American ginseng suppresses colitis through p53-mediated apoptosis of inflammatory cells

Abstract

Ulcerative colitis is a dynamic, chronic inflammatory condition associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism regulating ulcerative colitis. American ginseng (AG) is a putative antioxidant that can suppress hyperactive immune cells. We have recently shown that AG can prevent and treat mouse colitis. Because p53 levels are elevated in inflammatory cells in both mouse and human colitis, we tested the hypothesis that AG protects from colitis by driving inflammatory cell apoptosis through a p53 mechanism. We used isogenic p53(+/+) and p53(-/-) inflammatory cell lines as well as primary CD4(+)/CD25(-) effector T cells from p53(+/+) and p53(-/-) mice to show that AG drives apoptosis in a p53-dependent manner. Moreover, we used a dextran sulfate sodium (DSS) model of colitis in C57BL/6 p53(+/+) and p53(-/-) mice to test whether the protective effect of AG against colitis is p53 dependent. Data indicate that AG induces apoptosis in p53(+/+) but not in isogenic p53(-/-) cells in vitro. In vivo, C57BL/6 p53(+/+) mice are responsive to the protective effects of AG against DSS-induced colitis, whereas AG fails to protect from colitis in p53(-/-) mice. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of inflammatory cells within the colonic mesenteric lymph nodes is elevated in p53(+/+) mice consuming DSS + AG but not in p53(-/-) mice consuming DSS + AG. Results are consistent with our in vitro data and with the hypothesis that AG drives inflammatory cell apoptosis in vivo, providing a mechanism by which AG protects from colitis in this DSS mouse model.

Fig. 1

AG drives apoptosis of lymphoblasts through the p53 pathway. The TK6 human lymphoblast line, and NH32 isogenic p53 knock out cell line were cultured in RPMI + 10% FCS. Cells were exposed to indicated doses of AG for 12 hours, then harvested for protein. Following separation by 10% SDS-Page electrophoresis, gels were blotted onto nitrocellulose, then probed with the indicated antibodies. Results indicate that caspase 3, 7, 9 and PARP cleavage, and induction of Bax by AG are p53 dependent. p53 and phosphorylated p53 are also shown, indicating that p53 is activated by AG.

Fig. 2

Effects of AG on the Disease Activity Index (DAI) in p53^+/+ and p53^−/− mice in the chronic dextran sulfate sodium (DSS)-colitis model. Mice received 2½ (7 days DSS followed by 7 days water per cycle) of DSS with and without AG in chow, as indicated. (◦, indicates p53^−/− mice on DSS and on chow without AG; ●, indicates p53^−/− mice on DSS and on chow with 75 ppm AG; □, indicates p53^+/+ mice on DSS and on chow without AG; ■, indicates p53^+/+ mice on DSS and on chow with 75 ppm AG). *, indicates significant difference from other groups (p < 0.005) from day 21 to the end of the experiment.

Fig. 3

Effects of AG on the colon histology score in the acute DSS-colitis model. 10 mice from each group described in Fig. 2 were sacrificed on day 35, and the colon was harvested from each animal and the histology score was determined. Values represent the mean ± standard error of the mean. Representative H&E stained colons are shown for each group. Significant differences are indicated (p < 0.05).

Fig. 4

Effects of AG on apoptosis in cells of the epithelium and the MLNs. (A) IRS (TUNEL staining) in epithelial cells of indicated groups. (B) IRS (TUNEL staining) in MLNs of indicated groups. (C) Representative staining in the epithelium of indicated groups. (D) Representative staining of MLNs in indicated groups.