Evaluation of the anti-inflammatory activity of the alkaloid isatin in rats submitted to ulcerative colitis model induced by trinitrobenzene sulfonic acid

Orientadores: Alba Regina Monteiro Souza Brito, Anderson Luiz FerreiraTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: Retocolite ulcerativa inespecífica (RCUI) e doença de Crohn (DC) são as principais doenças inflamatórias intestinais (DII). São caracterizadas por inflamação crônica da mucosa do trato gastrintestinal, resultando em diarreia, fezes sanguinolentas, dores abdominais, anemia, febre, fadiga e perda de peso, tanto em homens quanto em mulheres. Acredita-se que tais manifestações sejam resultado de uma interação entre indivíduos geneticamente susceptíveis, fatores ambientais específicos, desbalanço na microflora intestinal e uma resposta imunológica exacerbada. Atualmente, as terapias vigentes visam atingir um estado remissivo, reduzindo a intensidade do processo inflamatório, bem como seus sintomas. Contudo, estas terapias apresentam custo elevado e uma série de efeitos adversos importantes. Neste contexto, pesquisas com produtos naturais mostram-se como alternativas promissoras no tratamento de DII. A isatina é um alcaloide indólico com atividades biológicas e farmacológicas já testadas que incluem atividades antimicrobiana, antitumoral, antioxidante e anti-inflamatória. Em vista disso, este trabalho avaliou os efeitos de isatina em dois protocolos de modelo de colite aguda induzida pelo ácido 2,4,6-trinitrobenzeno sulfônico (TNBS). No primeiro protocolo, os animais foram divididos nos seguintes grupos: não-colítico, colítico não-tratado e colíticos tratados com isatina (3, 6, 12.5, 18.75 e 25 mg/Kg) 72, 48, 24 e 2h antes do desafio pelo TNBS. A eutanásia dos animais ocorreu 96h após o desafio com TNBS, seguido de remoção do cólon para realização das análises macroscópicas e bioquímicas. Os resultados demonstraram que as doses de 6 e 25 mg/Kg foram eficazes em proteger a mucosa cólica contra o aumento de TNF-?, IFN-?, PGE2 e COX-2, bem como contra a redução dos níveis de IL-10. Estas doses foram efetivas, ainda, em prevenir a queda significativa dos níveis de GSH, bem como contra o aumento da atividade da GSH-Px. A dose de 6 mg/Kg também foi efetiva ao evitar a redução da atividade da SOD e o aumento da atividade da GSH-Gr. Em vista destes resultados, a dose de 6 mg/Kg foi novamente testada em um segundo protocolo experimental. Neste, os animais foram desafiados e em seguida tratados uma única vez com a dose de 6 mg/Kg. Os animais foram eutanaziados três e sete dias após o desafio. Os resultados macroscópicos revelaram que a dose única do alcaloide reduziu de forma significativa os parâmetros macroscópicos da lesão. As análises bioquímicas revelaram que o alcaloide foi eficaz na redução do processo inflamatório a partir do terceiro dia, sendo este efeito prolongado até o sétimo dia. A Isatina foi eficaz em aumentar os níveis de GSH e aumentar a atividade da GSH-Px, além de reduzir a atividade da MPO. Além disso, o alcaloide foi efetivo na redução dos níveis de TNF-?, IFN-?, IL-1?, IL-12, PGE2 e aumento dos níveis de IL-10. Os resultados obtidos em ambos os protocolos experimentais revelaram que a isatina, alcaloide indólico encontrado em certas plantas e em tecidos de mamíferos, apresenta uma atividade protetora significativa contra danos na mucosa intestinal. Este efeito dá-se, possivelmente, devido à uma combinação entre suas propriedades antioxidantes e anti-inflamatórias. No entanto, ainda são necessárias maiores investigações no intuito de se estabelecer todos os mecanismos envolvendo a isatina e seus efeitos farmacológicosAbstract: Ulcerative colitis (UC) and Crohn's disease (CD) are the major forms of inflammatory bowel diseases (IBD). They are characterized by chronic mucosal inflammation of gastrointestinal tract, which results in diarrhea, blood stools, abdominal pain, anemia, fever, fatigue and weight loss, both in man and in woman. It is believed that such manifestations are the result of an interaction between genetically susceptible individuals, specific environmental conditions, imbalance in the intestinal microflora and imbalance of the immune response. Currently, therapies aim to achieve a remissive state, reducing the intensity of the inflammatory process, as well as its symptoms. However, these therapies have high cost and present many significant adverse effects. In this context, research on natural products appear as promising alternative in the treatment of IBD. Isatin is an indole alkaloid with biological and pharmacological activities already tested, such as antimicrobial, anti-tumor, antioxidant and anti-inflammatory activity. In view of this, this study evaluated the effects of isatin in two acute colitis model protocols induced by 2,4,6-trinitrobenzene sulfonic acid. In the first protocol, the animals were divided into seven groups: non-colitic, untreated colitic and five treated colitic groups. Doses of 3, 6, 12.5, 18.75 and 25 mg/Kg of isatin were tested, and the animals were pre-treated on times 72, 48, 24 and 2h prior the challenge. The euthanasia occurred 96h after challenge with TNBS, followed by colon removal for macroscopic and biochemical analyses. The results showed that both 6 and 25 mg/Kg doses were effective in protecting the colonic mucosa against the increase of TNF-?, IFN-?, PGE2 and COX-2, as well as over IL-10 content reduction. These doses were also effective in preventing a significant reduction of GSH levels and the increase of GSH-PX activity. The dose of 6 mg/Kg was effective also in prevent the reduction of SOD activity and the increase of GSH-Gr activity. In view of these results, 6 mg/Kg dose was again tested in a second experimental protocol. In this, the animals were challenged and then treated once with 6 mg/Kg dose. The animals were sacrificed three and seven days after challenge. The macroscopic results shows that a single dose of de alkaloid significantly reduced the macroscopic lesion. Biochemical analyses revealed that the alkaloid was effective in reducing the inflammation from the third day after induction, which sustained until the seventh day. Isatin was effective by increasing the GSH content and the GSH-PX activity, besides reducing MPO activity. The alkaloid was effective also in reduce the levels of TNF-?, IFN-?, IL-1?, IL-12, PGE2 and increase IL-10 content. The results obtained in both experimental protocols showed that isatin, an indole alkaloid found in some plants and mammalian tissues, presents a significant protective effect against damage to the intestinal mucosa. This effect is possible due to a combination of its antioxidant and anti-inflammatory properties. However, it is still needed further investigation in order to establish all mechanisms involving the isatin and its pharmacological effectsDoutoradoFarmacologiaDoutor em FarmacologiaCAPE

Royal Jelly And Its Dual Role In Tnbs Colitis In Mice.

Royal Jelly (RJ) is widely consumed in diets throughout the world due to its beneficial effects: antioxidant, antitumor and anti-inflammatory. We have investigated the role of RJ in the development of TNBS colitis in mice. Colitis was induced by a rectal instillation of TNBS at 0.1 mL per mouse. Intestine samples of the animals orally treated with RJ (100, 150, and 200 mg/kg) were collected for antioxidant assays (GSH and GSH-Px), proinflammatory protein quantification (COX-2 and NF-κB), and histological analyses. RJ 100 mg/kg maintained GSH levels and increased the activity of GSH-Px, downregulated key inflammatory mediators (COX-2 and NF-κB), and decreased the lesions caused by TNBS as shown by the histological analyses. In conclusion, RJ showed anti-inflammatory and antioxidant properties in experimental colitis, resulting in the amelioration of the macroscopic and histological analyses. These results corroborate with the RJ supplementation in diets.201595623

Applications of the hexanic fraction of Agave sisalana Perrine ex Engelm (Asparagaceae): control of inflammation and pain screening

The present study evaluated the anti-inflammatory and analgesic properties of Agave sisalana Perrine in classic models of inflammation and pain. The hexanic fraction of A. sisalana (HFAS) was obtained by acid hydrolysis followed by hexanic reflux. Anti-inflammatory properties were examined in three acute mouse models (xylene ear oedema, hind paw oedema and pleurisy) and a chronic mouse model (granuloma cotton pellet). The antinociceptive potential was evaluated in chemical (acetic-acid) and thermal (tail-flick and hot-plate test) models of pain. When given orally, HFAS (5, 10, 25 and 50 mg/kg) reduced ear oedema (p < 0.0001; 52%, 71%, 62% and 42%, respectively). HFAS also reduced hind paw oedema at doses of 10 mg/kg and 25 mg/kg (p < 0.05; 42% and 58%, respectively) and pleurisy at doses of 10 mg/kg and 25 mg/kg (41% and 50%, respectively). In a chronic model, HFAS reduced inflammation by 46% and 58% at doses of 10 mg/kg and 25 mg/kg, respectively. Moreover, this fraction showed analgesic properties against the abdominal writhing in an acetic acid model (at doses of 5-25 mg/kg) with inhibitory rates of 24%, 54% and 48%. The HFAS also showed an increased latency time in the hot-plate (23% and 28%) and tail-flick tests (61% and 66%) for the 25 mg/kg and 50 mg/kg doses, respectively. These results suggest that HFAS has anti-inflammatory and analgesic properties.26327

Effects of Sambucus nigra L. monomeric anthocyanins on TNBS induced ulcerative colitis

Orientador: Alba Regina Monteiro Souza BritoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Retocolite ulcerativa idiopática e doença de Crohn são doenças inflamatórias intestinais caracterizadas por inflamação crônica da mucosa, resultando em diarréia, fezes sanguinolentas, dores abdominais, anemia, febre, fadiga e perda de peso, tanto em homens quanto em mulheres. Acredita-se que essas manifestações sejam resultado de uma interação multifatorial envolvendo indivíduos geneticamente susceptíveis, condições ambientais específicas, desbalanço na microflora intestinal e desajuste da resposta imune. Drogas derivadas do acido 5-aminossalicilico (sulfassalazina, mesalamina), corticosteroides e agentes imunomoduladores são utilizadas, em conjunto no tratamento dessas patologias. No entanto tais drogas apresentam efeitos adversos importantes, o que acaba por motivar pesquisas envolvendo produtos naturais como alternativas de tratamento. Neste contexto, este trabalho avaliou os efeitos de antocianinas presentes nos frutos de Sambucus nigra L. (sabugueiro), espécie arbustiva pertencente a família Adoxaceae, em modelo de colite ulcerativa induzida pelo acido 2,4,6-trinitrobenzeno sulfônico (TNBS). Os frutos do sabugueiro apresentam grandes concentrações de metabolitos secundários como antocianinas, alem de outros compostos fenólicos, que acabam por conferir aos frutos propriedades antioxidantes, anti-inflamatorias, imunomoduladoras e laxativas. Neste trabalho, foram avaliadas as propriedades antioxidantes e anti-inflamatorias da fração de antocianinas monoméricas presentes nos frutos de S. nigra. Os resultados comprovaram o efeito antioxidante in vitro das antocianinas, sendo elas eficazes em reduzir tanto o radical 2,2-difenil-1-picrilhidrazil (DPPH), teste que avalia a redução do radical via transferência de elétrons, quanto a taxa de oxidação do radical 2,2 azobis amidinopropano (AAPH), teste que verifica a oxidação do radical via transferência de átomos de hidrogênio. Nos ensaios in vivo concluiu-se que a dose de 5mg.Kg-1 de antocianinas apresentou a melhor resposta em reduzir a lesão causada pelo TNBS, sendo efetiva em manter os níveis de GSH (5,709 ± 0,931) comparado ao grupo TNBS (0,8525 ± 0,298) e ao grupo salina (6,610 ± 3,926). Essas antocianinas foram efetivas ainda em aumentar a atividade da SOD (8,487 ± 2,505), quando comparada com o grupo TNBS (3,884 ± 0,925) e ao grupo salina (12,240 ± 4,199), e reduzir a atividade da MPO (4,519 ± 2,016), quando comparada ao grupo TNBS (7,572 ± 2,572) e ao grupo salina (1,314 ± 0,319). Os resultados obtidos na avaliação da atividade das enzimas GPx (17,03 ± 3,951 no grupo tratado e 22,13 ± 11,510 no grupo TNBS) e GR (0,6524 ± 0,1180 no grupo tratado e 0,7249 ± 0,3968 no grupo TNBS), comparados ao grupo salina (84,22 ± 41,88) e (2,131 ± 0,9858) respectivamente, indicam que houve queda na ativação destas enzimas apos 24h de indução da colite, sendo que esta situação não foi revertida apos administração das antocianinas. Do mesmo modo não foram encontradas alterações nos níveis de LPO (5,756 ± 1,884 no grupo tratado e 5,113 ± 0,8254 no grupo TNBS) comparados ao grupo salina (4,688 ± 1,126) . Já os ensaios anti-inflamatórios revelaram atividade antiinflamatória promissora, visto que as antocianinas foram capazes de manter os níveis de IL-10 (466,7 ± 56,32) próximos aqueles obtidos no grupo não-colitico (492,4 ± 154,5), quando comparados ao grupo TNBS (264,8 ± 66,35), e de reduzir a produção de IL-12 (202,3 ± 53,33) quando comparados com os animais não tratados (319,3 ± 111,5) e ao grupo salina (149,8 ± 51,76). Neste estudo concluiu-se que as antocianinas presentes nos frutos de sabugueiro apresentam atividade antioxidante, alem de aumentar os níveis de IL-10, citocina essa que, provavelmente, participa na redução dos níveis de citocinas pro - inflamatórias como IL-12 e, consequentemente, a expressão de mediadores inflamatóriosAbstract: Ulcerative colitis and Crohn's disease are inflammatory bowel disease characterized by chronic inflammation of the mucosa, resulting in diarrhea, bloody stools, abdominal pain, anemia, fever, fatigue and weight loss in both men and women. It is believed that these manifestations are the result of a multifactorial interaction involving genetically susceptible individuals, environmental conditions, imbalance in intestinal microflora and immune response imbalance. Drugs derived from 5-aminosalicylic acid (sulfasalazine, mesalamine), corticosteroids and immunomodulatory agents are used together to treat these diseases. However, such drugs have significant adverse effects, which ultimately motivate research involving natural products as alternative treatments. In this context, this study evaluated the effects of anthocyanins in the fruits of Sambucus nigra L. (Elderberry), shrub species belonging to the family Adoxaceae, in a model of ulcerative colitis induced by 2,4,6-trinitrobenzenes sulfonic acid (TNBS). The fruits of elderberry have large concentrations of secondary metabolites such as anthocyanins and other phenolic compounds, which ultimately give the fruit antioxidant, anti-inflammatory, immunomodulatory and laxative properties. In this study we investigated the antioxidant and anti-inflammatory properties of the monomeric anthocyanins fraction in the fruits of S. nigra. The results confirmed the in vitro antioxidant effect of anthocyanins, which were effective in reducing both the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test to evaluate the reduction of the radical via electron transfer and the rate of oxidation 2,2 azobis amidinopropane (AAPH), a test that checks radical oxidation via the transfer of hydrogen atoms. In vivo tests concluded that the dose of anthocyanins 5mg.Kg-1 had the best response to reduce the damage caused by TNBS, being effective in maintaining the levels of GSH (5.709 ± 0.931) compared to TNBS group (0, 8525 ± 0.298) and the saline group (6.610 ± 3.926). These anthocyanins were also effective in increasing the activity of SOD (8.487 ± 2.505) compared with the TNBS group (3.884 ± 0.925) and the saline group (12.240 ± 4.199), and reduce the activity of MPO (4.519 ± 2.016) when compared to TNBS group (7.572 ± 2.572) and the saline group (1.314 ± 0.319). The results obtained in the enzymatic activities of GPx (17.03 ± 3.951 in the treated group and 22.13 ± 11.510 in group TNBS) and GR (0.6524 ± 0.1180 in the treated group and 0.7249 ± 0.3968 TNBS group) compared to saline group (84.22 ± 41.88) and (2.131 ± 0.9858) respectively, indicate that there was a decrease in the activation of these enzymes after 24h of colitis induction. Likewise there were no changes in the levels of LPO (5.756 ± 1.884 in the treated group and 5.113 ± 0.8254 in group TNBS) compared to saline group (4.688 ± 1.126). The anti-inflammatory assays have shown promising antiinflammatory activity, whereas anthocyanins were able to maintain levels of IL-10 (466.7 ± 56.32) than those obtained in non-colitis group (492.4 ± 154 , 5), when compared to TNBS group (264.8 ± 66.35), and reduce the production of IL-12 (202.3 ± 53.33) compared with untreated animals (319.3 ± 111 , 5) and the saline group (149.8 ± 51.76). In this study it was concluded that anthocyanins present in elderberry fruits have antioxidant activity, and increased levels of IL-10, this cytokine, which probably participates in reducing levels of proinflammatory cytokines such as IL-12 and consequently the expression of inflammatory mediatorsMestradoFisiologiaMestre em Biologia Funcional e Molecula

Probiotic supplementation attenuates the aggressiveness of chemically induced colorectal tumor in rats

To evaluate the effect of a probiotic on the aggressiveness of a chemically induced colorectal tumor in rats. Twenty-five male Fisher 344 rats, 250 g, provided with feed and water ad libitum, were randomly divided into 5 groups (5 rats/group): GControl, no treatment; GTumor, tumor induction; GTumor+5FU, tumor induction, 5-Fluorouracil applied; GTumor+Prob, induction of the tumor, supplemented with probiotic; GTumor+5-FU+Prob, tumor induction, 5-Fluorouracil applied, supplemented with probiotic. For tumor induction 20 mg/kg of 1,2-dimethylhydrazine was applied intraperitoneally over 4 weeks, followed by an interval of 15 days, and then repeated for a further 4 weeks. Five weeks after the final dose of the carcinogen, treatment was initiated with 5-Fluorouracil (15 mg/kg, intraperitoneally/week) and a commercial probiotic (1 × 109 CFU, daily/gavage). Data were analyzed by One Way Variance Analysis and means compared by Dunnett's test. GraphPad Prism statistical software was used. The histopathological analyzes were evaluated by the chi-square test. A 5% type-I error was considered statistically significant. Compared with the GTumor, the GTumor+Prob (p < 0.0373) and GTumor+5-FU+Prob (p < 0.0003) demonstrated an attenuated effect on the aggressiveness of the colorectal tumor, with a reduction in the count of Aberrant Crypt foci; and a lower percentage of malignant neoplastic lesions in the GTumor+Prob (40% low grade tubular adenoma, 40% carcinoma in situ, 20% low grade adenocarcinoma) and GTumor+5-FU+Prob (40% low grade tubular adenoma and 60% carcinoma in situ). Probiotic supplementation has the potential to decrease the formation of aberrant crypts and ameliorate tumor malignancy, enhancing the antitumor effect of 5-Fluorouracil chemotherapy in colic segments237The authors would like to thank the NANOBIOSS/UNICAMP Laboratories, INOMAT/UNICAMP, the NANOREG-MCTIC Program, and the Surgery Science Program/UNICAMP and University of Western São Paulo for financial support and research viabilit

P-mapa, a promisor immunomodulator against tumor cells of colonic tissues : an investigation of the action mechanism over the TLR4 signaling pathway

Colorectal cancer (CRC) is a multifactorial syndrome that drives to uncontrollable cell division, genetic alterations, and functional alteration. In the present work, we evaluated the immunomodulatory properties of P-mapa, a compound extracted from Aspergillus oryzae fungus, versus Fluorouracil (5-FU) treatment in chemically induced CRC. CRC was induced by DMH in F344 rats. Animals of treated groups receive weekly 15 mg/Kg of 5-FU or 5 mg/Kg of P-mapa, over 10 weeks. Tissues were stained for aberrant crypt foci (ACF) counting and histopathology evaluation, immunostained for TLR4 pathways and quantified for TNF alpha Cytokine assay. DMH was efficient to induce hyperplastic lesions and ACF. Both treatments reduced significantly ACF formation and tumor aggressiveness. Immunohistochemistry for TLR4 signaling reveals that both treatments had no effect over the TLR4-NF kappa B signaling pathway. On the other hand, both succeed in increase interferon signaling, with activation of the TRIF-IRF3 pathway and consequently inducing IFN gamma synthesis. The present results show the immunomodulatory properties of P-mapa in chemically induced CRC model. P-mapa induced a significant increase in Type-I IFNs synthesis and subsequently immune cell recruitment, resulting in an increase of IFN gamma concentration in colorectal mucosa and its inhibitory effects over tumoral growth. In this scenario, P-mapa showed an interesting antitumoral effect by inhibiting tumor growth242CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES380026/2017-1; 381017/2017-688887.161316/2017-0

Chemical composition of Rosmarinus officinalis essential oil and antioxidant action against gastric damage induced by absolute ethanol in the rat

Objective: To evaluate the antioxidant activity of the essential oil obtained from Rosmarinus officinalis (R. officinalis) in ethanol-induced gastric ulcer model in vivo. Methods: The antioxidant properties of the essential oil obtained from R. officinalis were evaluated against gastric injury induced by absolute ethanol. Gastric tissues were prepared to enzymatic assays. The levels of glutathione, lipid peroxides, and the activities of glutathione peroxidase, superoxide dismutase and myeloperoxidase were measured. Results: Ethanol produced severe hemorrhagic lesions in the stomach with ulcerative lesion of (140.2 ± 37.2) mm2. In animals pretreated with essential oil of R. officinalis (50 mg/kg, p.o.), a significant inhibition of mucosal injury of (21.2 ± 7.1) mm2 (84% inhibition) was observed. The essential oil of R. officinalis protected the gastric mucosa probably by modulating the activities of the enzymes (superoxide dismutase and glutathione peroxidase) and increasing or maintaining the levels of glutathione. In addition, lipid peroxides levels were reduced. The essential oil of R. officinalis was analyzed by gas chromatography–mass spectrometer and the main constituents were cineole (28.5%), camphor (27.7%) and alpha-pinene (21.3%). Conclusions: We suggest that the monoterpenes present in the essential oil obtained from R. officinalis may be among the active principles responsible for the antioxidant activity shown by essential oil of R. officinalis

Applications of the hexanic fraction of Agave sisalana Perrine ex Engelm (Asparagaceae): control of inflammation and pain screening

The present study evaluated the anti-inflammatory and analgesic properties of Agave sisalana Perrine in classic models of inflammation and pain. The hexanic fraction of A. sisalana (HFAS) was obtained by acid hydrolysis followed by hexanic reflux. Anti-inflammatory properties were examined in three acute mouse models (xylene ear oedema, hind paw oedema and pleurisy) and a chronic mouse model (granuloma cotton pellet). The antinociceptive potential was evaluated in chemical (acetic-acid) and thermal (tail-flick and hot-plate test) models of pain. When given orally, HFAS (5, 10, 25 and 50 mg/kg) reduced ear oedema (p < 0.0001; 52%, 71%, 62% and 42%, respectively). HFAS also reduced hind paw oedema at doses of 10 mg/kg and 25 mg/kg (p < 0.05; 42% and 58%, respectively) and pleurisy at doses of 10 mg/kg and 25 mg/kg (41% and 50%, respectively). In a chronic model, HFAS reduced inflammation by 46% and 58% at doses of 10 mg/kg and 25 mg/kg, respectively. Moreover, this fraction showed analgesic properties against the abdominal writhing in an acetic acid model (at doses of 5-25 mg/kg) with inhibitory rates of 24%, 54% and 48%. The HFAS also showed an increased latency time in the hot-plate (23% and 28%) and tail-flick tests (61% and 66%) for the 25 mg/kg and 50 mg/kg doses, respectively. These results suggest that HFAS has anti-inflammatory and analgesic properties