Molecules from American Ginseng Suppress Colitis through Nuclear Factor Erythroid-2-Related Factor 2

Abstract

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that affects millions of people worldwide and increases the risk of colorectal cancer (CRC) development. We have previously shown that American ginseng (AG) can treat colitis and prevent colon cancer in mice. We further fractionated AG and identified the most potent fraction, hexane fraction (HAG), and the most potent compound in this fraction, panaxynol (PA). Because (1) oxidative stress plays a significant role in the pathogenesis of colitis and associated CRC and (2) nuclear factor erythroid-2-related factor 2 (Nrf2) is the master regulator of antioxidant responses, we examined the role of Nrf2 as a mechanism by which AG suppresses colitis. Through a series of in vitro and in vivo Nrf2 knockout mouse experiments, we found that AG and its components activate the Nrf2 pathway and decrease the oxidative stress in macrophages (mΦ) and colon epithelial cells in vitro. Consistent with these in vitro results, the Nrf2 pathway is activated by AG and its components in vivo, and Nrf2^-/- mice are resistant to the suppressive effects of AG, HAG and PA on colitis. Results from this study establish Nrf2 as a mediator of AG and its components in the treatment of colitis.

Keywords: NFE2L2; Nrf2; colitis; falcarinol; ginseng; inflammation; inflammatory; mice; panaxynol; ulcerative.

Figure 1

American ginseng (AG), hexane fraction of American ginseng (HAG), and panaxynol (PA) activate Nrf2 pathway and decrease reactive oxygen species (ROS) in vitro. (A) An oxidative burst in ANA-1 mouse mΦ is suppressed by pretreatment with AG (100 μg/mL), HAG (100 μg/mL), and PA (0.25 μg/mL). The protocol is described in the Methods section. (B) AG, HAG, and PA induce translocation of Nrf2 into the nucleus. ANA-1 cells were treated with AG (100 μg/mL), HAG (100 μg/mL), and PA (0.25 µg/mL = 1 µM) for 12 h. Representative images of immunofluorescence (n = 3). Green arrows indicate nuclei with Nrf2 expression. (C) AG, HAG, and PA increase the expression of HO-1. Western blot image of ANA-1 cells treated with indicated doses of AG, HAG, and PA for 12 h. C—control sample: non-treated ANA-1 cells. (D,E) Twelve-hour incubation with AG (100 μg/mL), HAG (100 μg/mL), and PA (0.25 µg/mL) increases the expression of HO-1 (D) and Nrf2 (E) in ANA-1 cells. RT-qPCR data is cumulative of three separate experiments. (F,G) AG, HAG, and PA activate Nrf2 pathway in HCT-116 cells in the presence of activated mΦ. HCT-116 cells were pretreated with AG (100 μg/mL), HAG (100 μg/mL) and PA (0.25 µg/mL) and co-cultured with activated ANA-1 (10 ng/mL IFN-γ) for 3 h and separated for qPCR. p-values—*—>0.05, **—>0.005, ***—>0.001, ****—>0.0001. Values represent the mean ±S.D. The significance is compared with the control group.

Figure 2

AG, HAG, and PA activate Nrf2 pathway and decrease ROS in vivo. Effect of AG, HAG, and PA on oxidative stress and Nrf2 pathway activation. Dextran Sodium Sulphate (DSS)-induced colitis mice were treated with AG (75 mg/kg/day), HAG (75 mg/kg/day), or PA (1 mg/kg/day). Colons from these mice were probed for (A) 4-HNE and (B) HO-1 to indicate oxidative stress and Nrf2 pathway activation, respectively. Values represent the mean ±S.E. N = 8. The significance is compared with the DSS-only group. p-values—*—>0.05, **—>0.005, ***—>0.001, ****—>0.0001.

Figure 3

AG, HAG, and PA decrease the clinical disease index (CDI) in WT mice but not Nrf2^-/- mice. (A,B) Schematic and groups of the experiment. (C) Effect of AG (75 mg/kg/day), HAG (75 mg/kg/day), and PA (1 mg/kg/day) on the clinical disease index, which accounts for weight loss, blood in stool, and stool consistency. Values represent the mean ±S.E. Significance is compared with the DSS only sub-group within WT and Nrf2^-/- groups. p-values—*>0.05, **—>0.005, ***—>0.001, ****—>0.0001.

Figure 4

AG, HAG, and PA decrease the inflammation in WT mice but not Nrf2^-/- mice. Seven to ten mice from each group from Figure 3 were euthanized on day 14, and colons were harvested. (A,B) Effects of AG, HAG, and PA on the colon histology score in the acute DSS colitis model. The histology score was determined as described in Materials and Methods. Values represent the mean ±S.E. of the mean. Representative H&E-stained colons are shown for each group (A). Sections of the colon were probed for cyclooxygenase-2 (COX2). (C) Representative IHC images probed for COX2. (D) Immunoreactivity score. * Significant difference from the DSS group p-values *—>0.05, **—>0.005, ***—>0.001, ****—>0.0001. A and C are representative images (400× magnification).

Figure 5

AG, HAG, and PA suppress colitis by activation of the Nrf2 pathway. Schematic representing the conclusions: oxidative stress is one of the factors that play a significant role in the progression of colitis. AG, HAG, and PA activate the transcription factor, Nrf2, which in turn activates antioxidant genes that decrease oxidative stress, thereby suppressing colitis.