Preventive and Therapeutic Euphol Treatment Attenuates Experimental Colitis in Mice

BACKGROUND: The tetracyclic triterpene euphol is the main constituent found in the sap of Euphorbia tirucalli. This plant is widely known in Brazilian traditional medicine for its use in the treatment of several kinds of cancer, including leukaemia, prostate and breast cancers. Here, we investigated the effect of euphol on experimental models of colitis and the underlying mechanisms involved in its action. METHODOLOGY/PRINCIPAL FINDINGS: Colitis was induced in mice either with dextran sulfate sodium (DSS) or with 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the effect of euphol (3, 10 and 30 mg/kg) on colonic injury was assessed. Pro-inflammatory mediators and cytokines were measured by immunohistochemistry, enzyme-Linked immunoabsorbent assay (ELISA), real time-polymerase chain reaction (RT-PCR) and flow cytometry. Preventive and therapeutic oral administration of euphol attenuated both DSS- and TNBS-induced acute colitis as observed by a significant reduction of the disease activity index (DAI), histological/microscopic damage score and myeloperoxidase (MPO) activity in colonic tissue. Likewise, euphol treatment also inhibited colon tissue levels and expression of IL-1β, CXCL1/KC, MCP-1, MIP-2, TNF-α and IL-6, while reducing NOS2, VEGF and Ki67 expression in colonic tissue. This action seems to be likely associated with inhibition of activation of nuclear factor-κB (NF-κB). In addition, euphol decreased LPS-induced MCP-1, TNF-α, IL-6 and IFN-γ, but increased IL-10 secretion from bone marrow-derived macrophages in vitro. Of note, euphol, at the same schedule of treatment, markedly inhibited both selectin (P- and E-selectin) and integrin (ICAM-1, VCAM-1 and LFA-1) expression in colonic tissue. CONCLUSIONS/SIGNIFICANCE: Together, these results clearly demonstrated that orally-administered euphol, both preventive or therapeutic treatment were effective in reducing the severity of colitis in two models of chemically-induced mouse colitis and suggest this plant-derived compound might be a potential molecule in the management of inflammatory bowel diseases

The transition of proembryogenic masses to somatic embryos in Araucaria angustifolia (Bertol.) Kuntze is related to the endogenous contents of IAA, ABA and polyamines

In somatic embryogenesis (SE) of conifers, the inability of many embryogenic cell lines to form well-developed somatic embryos may results from failure and constraints during the transition of proembryogenic masses (PEMs) to early somatic embryos. In the present work, we propose the inclusion of a preculture and prematuration steps looking at enhancing PEM III-to-early somatic embryos transition. It was further hypothesized that these results would correlate with the contents of endogenous indole-3-acetic acid (IAA), abscisic acid (ABA) and polyamines (PA). To test these hypotheses, the embryogenic culture was subjected to preculture with fluridone (FLD) and prematuration treatments with different combinations of carbon source and polyethylene glycol (PEG). The frequency of PEM III was increased after FLD preculture and the contents of IAA and ABA decreased, while the contents of PA increased. Putrescine (Put) was the most abundant PA present at this stage, followed by spermidine (Spd) and spermine (Spm). In early embryogenesis, prematuration treatments supplemented with maltose or lactose plus PEG enhanced the PEM III-to-early somatic embryos transition. IAA and ABA contents increased at this stage, while a decrease of the total free PA levels was observed. Put was the most abundant PA, followed by Spd and Spm, mainly in the treatment supplemented with PEG. This resulted in a decrease of PA ratio (Put/Spd + Spm) and, hence, PEM III-to-early somatic embryos transition. It was concluded that the preculture with FLD and prematuration treatments promote the PEM III-to-early somatic embryos transition throughout the whole early developmental process in Araucaria angustifolia.This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) and Fundação de Apoio à Pesquisa Cientifica e Inovação Tecnológica do Estado de Santa Catarina (FAPESC). This study is part of the Ph.D. thesis of the first author

Effects of UV-B radiation on germlings of the red macroalga Nemalion helminthoides (Rhodophyta)

AbstractStudies have clearly demonstrated the damaging effects of UV-B exposure on macroalgae, but few have reported the impact of UV-B on spore germination and development at juvenile stages. Therefore, this work aimed to analyze the effects of UV-B radiation on germlings of Nemalion helminthoides at the tetrasporophytic phase. To accomplish this, germlings of N. helminthoides were cultivated in the laboratory and separated into two groups. The control group was exposed onlyto photosynthetic radiation, while the treatment group was exposed to photosynthetic radiation + UV-B for 2hours during a period of 12 days. Control germlings showed increasing cellular proliferation and accumulation of reserve substances, as well as intense ramification in the last observed stages between 9 days and 12 days of development. Moreover, the chloroplasts presented a typical globular pyrenoid, profusely traversed by thylakoid membranes. Treated germlings, by contrast, showed intracellular damage, such as cell wall thickness, loss of chloroplast organization, changes in mitochondrial cristae, and increasing atrophy of the Golgi bodies. Additionally, changes in developmental patterns were observed, including loss of polarity in the first divisions of carpospores and abnormal stem ramification. The quantification of autofluorescence data coincided with the ultrastructural changes observed in the chloroplasts of cells exposed to UV-B. It can be concluded that exposure to radiation changed the developmental pattern and morphology of the germlings of N. helminthoides

Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze

Somatic embryogenesis is a morphogenetic route useful for the study of embryonic development, as well as the large-scale propagation of endangered species, such as the Brazilian pine (Araucaria angustifolia). In the present study, we investigated the morphological and ultrastructural organization of A. angustifolia somatic embryo development by means of optical and electron microscopy. The proembryogenic stage was characterized by the proliferation of proembryogenic masses (PEMs), which are cellular aggregates composed of embryogenic cells (ECs) attached to suspensor-like cells (SCs). PEMs proliferate through three developmental stages, PEM I, II, and III, by changes in the number of ECs and SCs. PEM III-to-early somatic embryo (SE) transition was characterized by compact clusters of ECs growing out of PEM III, albeit still connected to it by SCs. Early SEs showed a dense globular embryonic mass (EM) and suspensor region (SR) connected by embryonic tube cells (TCs). By comparison, early somatic and zygotic embryos showed similar morphology. ECs are round with a large nucleus, nucleoli, and many cytoplasmic organelles. In contrast, TCs and SCs are elongated and vacuolated with cellular dismantling which is associated with programmed cell death of SCs. Abundant starch grains were observed in the TCs and SCs, while proteins were more abundant in the ECs. Based on the results of this study, a fate map of SE development in A. angustifolia is, for the first time, proposed. Additionally, this study shows the cell biology of SE development of this primitive gymnosperm which may be useful in evolutionary studies in this area.Fil: Steiner, Neusa. Universidade Federal de Santa Catarina; BrasilFil: Farias-Soares, Francine L.. Universidade Federal de Santa Catarina; BrasilFil: Schmidt, Éder C.. Universidade Federal de Santa Catarina; BrasilFil: Pereira, Maria L. T.. Universidade Federal de Santa Catarina; BrasilFil: Scheid, Bruna. Universidade Federal de Santa Catarina; BrasilFil: Rogge-Renner, Gladys D.. Universidade Da Regiao de Joinville; Brasil. Universidade Federal de Santa Catarina; BrasilFil: Bouzon, Zenilda L.. Universidade Federal de Santa Catarina; BrasilFil: Schmidt, Daniela. Universidade Federal de Santa Catarina; BrasilFil: Maldonado, Sara Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Guerra, Miguel P.. Universidade Federal de Santa Catarina; Brasi

Euphol ameliorates DSS-induced acute colitis.

<p>(A), Chemical structure of euphol. Mice received DSS for 5 days and drinking water for the next 2 days. Animals were orally treated by gavage with 3, 10, or 30 mg/kg of euphol twice a day from day 0 to day 7 (preventive treatment) or with 30 mg/kg from day 3 to day 7 (therapeutic treatment). Preventive or therapeutic oral treatment with euphol improved the disease activity index (DAI) score (B), reduced body weight loss (C) and colon macroscopic damage (D), and enhanced colon length (E) when compared with mice from the DSS group. Data are reported as means ± S.E.M. of 8 to 10 mice per group and is representative of three independent experiments. ^#P<0.05 vs. control healthy group; *P<0.05 vs. DSS-treated group.</p

Preventive treatment with euphol changes colonic protein levels and mRNA expression of inflammatory mediators.

<p>At the end of 7 days, colon tissue was collected and processed for cytokine levels and mRNA expression. (A–D) Enzyme-linked immunosorbent assay. Preventive treatment with euphol (30 mg/kg, p.o.) reduced colonic levels of interleukin-1β (IL-1β) (A), keratinocyte-derived chemokine (CXCL1/KC) (B), macrophage inflammatory protein-2 (MIP-2) (C) and monocyte chemoattractant protein-1 (MCP-1) (D). (E–H) Real-time PCR. The same scheme of treatment with euphol also impaired the increase colonic mRNA expression of IL-1β (E), CXCL1/KC (F), tumor necrosis factor-α (TNF-α) (G) and interleukin-6 (IL-6) (H). The real-time PCR assay was performed in duplicate and GAPDH mRNA was used to normalize the relative amount of mRNA. Data are reported as means ± S.E.M. of 8 to 10 mice per group and is representative of three independent experiments. ^#P<0.05 vs. control healthy group (non colitic); *P<0.05 vs. DSS-treated group.</p

Preventive treatment with euphol blocks integrins and selectins expression in the colonic tissue after DSS-induced colitis.

<p>At the end of 7 days, colon tissue was collected and processed for mRNA expression and immunofluorescence. Preventive treatment with euphol (30 mg/kg, p.o.) reduced colonic mRNA expression of inter-cellular adhesion molecule 1 (ICAM-1) (A), vascular cell adhesion molecule-1 (VCAM-1) (B) and lymphocyte function-associated antigen 1 (LFA-1) (C). The real-time PCR assay was performed in duplicate and GAPDH mRNA was used to normalize the relative amount of mRNA. The same scheme of treatment with euphol also impaired the increase of P-selectin (D) and E-selectin (E). Representative images of P-selectin and E-selectin immunofluorescent stains were obtained on day 7 from control healthy mice, DSS-treated group and euphol (30 mg/kg, p.o.) treated group. Nuclei were stained with Hoechst (0.5 µl/ml). Scale bar corresponds to 50 µm and applies throughout. Data are reported as means ± S.E.M. of 8 to 10 mice per group and is representative of three independent experiments. ^#P<0.05 vs. control healthy group (non colitic); *P<0.05 vs. DSS-treated group.</p

Treatment with euphol reduces cell influx and microscopic colon damage after DSS-induced acute colitis.

<p>At 7 days after euphol oral treatment, colon tissues were processed for histological evaluation, measurement of myeloperoxidase (MPO) activity and scanning electron microscopy. Preventive (3, 10, and 30 mg/kg, p.o.) or therapeutic (30 mg/kg, p.o.) treatment with euphol reduced MPO (A) activity. (B) Representative histological sections of colon from control healthy mice (non colitic), DSS-treated and euphol-treated mice (30 mg/kg, p.o.) were examined microscopically after H&E staining with original magnification x20. The images are representative of at least four mice per group. (C) Preventive treatment with euphol (30 mg/kg, p.o.) decreased the microscopic damage score in mouse colon. (D) Scanning electron microscopy photographs of the colon of the colon surfaces of control healthy mice, DSS-treated group, and DSS plus euphol (30 mg/kg, p.o.) treated mice after 7 days following DSS administration. Original magnification: x750 and x6,000, respectively. Each column represents the mean ± S.E.M. of 8 to 10 mice per group and is representative of two independent experiments. ^#P<0.05 vs. control healthy group (non colitic); *P<0.05 vs. DSS-treated group.</p

Therapeutic treatment with euphol protects mice against TNBS-induced acute colitis.

<p>Mice were given 100 µL of the TNBS (in 35% ethanol) and after 24 h, treated with euphol (30 mg/kg, p.o.). (A) The time-course of body weight changes on day 3 after TNBS-induced colitis. (B) Macroscopic score; (C) colon length after TNBS-induced colitis. (D) Representative photograph of colons from day 3 after the induction of TNBS-colitis. 1, Control healthy mice; 2, TNBS-treated (only vehicle administration); 3, TNBS plus euphol (30 mg/kg, p.o.). Each column represents the mean ± S.E.M. of 8 to 10 mice per group and is representative of two independent experiments. ^#P<0.05 vs. control healthy group (non colitic); *P<0.05 vs. TNBS-treated group. Vehicle corresponds to 5% Tween 80 in saline 0.9% NaCl.</p