RESUMO
Natural smectites have demonstrated efficacy in the treatment of diarrhea. The present study evaluated the prophylactic effect of a diosmectite (FI5pp) on the clinical course, colon damage, expression of tight junction (TJ) proteins and the composition of the gut microbiota in dextran sulfate sodium (DSS) colitis. Diosmectite was administered daily to Balb/c mice from day 1 to 7 by oral gavage, followed by induction of acute DSS-colitis from day 8 to 14 ("Control", n = 6; "DSS", n = 10; "FI5pp + DSS", n = 11). Mice were sacrificed on day 21. Clinical symptoms (body weight, stool consistency and occult blood) were checked daily after colitis induction. Colon tissue was collected for histological damage scoring and quantification of tight junction protein expression. Stool samples were collected for microbiome analysis. Our study revealed prophylactic diosmectite treatment attenuated the severity of DSS colitis, which was apparent by significantly reduced weight loss (p = 0.022 vs. DSS), disease activity index (p = 0.0025 vs. DSS) and histological damage score (p = 0.023 vs. DSS). No significant effects were obtained for the expression of TJ proteins (claudin-2 and claudin-3) after diosmectite treatment. Characterization of the microbial composition by 16S amplicon NGS showed that diosmectite treatment modified the DSS-associated dysbiosis. Thus, diosmectites are promising candidates for therapeutic approaches to target intestinal inflammation and to identify possible underlying mechanisms of diosmectites in further studies.
Assuntos
Bactérias/classificação , Colite/tratamento farmacológico , Sulfato de Dextrana/efeitos adversos , Microbiota/efeitos dos fármacos , Silicatos/administração & dosagem , Administração Oral , Animais , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Peso Corporal/efeitos dos fármacos , Colite/induzido quimicamente , Colite/metabolismo , Colite/microbiologia , DNA Bacteriano/genética , DNA Ribossômico/genética , Fezes/microbiologia , Masculino , Camundongos Endogâmicos BALB C , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Índice de Gravidade de Doença , Silicatos/farmacologia , Proteínas de Junções Íntimas/metabolismo , Resultado do TratamentoRESUMO
Colorectal cancer is one of the most leading death-causing cancers in the world. Vernodalin, a cytotoxic sesquiterpene, has been reported to possess anticancer properties against human breast cancer cells. We aimed to examine the anticancer mechanism of vernodalin on human colon cancer cells. Vernodalin was used on human colon cancer cells, HT-29 and HCT116. The cytotoxicity of vernodalin on human colon cancer cells was determined through in vitro 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Small interfering RNA was used to analyze the cascade activation of mitogen-activated protein kinase (MAPK) pathway, c-Jun N-terminal kinase (JNK) in HT-29, and HCT116 cells against vernodalin treatment. The protein expressions of caspase 3, Bcl-2, and Bax were examined through Western blot analysis. Immunoblot analysis on the JNK, ERK, and p38 MAPK pathways showed increased activation due to vernodalin treatment. It was proven from the JNK and p38 inhibition test that both pathways are significantly activated by vernodalin to induce apoptosis. Our results, collectively, showed the apoptosis-induced anticancer mechanism of vernodalin on human colon cancer cells that was mediated through the activation of JNK pathway and apoptotic regulator proteins. These results suggest that vernodalin could be developed as a potent chemotherapeutic agent for human colorectal cancer treatment.
Assuntos
Apoptose/efeitos dos fármacos , MAP Quinase Quinase 4/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ativação Enzimática , Células HCT116 , Células HT29 , Humanos , Estresse Oxidativo/efeitos dos fármacos , Sesquiterpenos/farmacologiaRESUMO
Ulcerative colitis (UC) is a severe inflammatory bowel disease (IBD) characterized by multifactorial complex disorders triggered by environmental factors, genetic susceptibility, and also gut microbial dysbiosis. Faecalibacterium prausnitzii, Bacteroides faecis, and Roseburia intestinalis are underrepresented species in UC patients, leading to the hypothesis that therapeutic application of those bacteria could ameliorate clinical symptoms and disease severity. Acute colitis was induced in mice by 3.5% DSS, and the commensal bacterial species were administered by oral gavage simultaneously with DSS treatment for up to 7 days. The signs of colonic inflammation, the intestinal barrier integrity, the proportion of regulatory T cells (Tregs), and the expression of pro-inflammatory and anti-inflammatory cytokines were quantified. The concentrations of SCFAs in feces were measured using Gas-liquid chromatography. The gut microbiome was analyzed in all treatment groups at the endpoint of the experiment. Results were benchmarked against a contemporary mesalazine treatment regime. We show that commensal species alone and in combination reduced disease activity index scores, inhibited colon shortening, strengthened the colonic epithelial barrier, and positively modulated tight junction protein expression. The expression level of pro-inflammatory cytokines was significantly reduced. Immune modulation occurred via inhibition of the loss of CD4 +CD25 +Treg cells in the spleen. Our study proofed that therapeutic application of F. prausnitzii, B. faecis, and R. intestinalis significantly ameliorated DSS-induced colitis at the level of clinical symptoms, histological inflammation, and immune status. Our data suggest that these positive effects are mediated by immune-modulatory pathways and influence on Treg/Th17 balance.
Assuntos
Colite Ulcerativa , Colite , Humanos , Camundongos , Animais , Linfócitos T Reguladores , Faecalibacterium prausnitzii/metabolismo , Células Th17 , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Colite Ulcerativa/tratamento farmacológico , Colo , Citocinas/metabolismo , Bactérias/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
Ulcerative colitis (UC) is a serious health condition and defined as inflammation in the colon. Untreated, UC can develop into colitis-associated cancer (CAC), for which effective medicines are not available. Natural products are a better choice to treat UC by alleviating the inflammation. Caffeic acid phenethyl ester (CAPE) is a phenolic compound and known for its beneficial effects, including antibacterial, anti-inflammatory, anti-diabetic, and anticancer. We aimed to study the effect of CAPE on dextran sulfate sodium (DSS)-induced UC in mouse model. Administration of CAPE to DSS-induced mice protected against colon damage by improving body weight of mice, reducing the weight of spleen, and increased colon length. In addition, administration of CAPE resulted reduced the activity of myeloperoxidase (MPO) and CD68+ positive cells. Furthermore, a significant decrease in the production of key cytokines and the expression of nuclear factor (p65-NF)-κB. Moreover, p65-NF-κB activation was reduced in lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells from mouse origin. CAPE treatment leads to the reduced expressions of intercellular adhesion molecules (ICAM)-1 and vascular cell adhesion molecules (VCAM), both are key cell adhesion molecules. The results of this study clearly indicate that CAPE can potentially control inflammation in the colon and can be used as a therapy for UC.
Assuntos
NF-kappa BRESUMO
Alterations in the gut microbiota composition play a crucial role in the pathogenesis of inflammatory bowel disease (IBD) as specific commensal bacterial species are underrepresented in the microbiota of IBD patients. In this study, we examined the therapeutic potential of three commensal bacterial species, Faecalibacterium prausnitzii (F. prausnitzii), Roseburia intestinalis (R. intestinalis) and Bacteroides faecis (B. faecis) in an in vitro model of intestinal inflammation, by using differentiated Caco-2 and HT29-MTX cells, stimulated with a pro-inflammatory cocktail consisting of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and lipopolysaccharide (LPS). Results obtained in this work demonstrated that all three bacterial species are able to recover the impairment of the epithelial barrier function induced by the inflammatory stimulus, as determined by an amelioration of the transepithelial electrical resistance (TEER) and the paracellular permeability of the cell monolayer. Moreover, inflammatory stimulus increased claudin-2 expression and decreased occludin expression were improved in the cells treated with commensal bacteria. Furthermore, the commensals were able to counteract the increased release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) induced by the inflammatory stimulus. These findings indicated that F. prausnitzii, R. intestinalis and B. faecis improve the epithelial barrier integrity and limit inflammatory responses.
Assuntos
Bacteroides , Clostridiales , Células Epiteliais/microbiologia , Faecalibacterium prausnitzii , Microbioma Gastrointestinal/fisiologia , Doenças Inflamatórias Intestinais/microbiologia , Células CACO-2 , Quimiocina CCL2/metabolismo , Claudina-2 , Impedância Elétrica , Células HT29 , Humanos , Interferon gama/administração & dosagem , Interleucina-1beta/administração & dosagem , Interleucina-8/metabolismo , Mucosa Intestinal/microbiologia , Lipopolissacarídeos/administração & dosagem , Ocludina/metabolismo , Permeabilidade , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
The aim of the present study is to isolate bioactive compounds from the roots of Piper sarmentosum and examine the mechanism of action using human breast cancer cell line (MDA-MB-231). Bioassay guided-fractionation of methanolic extract led to the isolation of asaricin (1) and isoasarone (2). Asaricin (1) and isoasarone (2) had significant cytotoxicity towards MDA-MB-231. MCF-10A (human normal breast epithelial cells) cells are less sensitive than MDA-MB-231, but they respond to the treatment with the same unit of measurement. Both compounds increase reactive oxygen species (ROS), decrease mitochondrial membrane potential (MMP) and enhance cytochrome c release in treated MDA-MB-231 cells. Isoasarone (2) markedly elevated caspase -8 and -3/7 activities and caused a decline in nuclear NF-κB translocation, suggesting extrinsic, death receptor-linked apoptosis pathway. Quantitative PCR results of MDA-MB-231 treated with asaricin (1) and isoasarone (2) showed altered expression of Bcl-2: Bax level. The inhibitory potency of these isolates may support the therapeutic uses of these compounds in breast cancer.
Assuntos
Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fenilpropionatos/farmacologia , Piper/química , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos Fitogênicos , Neoplasias da Mama , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Mitocôndrias/metabolismo , Estrutura Molecular , Fenilpropionatos/químicaRESUMO
Ulcerative colitis (UC) is a nonspecific inflammatory disorder characterized by oxidative and nitrosative stress, leucocyte infiltration, and upregulation of inflammatory mediators. Boldine is an alkaloid compound found in Boldo tree, with multiple pharmacological actions, mainly anti-inflammatory, antioxidant, antitumor, and immunomodulatory activities. Hence, the effect of boldine for its anti-inflammatory properties against dextran sulfate sodium (DSS)-induced UC in BALB/c mice was studied. Administration of boldine to DSS-induced mice protects colon damage by reduced disease activity index, spleen weight, and increased colon length. Also administration of boldine showed a reduction in the activity of myeloperoxidase (MPO) and CD 68+ expression. Boldine reduced the colon damage, with significant reductions in both the extent and the severity of the inflammation as well as in crypt damage and leukocyte infiltration in the mucosa. Analysis in vivo showed clear decrease in the production of tumor necrosis factor (TNF)-α, Interleukin (IL)-6, IL-17, and signal transducer and activator of transcription-(p-STAT3)(Y705) with nuclear factor (p65-NF-κB) production being reduced significantly. Moreover, p65-NF-κB activation was reduced in mouse macrophage RAW 264.7 cells in vitro. The data demonstrated that boldine may be beneficial in colitis through selective immunomodulatory effects, which may be mediated, at least in part, by inhibition of p65-NF-κB and STAT3 signaling pathways. © 2016 BioFactors, 42(3):247-258, 2016.
Assuntos
Aporfinas/administração & dosagem , Colite/tratamento farmacológico , NF-kappa B/genética , Fator de Transcrição STAT3/biossíntese , Fator de Transcrição RelA/biossíntese , Animais , Anti-Inflamatórios/administração & dosagem , Antioxidantes/administração & dosagem , Colite/induzido quimicamente , Colite/genética , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/administração & dosagem , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Camundongos , NF-kappa B/biossíntese , Células RAW 264.7 , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/antagonistas & inibidoresRESUMO
Gallic acid (GA) is a polyhydroxy phenolic compound that has been detected in various natural products, such as green tea, strawberries, grapes, bananas, and many other fruits. In inflammatory bowel disease, inflammation is promoted by oxidative stress. GA is a strong antioxidant; thus, we evaluated the cytoprotective and anti-inflammatory role of GA in a dextran sulfate sodium (DSS)-induced mouse colitis model. Experimental acute colitis was induced in male BALB/c mice by administering 2.5% DSS in the drinking water for 7 days. The disease activity index; colon weight/length ratio; histopathological analysis; mRNA expressions of IL-21 and IL-23; and protein expression of nuclear erythroid 2-related factor 2 (Nrf2) were compared between the control and experimental mice. The colonic content of malondialdehyde and the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity were examined as parameters of the redox state. We determined that GA significantly attenuated the disease activity index and colon shortening, and reduced the histopathological evidence of injury. GA also significantly (P<0.05) reduced the expressions of IL-21 and IL-23. Furthermore, GA activates/upregulates the expression of Nrf2 and its downstream targets, including UDP-GT and NQO1, in DSS-induced mice. The findings of this study demonstrate the protective effect of GA on experimental colitis, which is probably due to an antioxidant nature of GA.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Colite/prevenção & controle , Colo/efeitos dos fármacos , Sulfato de Dextrana , Ácido Gálico/farmacologia , Fármacos Gastrointestinais/farmacologia , Animais , Antioxidantes/metabolismo , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Citoproteção , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Masculino , Camundongos Endogâmicos BALB C , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/metabolismo , UDP-Glucuronosiltransferase 1ARESUMO
Inflammatory bowel diseases (IBD) encompass at least two forms of intestinal inflammation: Crohn's disease and ulcerative colitis (UC). Both conditions are chronic and inflammatory disorders in the gastrointestinal tract, with an increasing prevalence being associated with the industrialization of nations and in developing countries. Patients with these disorders are 10 to 20 times more likely to develop cancer of the colon. The aim of this study was to characterize the effects of a naturally occurring polyphenol, gallic acid (GA), in an experimental murine model of UC. A significant blunting of weight loss and clinical symptoms was observed in dextran sodium sulfate (DSS)-exposed, GA-treated mice compared with control mice. This effect was associated with a remarkable amelioration of the disruption of the colonic architecture, a significant reduction in colonic myeloperoxidase (MPO) activity, and a decrease in the expression of inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and pro-inflammatory cytokines. In addition, GA reduced the activation and nuclear accumulation of p-STAT3(Y705), preventing the degradation of the inhibitory protein IκB and inhibiting of the nuclear translocation of p65-NF-κB in colonic mucosa. These findings suggest that GA exerts potentially clinically useful anti-inflammatory effects mediated through the suppression of p65-NF-κB and IL-6/p-STAT3(Y705) activation.
Assuntos
Colite/tratamento farmacológico , Ácido Gálico/administração & dosagem , Imunossupressores/administração & dosagem , Doenças Inflamatórias Intestinais/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Animais , Colite/induzido quimicamente , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/metabolismo , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
BACKGROUND: Natural compounds have been demonstrated to lower breast cancer risk and sensitize tumor cells to anticancer therapies. Recently, we demonstrated that vernodalin (the active constituent of the medicinal herb Centratherum anthelminticum seeds) induces apoptosis in breast cancer cell-lines. The aim of this work was to gain an insight into the underlying anticancer mechanism of vernodalin using in vitro and in vivo model. METHODS: Vernodalin was isolated through the bioassay guided fractionation from the seeds. The protein expression of p-Akt, PI3K, FOXO3a, Bim, p27kip1, cyclinD1, and cyclinE was examined by the Western blot analysis. Immunoprecipitation assays were performed to analyse Akt kinase activity. Small interfering RNA (siRNA) was used to study the role of FOXO3a upregulation and their targets during vernodalin treatment. Immunofluorescence, subcellular localisation of FOXO3a by Western blot was performed to analyse FOXO3a localisation in nucleus of breast cancer cells. Immunohistochemical analysis of PCNA, Ki67, p27kip1, FOXO3a and p-FOXO3a in the LA7-induced mammary gland tumor model was performed. RESULTS: Our results showed that vernodalin regulates cancer cell apoptosis through activation of FOXO transcription factors and its downstream targets (Bim, p27Kip1, p21Waf1/cip1, cyclin D1, cyclin E) as examined by Western blots. Furthermore, we showed that FOXO3a/PI3K-Akt played a significant role in vernodalin induced apoptosis in breast cancer cells. Immunoprecipitation assays showed Akt kinase activity was downregulated. Immunofluorescence, subcellular fractionation and Western blot showed FOXO3a accumulation in the nucleus of breast cancer cells after vernodalin treatment. Silencing of FOXO3a protected breast cancer cells against vernodalin induced apoptosis. The anti-tumor action of vernodalin was further confirmed by examining cell proliferative markers, PCNA and Ki67 in the LA7-induced mammary gland tumor model. We also corroborated our findings in vivo by showing upregulation of p27Kip1, FOXO3a and decrease in the p-FOXO3a level in vernodalin-treated breast tumor tissue. CONCLUSIONS: Our results suggest that PI3K-Akt/FOXOa pathway is a critical mediator of vernodalin-induced cytotoxicity and this compound could be further developed as a potential chemopreventive or chemotherapeutic agent for breast cancer therapy.