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.
Project description:Resveratrol is a naturally occurring polyphenol that exhibits pleiotropic health beneficial effects, including anti-inflammatory, cardio-protective, and cancer-protective activities. It is recognized as one of the more promising natural molecules in the prevention and treatment of chronic inflammatory and autoimmune disorders. Ulcerative colitis is an idiopathic, chronic inflammatory disease of the colon associated with a high colon cancer risk. Here, we used a dextran sulfate sodium (DSS) mouse model of colitis, which resembles human ulcerative colitis pathology. Resveratrol mixed in food ameliorates DSS-induced colitis in mice in a dose-dependent manner. Resveratrol significantly improves inflammation score, downregulates the percentage of neutrophils in the mesenteric lymph nodes and lamina propria, and modulates CD3(+) T cells that express tumor necrosis factor-alpha and IFN-gamma. Markers of inflammation and inflammatory stress (p53 and p53-phospho-Ser(15)) are also downregulated by resveratrol. Because chronic colitis drives colon cancer risk, we carried out experiments to determine the chemopreventive properties of resveratrol. Tumor incidence is reduced from 80% in mice treated with azoxymethane (AOM) + DSS to 20% in mice treated with AOM + DSS + resveratrol (300 ppm). Tumor multiplicity also decreased with resveratrol treatment. AOM + DSS-treated mice had 2.4 +/- 0.7 tumors per animal compared with AOM + DSS + 300 ppm resveratrol, which had 0.2 +/- 0.13 tumors per animal. The current study indicates that resveratrol is a useful, nontoxic complementary and alternative strategy to abate colitis and potentially colon cancer associated with colitis.
Project description:Intestinal epithelial cell (IEC) apoptosis contributes to the development of ulcerative colitis (UC), an inflammatory bowel disease (IBD) that affects the colon and rectum. Therapies that target the inflammatory cytokine TNF have been found to inhibit IEC apoptosis in patients with IBD, although the mechanism of IEC apoptosis remains unclear. We therefore investigated the role of p53-upregulated modulator of apoptosis (PUMA), a p53 target and proapoptotic BH3-only protein, in colitis and IEC apoptosis, using patient samples and mouse models of UC. In UC patient samples, PUMA expression was elevated in colitis tissues relative to that in uninvolved tissues, and the degree of elevation of PUMA expression correlated with the severity of colitis and the degree of apoptosis induction. In mice, PUMA was markedly induced in colonic epithelial cells following induction of colitis by either dextran sulfate sodium salt (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). The induction of PUMA was p53-independent but required NF-?B. Absence of PUMA, but neither absence of p53 nor that of another BH3-only protein (Bid), relieved DSS- and TNBS-induced colitis and inhibited IEC apoptosis. Furthermore, treating mice with infliximab (Remicade), a clinically used TNF-specific antibody, suppressed DSS- and TNBS-induced PUMA expression and colitis. These results indicate that PUMA induction contributes to the pathogenesis of colitis by promoting IEC apoptosis and suggest that PUMA inhibition may be an effective strategy to promote mucosal healing in patients with UC.
Project description:Inflammatory bowel disease (IBD) is associated with increased apoptosis of intestinal epithelial cells (IECs). Mutations in the tumor suppressor p53 appear during early stages of progression from colitis to cancer. We investigated the role of p53 and its target, p53-upregulated modulator of apoptosis (PUMA), in inflammation-induced apoptosis of IECs.Apoptosis was induced in mouse models of mucosal inflammation. Responses of IECs to acute, T-cell activation were assessed in wild-type, p53?/?, Bid?/?, Bim?/?, Bax3?/?, Bak?/?, PUMA?/?, and Noxa?/? mice. Responses of IECs to acute and chronic colitis were measured in mice following 1 or 3 cycles of dextran sulfate sodium (DSS), respectively. Apoptosis was assessed by TUNEL staining and measuring activity of caspases 3 and 9; levels of p53 and PUMA were assessed in colon tissue from patients with and without ulcerative colitis.Apoptosis of IECs occurred in the lower crypts of colitic tissue from humans and mice. Colitis induction with anti-CD3 or 3 cycles of DSS increased apoptosis and protein levels of p53 and PUMA in colonic crypt IECs. In p53?/? and PUMA?/? mice, apoptosis of IECs was significantly reduced but inflammation was not. Levels of p53 and PUMA were increased in inflamed mucosal tissues of mice with colitis and in patients with UC, compared with controls. Induction of PUMA in IECs of p53?/? mice indicated that PUMA-mediated apoptosis was independent of p53.In mice and humans, colon inflammation induces apoptosis of IECs via p53-dependent and - independent mechanisms; PUMA also activates an intrinsic apoptosis pathway associated with colitis.
Project description:Ulcerative colitis is a chronic inflammatory condition associated with a high colon cancer risk. We have previously reported that American ginseng extract significantly reduced the inflammatory parameters of chemically induced colitis. The aim of this study was to further delineate the components of American ginseng that suppress colitis and prevent colon cancer. Among five different fractions of American ginseng (butanol, hexane, ethylacetate, dichloromethane, and water), a hexane fraction has particularly potent antioxidant and proapoptotic properties. The effects of this fraction were shown in a mouse macrophage cell line (ANA-1 cells), in a human lymphoblastoid cell line (TK6), and in an ex vivo model (CD4(+)/CD25(-) primary effector T cells). A key in vivo finding was that compared with the whole American ginseng extract, the hexane fraction of American ginseng was more potent in treating colitis in a dextran sodium sulfate (DSS) mouse model, as well as suppressing azoxymethane/DSS-induced colon cancer. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) labeling of inflammatory cells within the colonic mesenteric lymph nodes was elevated in mice consuming DSS + the hexane fraction of American ginseng. Results are consistent with our in vitro data and with the hypothesis that the hexane fraction of American ginseng has anti-inflammatory properties and drives inflammatory cell apoptosis in vivo, providing a mechanism by which this fraction protects from colitis in this DSS mouse model. This study moves us closer to understanding the molecular components of American ginseng that suppress colitis and prevent colon cancer associated with colitis.
Project description:Background:Rumex japonicus Houtt. (RJ) is widely distributed in Korea, Japan, and China. The root of RJ has traditionally been used to treat constipation, jaundice, hematemesis, dysfunctional uterine bleeding, and gastrointestinal diseases. According to recent studies, plants of the genus Rumex have beneficial functionalities such as anti-microbial, antioxidative, and anti-inflammatory effects. Inflammatory bowel disease, including Crohn's disease and ulcerative colitis, is a chronic inflammatory disease characterized by an abnormal immune response and epithelial barrier dysfunction. This study evaluates the protective effect of RJ against dextran sulfate sodium (DSS)-induced colitis. Methods:Male 8-week-old C57BL/6?N mice were treated with methanolic extract of RJ for 14 days, and DSS-induced groups were administered 2.5% DSS for last 7 days. After sacrifice, the length and weight of the colon were measured, and colon sections were subjected to H&E staining, immunohistochemistry and Western blotting to investigate the changes of inflammatory cytokines, tight junction and apoptosis-related factors. Results:The colon of DSS-treated mouse was significantly shorter and heavier than the normal mouse. Moreover, DSS exposure induced an increase of tumor necrosis factor-?, interleukin (IL)-1?, IL-6, occludin, zonula occludens-1, p21, p53 and Bcl-2, and decreased the expressions of IL-10, claudin-2 and cleaved caspase-3 in the colon tissue. These DSS-induced changes were inhibited by RJ treatment. Conclusion:Our results indicate that RJ effectively suppresses DSS-induced colitis by protecting tight junction connections in the colonic tissue. We therefore infer that RJ has the potential as a medicine or ingredient for treating colitis.
Project description:We have recently shown that American ginseng (AG) prevents and treats mouse colitis. Because both mice and humans with chronic colitis have a high colon cancer risk, we tested the hypothesis that AG can be used to prevent colitis-driven colon cancer. Using the azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model of ulcerative colitis, we show that AG can suppress colon cancer associated with colitis. To explore the molecular mechanisms of the anticancer effects of AG, we also carried out antibody array experiments on colon cells isolated at a precancerous stage. We found there were 82 protein end points that were either significantly higher (41 proteins) or significantly lower (41 proteins) in the AOM + DSS group compared with the AOM-alone (control) group. In contrast, there were only 19 protein end points that were either significantly higher (10 proteins) or significantly lower (9 proteins) in the AOM + DSS + AG group compared with the AOM-alone (control) group. Overall, these results suggest that AG keeps the colon environment in metabolic equilibrium when mice are treated with AOM + DSS and gives insight into the mechanisms by which AG protects from colon cancer associated with colitis.
Project description:Disruption of the healthy intestinal microbiome and homeostasis of the intestinal immune system, which are closely interactive, are two key factors for ulcerative colitis. Here, we show that MI-2, a selective inhibitor of mucosa-associated lymphoid tissue lymphoma translocation-1 (MALT1), alleviated excessive inflammatory responses and was associated with restoration of healthy intestinal microbiome in mice suffering from dextran sulfate sodium (DSS)-induced colitis. We found that the diversity of intestinal microbiome of mice with DSS-induced colitis was significantly lower than that of healthy mice. However, MI-2 treatment in mice with DSS-induced colitis resulted in restored microbially diverse populations. To understand the possibility of the beneficial effect of the restored microbially diverse populations of MI-2-treated mice with DSS-induced colitis, we showed that inserting fecal microbiota from MI-2-treated mice with DSS-induced colitis and healthy control mice into mice with DSS-induced colitis could alleviate symptoms of colitis. The possibility of MI-2 treatment in DSS-induced colitis, associated with restoration of healthy microbially diverse populations in addition to reshaping host immune modulating capacity by reducing inflammatory cytokines (tumor necrosis factor alpha, interleukin-1? [IL-1?], IL-17?, and IL-22), may be considered therapeutic for ulcerative colitis.
Project description:Ulcerative colitis (UC) is a multifactorial inflammatory disease, and increasing evidence has demonstrated that the mechanism of UC pathogenesis is associated with excessive cellular apoptosis and reactive oxygen species (ROS) production. However, their function and molecular mechanisms related to UC remain unknown. In this study, Rab27A mRNA and protein were proven to be overexpressed in intestinal epithelial cells of UC patients and DSS-induced colitis mice, compared with control (P < 0.05). And Rab27A silencing inhibits inflammatory process in DSS-induced colitis mice (P < 0.05). Then, it was shown that knockdown of Rab27A suppressed apoptosis and ROS production through modulation of miR-124-3p, whereas overexpression of Rab27A promoted apoptosis and ROS production in LPS-induced colonic cells. In addition, enhanced expression of miR-124-3p attenuated apoptosis and ROS production by targeting regulation of STAT3 in LPS-induced colonic cells. Mechanistically, we found Rab27A reduced the expression and activity of miR-124-3p to activate STAT3/RelA signalling pathway and promote apoptosis and ROS production in LPS-induced colonic cells, whereas overexpression of miR-124-3p abrogated these effects of Rab27A. More importantly, animal experiments illustrated that ectopic expression of Rab27A promoted the inflammatory process, whereas overexpression of miR-124-3p might interfere with the inflammatory effect in DSS-induced colitis mice. In summary, Rab27A might modulate the miR-124-3p/STAT3/RelA axis to promote apoptosis and ROS production in inflammatory colonic cells, suggesting that Rab27A as a novel therapeutic target for the prevention and treatment of UC patients.
Project description:Ulcerative colitis is a relatively frequent, chronic disease that impacts significantly the patient's quality of life. Although many therapeutic options are available, additional approaches are needed because many patients either do not respond to current therapies or show significant side effects. Cardiotrophin-1 (CT-1) is a cytokine with potent cytoprotective, anti-inflammatory, and antiapoptotic properties. The purpose of this study was to assess if the administration of CT-1 could reduce colon damage in mice with experimental colitis was induced with 5% dextran sulfate sodium (DSS) in the drinking water. Half of the mice received an i.v. dose of CT-1 (200 µg/kg) 2 h before and 2 and 4 days after DSS administration. Animals were followed during 7 days after DSS administration. The severity of colitis was measured by standard scores. Colon damage was assessed by histology and immunohistochemistry. Inflammatory mediators were measured by Western blot and PCR. CT-1 administration to DSS-treated mice ameliorated both the clinical course (disease activity index), histological damage, inflammation (colon expression of TNF-?, IL-17, IL-10, INF IFN-?, and iNOS), and apoptosis. Our results suggest that CT-1 administration before induction of colitis improves the clinical course, tissue damage, and inflammation in DSS-induced colitis in mice.
Project description:The bioactive sphingolipid sphingosine-1-phosphate (S1P) and the kinase that produces it have been implicated in inflammatory bowel diseases in mice and humans; however, little is known about the role of the 2 S1P-specific phosphohydrolase isoforms, SGPP1 and SGPP2, which catalyze dephosphorylation of S1P to sphingosine. To elucidate their functions, we generated specific knockout mice. Deletion of Sgpp2, which is mainly expressed in the gastrointestinal tract, significantly reduced dextran sodium sulfate (DSS)-induced colitis severity, whereas deletion of ubiquitously expressed Sgpp1 slightly worsened colitis. Moreover, Sgpp1 deletion enhanced expression of multifunctional proinflammatory cytokines, IL-6, TNF-?, and IL-1?, activation of the transcription factor signal transducer and activator of transcription 3, and immune cell infiltration into the colon. Conversely, Sgpp2-null mice failed to mount a DSS-induced systemic inflammatory response. Of interest, Sgpp2 deficiency suppressed DSS-induced intestinal epithelial cell apoptosis and improved mucosal barrier integrity. Furthermore, down-regulation of Sgpp2 attenuated LPS-induced paracellular permeability in cultured cells and enhanced expression of the adherens junction protein E-cadherin. Finally, in patients with ulcerative colitis, SGPP2 expression was elevated in colitis tissues relative to that in uninvolved tissues. These results indicate that induction of SGPP2 expression contributes to the pathogenesis of colitis by promoting disruption of the mucosal barrier function. SGPP2 may represent a novel therapeutic target in inflammatory bowel disease.-Huang, W.-C., Liang, J., Nagahashi, M., Avni, D., Yamada, A., Maceyka, M., Wolen, A. R., Kordula, T., Milstien, S., Takabe, K., Oravecz, T., Spiegel, S. Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.