Anti-Inflammatory Effects of Ursolic Acid Figure 4. Ursolic acid suppresses inducible expression of T cell and B cell activation markers and co-stimulatory molecules. Lymphocytes were treated with UA (5 mM, 4 h) and then stimulated with Con A (10 mg/ml) or LPS (50 mg/ml) for 24 h (A–B) or 48 h(C–H) at 37uC. In each group, 16106 cells were stained with PE conjugated anti-CD69 (A) or anti-CD25 (B) or anti-CD134 (C) or anti-CD28 (D) or anti-CD19 (E) or anti- CD80 (F) or anti-CD86 (G) or anti-I-A (H) mAbs. Representative flowcytometric histogram and corresponding bar diagram are shown. Data points represent mean6S.E.M. from three replicates and two such independent experiments were carried out. *p,0.01, as compared to vehicle treated cells and #p,0.01, as compared to Con A or LPS stimulated cells. doi:10.1371/journal.pone.0031318.g004 In a recent report by Liu et al., UA induced tolerance to allogenic cardiac transplant in mice was attributed to suppression of NF-kB [48]. They have shown that UA suppressed T cell responses including NF-kB inhibition at 25 mM whereas we have observed that a dose of 5 mM was sufficient to suppress immune cell (T cell, B cell and macrophage) activation. Apart from this, Tao Xu et al., have shown that anti-inflammatory effects of UA are mediated through suppression of transcription regulator RORct resulting in decreased IL-17 expression in Th17 cells [49]. Further, our studies demonstrate that besides NF-kB suppression UA also suppressed other immuno- logically important transcription factors AP-1, NF-AT as well as MAPKinases. In conclusion, the present report identified multiple cellular targets of ursolic acid and underlines its application as a potent anti-inflammatory agent with therapeutic potential. Figure 5. Ursolic acid Modulates cellular ROS levels. (A) UA increased ROS levels in lymphocytes. Lymphocytes were stained with DCF-DA (20 mM, 30 min at 37uC), treated with UA (0.5–5 mM) for 1 h and fluorescence emission was measured at 535 nm. *p,0.01, as compared to DCF treated cells (B) Effect of UA on intracellular GSH levels. Lymphocytes were treated with UA for 4 h at 37uC and stained with MCB (final concentration 40 mM) for 30 min. Fluorescence emission was measured at 490 nm following excitation at 394 nm. *p,0.01, as compared to MCB treated cells (C) Immunosuppressive effects of UA were independent of cellular redox status. Lymphocytes were incubated with different antioxidants (GSH or NAC or DTT or trolox) for 2 h. These cells were then stimulated with Con A in presence or absence of UA for 24 h at 37uC .The concentration of IL-2 and IFN-c in the culture supernatant was estimated by ELISA. (D&E) Lymphocytes were stained with CFSE and were incubated with different antioxidants (GSH 10 mM or NAC 10 mM or DTT 100 mM or trolox 100 mM) for 2 h. The cells were then stimulated with Con A in presence or absence of UA for 72 h at 37uC. Cell proliferation was measured from CFSE dye dilution using a flowcytometer. Representative flowcytometric histograms and corresponding bar diagram are shown (Fig. 5D&E). Each bar shows mean6S.E.M from three replicates and two such independent experiments were carried out. *p,0.01, as compared to vehicle treated cells and #p,0.01, as compared to Con A stimulated cells. doi:10.1371/journal.pone.0031318.g005 PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e31318