Anti-Inflammatory Effects of Ursolic Acid Figure 7. In vivo immunosuppressive effect of Ursolic acid. (A, B) Ursolic acid delayed mortality and weight loss in GVHD mice: Eight million lymphocytes from C57BL/6 donor mice were injected i.v. into immunocompromised Balb/c mice 48 h after WBI (600 cGy). Ten mice were included in each group. Control group mice received vehicle treated cells whereas the UA group received cells treated with 5 mM UA for 4 h. (A) Survival of the immunocompromised mice reconstituted with allogenic lymphocytes treated with UA or vehicle. *p,0.05, as compared to mice injected with vehicle treated allogenic lymphocytes. (B) Changes in the body weight of the mice after allo-transplantation. Data points represent mean6SEM from 10 mice. Changes in levels of IL-2 (C), IL-6 (D) and IFN-c (E) in serum separated from the blood collected on days 3 and 5 from recipient mice (Balb/c) injected with vehicle treated lymphocytes or UA treated lymphocytes isolated from C57BL/6 mice (F) Ursolic acid inhibits cytokine production in activated lymphocytes. Lymphocytes were stimulated with Con A (5 mg/ml) following which UA (5 mM) was added at the indicated time points and the cells were further cultured for 24 h at 37uC. The concentration of IL-2 cytokines in the culture supernatant was estimated using ELISA. (G) Ursolic acid inhibits Con A induced cytokine production even after washing. Lymphocytes were treated with UA (5 mM)) for 4 h and washed with normal RPMI twice, rested for 48 h and then stimulated with Con A (5 mg/ml) for 24 h at 37uC. The concentration of IL-2 in the culture supernatant was estimated using ELISA. Each bar represents 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.g007 1%BSA). Before staining with monoclonal antibody against Bcl-2 and Bcl-xl, cells were permeabilized with PBST (PBS cotain- ing0.02% tween-20) thrice for 5 min each at room temperature followed by 2 washes with wash buffer and then incubated with the indicated antibodies for 30 min at room temperature, washed twice and analyzed using a Partec Cyflow flowcytometer. Surface staining with PE labeled antibodies was done as described earlier [52]. In brief, splenic lymphocytes were treated with ursolic acid (5 mM, 4 h) and were further stimulated with Con A (10 mg/ml) or LPS (50 mg/ml) for 24 h or 48 h. Staining with PE conjugated CD25 antibody or CD69 antibody were done with cells (16106) obtained after 24 h treatment, while those with CD28, CD134, CD19, CD80, CD86, MHCII were done with cells (16106) obtained after 48 h treatment. A total 20,000 cells in each group were acquired and analyzed in a Partec Cyflow flowcytometer. Western blot analysis Splenic lymphocytes were treated with ursolic acid (5 mM, 4 h) and were stimulated with Con A (5 mg/ml) for 1 h at 37uC and cytosolic extract or whole cell extract was prepared as described earlier [54] . Vehicle treated cells served as control. Briefly, cells were washed with ice-cold phosphate buffered saline and suspended in 0.1 ml lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothre- itol, 0.5 mM PMSF, 2 mg/ml leupeptin, 2 mg/ml aprotinin, and 0.5 mg/ml benzamidine). Then cells were allowed to swell on ice for 15 min, after which 25 ml of 10% NP-40 was added and tubes were vortexed. The supernatants containing proteins from cytosolic fraction were collected by centrifuging the cells at 8000 rpm for 6 min at 4uC. The pellet were suspended in nuclear extraction buffer for performing EMSA as described below. Protein estimation was carried out by Bradford method using Bio- Rad Protein Assay Kit. Equal amounts of protein (50 mg) were resolved by SDS-PAGE (10%) and transferred to nitro cellulose membrane. After the membrane was blocked in 5% nonfat powdered milk, it was incubated overnight with the primary antibody specific to IkB-a or p-c-Raf or p-MEK or p-ERK or p- JNK and washed three times with Tris-buffer saline containing 0.05% tween 20 (TBST) and further incubated with horseradish peroxidase-labeled secondary antibody for 1 h. The membranes were washed, and specific bands were visualized on X-ray films using enhanced chemiluminiscence kit (Roche, Germany). The membrane was stripped and reprobed with actin-b or ERK or JNK antibody. Electrophoretic mobility shift assay Splenic lymphocytes were treated with ursolic acid (5 mM, 4 h) and were stimulated with Con A (5 mg/ml) for 1 h at 37uC and nuclear extracts were prepared. The nuclear pellets were resuspended in 25 ml of ice cold nuclear extraction buffer (20 mMHEPES, pH 7.9, 0.4MNaCl, 1 mM EDTA, 1 mMEGTA, 1 mMDTT, 1 mMPMSF, 2.0 pg/ml leupeptin, 2.0 mg/ml aprotinin, and 0.5 mg/ml benzamidine), and the tubes were incubated on ice for 60 min with intermittent agitation. Samples were microcentrifuged for 5 min at 12,000 rpm, and the supernatant was collected in fresh tubes and frozen at 270uC. EMSA was performed by incubating 10 mg of nuclear proteins with 16 fmol of 32 P-end-labeled, double stranded NF-kB oligonucleotides from the human immunodeficiency virus long terminal repeat (59-TTGTTACAAGGGACTTTCCGCTGGG- GACTTTCCAGGGAGGCGTGG-39; boldface indicates NF-kB binding sites) or AP-1(59-CGCTTGATGACTCAGCCGGAA-39; boldface indicates AP-1 binding site) or NF-AT(59-CGC CCA AAG AGG AAA ATT TGT TTC ATA-39; boldface indicates NF-AT binding site) in the presence of 0.5 mg of poly (29- deoxyinosinic-29-deoxycytidylic acid) (poly (dI–dC)) in binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol,1% NP 40, 5% glycerol, and 50 mMNaCl) for 30 min at 37uC. The DNA–protein complex formed was separated from free oligonucleotide on 6.6% native polyacryl- amide gels using buffer containing 50 mM Tris, 200 mM glycine, and 1 mM EDTA, pH8.5. The dried gel was exposed on phosphorimage plate and the radioactive bands were visualized Table 1. List of primer sequences used for real time quantitative PCR. Gene Cdc25a E2F Gadd45g Plcg2 Mcm7 IFN-gamma IL-2 b-actin Sequence Forward: ACAGCAGTCTACAGAGAATGGG Reverse: GATGAGGTGAAAGGTGTCTTGG Forward: CAGAACCTATGGCTAGGGAGT Reverse: GATCCAGCCTCCGTTTCACC Forward: GGGAAAGCACTGCACGAACT Reverse: AGCACGCAAAAGGTCACATTG Forward: GTGGACACCCTTCCAGAATATG Reverse: ACCTGCCGAGTCTCCATGAT Forward: AGTATGGGACCCAGTTGGTTC Reverse: GCATTCTCGCAAATTGAGTCG Forward: TGGAGGAACTGGCAAAAGGATGGT Reverse: TTGGGACAATCTCTTCCCCAC Forward: TGATGGACCTACAGGAGCTCCTGAG Reverse: GAGTCAAATCCAGAACATGCCGCAG Forward: GCGGGAAATCGTGCGTGACATT Reverse: GATGGAGTTGAAGGTAGTTTCGTG doi:10.1371/journal.pone.0031318.t001 PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e31318