (B) Each bar represents percentage of daughter cells in each treatment group. (C & D) UA inhibits anti-CD3 induced T cell proliferation. CFSE labelled lymphocytes were treated with UA (1 & 5 µM, 4 h) and stimulated with coated anti-CD3mAb (1 µg/ml) and soluble anti CD28mAb (1 µg/ml) at 37°C for 72 h. Percent daughter cells were estimated by CFSE dye dilution. Representative histogram and corresponding bar diagram are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031318#pone-0031318-g001" target="_blank">Fig. 1C and D</a> respectively. (E) UA inhibits Con A induced cell cycle progression: Lymphocytes were treated with UA (0.25–5 µM, 4 h) and stimulated with the Con A (5 µg/ml) at 37°C for 72 h. The cells were stained with propidium iodide and twenty thousand cells were acquired in a flowcytometer. The hollow bars represent percentage of cells containing less than 2n DNA (sub-G1/apoptotic cells), light gray bars show cells containing 2n DNA (in G1 phase) and the dark gray bars indicate the cells containing more than 2n DNA (in S+G2/M phase). (F) Evaluation of the potential cytotoxicity of UA to lymphocytes. Lymphocytes were cultured with UA (5 µM) for 24 h and cytotoxicity was measured by PI staining. Vehicle treated cells served as control. Percentage apoptosis (pre-G1 peak) in lymphocytes was estimated and is shown in the histograms. (G) Each bar represents percentage of apoptotic in each treatment group. Data points represent mean±S.E.M. from three replicates and three such experiments were carried out. *p<0.01, as compared to vehicle treated cells and #p<0.01, as compared to Con A or antiCD3/CD28 stimulated cells.