Preventive and therapeutic oral administration of the pentacyclic triterpene α,β-amyrin ameliorates dextran sulfate sodium-induced colitis in mice: The relevance of cannabinoid system.
Preventive and therapeutic oral administration of the pentacyclic triterpene α,β-amyrin ameliorates dextran sulfate sodium-induced colitis in mice: The relevance of cannabinoid system.
Source
Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, 88049-900, Florianópolis, SC, Brazil.
Abstract
The pentacyclic triterpene α,β-amyrin has been previously reported as an effective compound in the treatment of several inflammatory conditions. Recent evidence indicates that α,β-amyrin displayed its effects through interaction with the cannabinoid pathway. We assessed the anti-inflammatory effects of the α,β-amyrin in the dextran sulfate sodium (DSS)-induced colitis in mice and investigated whether its effects were associated with the interaction with the cannabinoid system. Our results showed that the oral preventive or therapeutic treatment with α,β-amyrin significantly reduced disease activity, body weight loss, colonic damage, as well as colonic myeloperoxidase and N-acetylglucosaminidase activities. Moreover, α,β-amyrin decreases the colonic pro-inflammatory mediators tumor necrosis factor (TNF)-α, interleukin (IL)-1β and keratinocyte-derived chemokine (CXCL1/KC), while up-regulating the IL-4 levels. Additionally, we also observed that the α,β-amyrin caused a significant reduction of the adhesion molecules mRNA expression for intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), platelet cell adhesion molecule 1 (PCAM-1), β2-integrin and protein expression for proliferation marker Ki67, the macrophage molecule CD68 and for adhesion molecule P-selectin. Interestingly, our results also showed that thecannabinoid receptor 1 (CB1), but not CB2, pharmacological blockade significantly reversed the beneficial effects of α,β-amyrin in DSS-induced colitis. Besides, our data demonstrated that mRNA expression for both the endocannabinoid hydrolase monoglyceride lipase 1 (MGL1) and fatty acid amide hydrolase (FAAH) were significantly reduced in the colon of α,β-amyrin-treated mice. Altogether, these results suggest that the α,β-amyrin might possess potential therapeutic interest for the treatment of IBD, and also provide new insights for the underlying mechanisms.
Copyright © 2013 Elsevier Ltd. All rights reserved.
- PMID:
23454360
[PubMed – as supplied by publisher]
Graphical abstract
Highlights
Abbreviations
- COX-2, cyclooxygenase-2;
- DAI, disease activity index;
- DSS, dextran sulfate sodium;
- IBD, inflammatory bowel disease;
- KC, keratinocyte-derived chemokine;
- MPO, myeloperoxidase;
- NAG, N-acetylglucosaminidase;
- PMN, polymorphonuclear;
- TNBS, 2,4,6-trinitrobenzene sulfonic acid;
- TNF, tumor necrosis factor interleukin (IL);
- IFN, interferon;
- ICAM, intercellular adhesion molecule;
- VCAM, vascular cell adhesion molecule;
- PCAM, platelet cell adhesion molecule;
- CB, cannabinoid receptor;
- MGL1,monoglyceride lipase 1;
- FAAH, fatty acid amide hydrolase;
- CD, Crohn’s disease;
- UC, ulcerative colitis
Keywords
- α,β-Amyrin;
- Inflammatory bowel disease;
- Dextran sodium sulfate;
- Cannabinoid system;
- Inflammatory cytokines
Figures and tables from this article:
- Fig. 1. α,β-Amyrin protects against DSS-induced colitis. Mice received DSS (3%) for 5 days and drinking water for the next 2 days. Animals were orally treated by gavage with 1, 3 or 10 mg/kg of α,β-amyrin twice a day from day 0 to day 7 (preventive treatment) or with 10 mg/kg from day 3 to day 7 (therapeutic treatment). Preventive or therapeutic oral treatment with α,β-amyrin improved the disease activity index (DAI) (A), reduced body weight loss (B), colon macroscopic damage (C), and enhanced colon length (D) when compared to mice from the DSS group. Each column represents the mean ± S.E.M. of 5–7 mice per group. #P < 0.05 versus the control group; *P < 0.05 versus the DSS-treated group.
- Fig. 2. α,β-Amyrin reduces cell infiltration and improves microscopic colon damage. Seven days after α,β-amyrin oral treatment, colon tissues were processed for histological evaluation or for the measurement of myeloperoxidase (MPO) activity and N-acetylglucosaminidase (NAG) activity. Preventive or therapeutic oral treatment with α,β-amyrin (10 mg/kg) reduced MPO (A) and NAG (B) activities. In addition, preventive or therapeutic oral treatment with α,β-amyrin (10 mg/kg) decreased the microscopic damage score in mouse colon (C). Each column represents the mean ± S.E.M. of 5–7 mice. #P < 0.05 versus the control group; *P < 0.05 versus the DSS-treated group. Representative paraffin sections of colons from control mice (Control), DSS-treated mice (DSS) and preventive or therapeutic α,β-amyrin-treated mice (10 mg/kg, p.o.) stained with H&E (D). Original magnifications 100×, 200× and 400×. PMN = black arrows.
- Fig. 3. Preventive or therapeutic oral treatment with α,β-amyrin changes protein levels of inflammatory mediators. At the end of the seventh day, colon tissue was collected and processed for cytokine levels. Preventive or therapeutic oral treatment with α,β-amyrin (10 mg/kg) reduced colonic levels of tumor necrosis factor-α (TNF-α) (A), interleukin-1β (IL-1β) (B), keratinocyte-derived chemokine (CXCL1/KC) (C), and increased interleukin-4 (IL-4) (D) levels as analyzed by ELISA. Each column represents the mean ± S.E.M. of five to seven mice. #P < 0.05 versus the control group; *P < 0.05 versus the DSS-treated group.
- Fig. 4. Oral treatment with α,β-amyrin decreases colonic adhesion molecules mRNA expression in DSS-induced colitis. At the end of the seven days, colon tissue was collected and processed for chemokine/cytokine mRNA expression. Preventive oral treatment with α,β-amyrin (10 mg/kg) reduced vascular cell adhesion molecule 1 (VCAM-1) (A), intercellular adhesion molecule 1 (ICAM-1) (B), platelet cell adhesion molecule 1 (PCAM-1) (C) and β2-integrin (D) mRNA expression as analyzed by real-time PCR. The real-time PCR assay was performed in duplicate and GAPDH mRNA was used to normalize the relative amount of mRNA. Each column represents the mean ± S.E.M. of 5–7 animals/group. #P < 0.05 versus the control group; *P < 0.05 versus the DSS-treated group.
- Fig. 5. α,β-Amyrin treatment reduces Ki67, CD68 and P-selectin immunostaining in colonic tissue. Following a 7-day α,β-amyrin treatment, colon samples were processed for immunohistochemistry analysis. Preventive oral treatment with α,β-amyrin (10 mg/kg) significantly reduced the proliferation marker Ki67 (A), macrophage marker CD68 (B) and the adhesion molecule P-selectin (C) immunostaining. The mean intensity of Ki67, CD68 and P-Selectin staining were determined from image analysis and are represented as arbitrary units (D). Each column represents the mean ± S.E.M. of 5–7 animals/group. #P < 0.05 versus the control group; *P < 0.05 versus the DSS-treated group.
- Fig. 6. Antagonism of CB1 receptor, but not CB2, reverses α,β-amyrin anti-inflammatory effects. Preventive oral treatment with α,β-amyrin (10 mg/kg) significantly ameliorated disease activity, but treatment with CB1 selective antagonist AM251 plus α,β-amyrin, impaired α,β-amyrin DAI improvement (A). However, previous treatment with CB2selective antagonist AM630 did not reverse beneficial effects of α,β-amyrin in DSS-induced colitis. At the end of seven days, DSS enhanced mRNA expression for endocannabinoid hydrolases monoglyceride lipase 1 (MGL1) (C) and fatty acid amide hydrolase (FAAH) (D); however, α,β-amyrin preventive treatment (10 mg/kg, p.o.) reduced the mRNA expression for these enzymes (C, D). DSS-treated mice showed a significant increase of CB1 mRNA expression on colonic tissue compared to the control animals (E). The real-time PCR assay was performed in duplicate and GAPDH mRNA was used to normalize the relative amount of mRNA. Each column represents the mean ± S.E.M. of 5–7 animals/group. #P < 0.05 versus the control group; *P < 0.05 versus the DSS-treated group; ΔP < 0.05 versus the α,β-amyrin-treated group.
- Supplementary Fig. I. α,β-Amyrin reduce CXCL1/KC and TNF-α, but not IFN-γ mRNA expression in colonic tissue with DSS-induced colitis. At the end of the seven days, colon tissue was collected and processed for chemokine/cytokine mRNA expression. Preventive oral treatment with α,β-amyrin (10 mg/kg) reduced colonic keratinocyte-derived chemokine (CXCL1/KC) (A) and tumor necrosis factor-α (TNF-α) (B), but not IFN-γ (C) mRNA expression as analyzed by real-time PCR. The real-time PCR assay was performed in duplicate and GAPDH mRNA was used to normalize the relative amount of mRNA. Each column represents the mean ± S.E.M. of 5–7 animals/group. #P < 0.05 versus the control group; *P < 0.05 versus the DSS-treated group.
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- This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Apoio à Pesquisa Científica Tecnológica do Estado de Santa Catarina (FAPESC) (Brazil). AFB and RFC are Postdoctoral students, RM is a PhD student and IM is an undergraduate student; all received grants from CNPq.
- Corresponding author at: Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina (UFSC), Campus Universitário, Trindade, 88049-900, Florianópolis, SC, Brazil. Tel.: +55 48-33313414.
- 1
- These authors contributed equally to this study.
Copyright © 2013 Elsevier Ltd. All rights reserved.
http://www.ncbi.nlm.nih.gov/pubmed/23454360
