Spirulina platensis aqueous extracts ameliorate colonic mucosal damage and modulate gut microbiota disorder in mice with ulcerative colitis by inhibiting inflammation and oxidative stress / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Ulcerative colitis (UC) is a chronic and recurrent inflammatory bowel disease (IBD) that has become a major gastroenterologic problem during recent decades. Numerous complicating factors are involved in UC development such as oxidative stress, inflammation, and microbiota disorder. These factors exacerbate damage to the intestinal mucosal barrier. Spirulina platensis is a commercial alga with various biological activity that is widely used as a functional ingredient in food and beverage products. However, there have been few studies on the treatment of UC using S. platensis aqueous extracts (SP), and the underlying mechanism of action of SP against UC has not yet been elucidated. Herein, we aimed to investigate the modulatory effect of SP on microbiota disorders in UC mice and clarify the underlying mechanisms by which SP alleviates damage to the intestinal mucosal barrier. Dextran sulfate sodium (DSS) was used to establish a normal human colonic epithelial cell (NCM460) injury model and UC animal model. The mitochondrial membrane potential assay 3-‍‍(4,5-dimethylthiazol-2-yl)-2,‍5-diphenyltetrazolium bromide (MTT) and staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) and Hoechst 33258 were carried out to determine the effects of SP on the NCM460 cell injury model. Moreover, hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR), western blot, and 16S ribosomal DNA (rDNA) sequencing were used to explore the effects and underlying mechanisms of action of SP on UC in C57BL/6 mice. In vitro studies showed that SP alleviated DSS-induced NCM460 cell injury. SP also significantly reduced the excessive generation of intracellular reactive oxygen species (ROS) and prevented mitochondrial membrane potential reduction after DSS challenge. In vivo studies indicated that SP administration could alleviate the severity of DSS-induced colonic mucosal damage compared with the control group. Inhibition of inflammation and oxidative stress was associated with increases in the activity of antioxidant enzymes and the expression of tight junction proteins (TJs) post-SP treatment. SP improved gut microbiota disorder mainly by increasing antioxidant enzyme activity and the expression of TJs in the colon. Our findings demonstrate that the protective effect of SP against UC is based on its inhibition of pro-inflammatory cytokine overproduction, inhibition of DSS-induced ROS production, and enhanced expression of antioxidant enzymes and TJs in the colonic mucosal barrier.

Enema de sulcralfato pode prevenir colite em segmentos colônicos sem trânsito fecal? / Can sucralfate enema prevent colitis in colonic segments without fecal transit?

RESUMO - RACIONAL: O estresse oxidativo é um dos principais mecanismos associados à ruptura dos mecanismos de defesa que formam a barreira epitelial cólica e reduz o conteúdo tecidual das proteínas claudina-3 e ocludina principais constituintes das junções de oclusão intercelulares. O sucralfato, possui atividade antioxidante e tem sido usado para tratar diferentes formas de colite. OBJETIVO: Mensurar o conteúdo tecidual de claudina-3 e ocludina da mucosa do cólon sem trânsito fecal, submetido à intervenção com sucralfato. MÉTODO: Trinta e seis ratos foram submetidos à colostomia do cólon esquerdo e fístula mucosa distal. Os animais foram divididos em dois grupos de acordo com a eutanásia ser realizada duas ou quatro semanas após a intervenção. Cada grupo foi dividido em três subgrupos de acordo com o tipo de intervenção realizada diariamente: solução salina isolada; sucralfato a 1 g/kg/dia ou sucralfato a 2g/kg/dia. A colite foi diagnosticada por análise histológica adotando escala de validação prévia. A expressão tecidual de ambas as proteínas foi identificada por imunoistoquímica. O conteúdo das proteínas foi quantificado por análise de imagem assistida por computador. RESULTADOS: O escore inflamatório foi maior nos segmentos cólicos sem trânsito fecal e os enemas com sucralfato reduziram o escore inflamatório nesses segmentos, principalmente nos animais submetidos à intervenção com sucralfato em maior concentração e por período mais longo de intervenção. Houve aumento no conteúdo tecidual das proteínas claudina-3 e ocludina, relacionado com a concentração de sucralfato. O conteúdo tecidual de ambas as proteínas não se modificou com a duração da intervenção. CONCLUSÃO: Enemas com sucralfato reduzem a inflamação e aumentam o conteúdo tecidual de claudina-3 e ocludina na mucosa cólica sem trânsito intestinal.

ABSTRACT - BACKGROUND: Oxidative stress is one of the main mechanisms associated with the rupture of the defense mechanisms of the colonic epithelial barrier; it reduces the tissue content of the claudin-3 and occludin proteins, which are the main constituents of intercellular tight junctions. Sucralfate (SCF) has antioxidant activity and has been used to treat different forms of colitis. AIM: This study aimed to measure the tissue claudin-3 and occludin content of the colon mucosa without fecal transit, subjected to intervention with SCF. METHODS: Thirty-six rats were subjected to left colon colostomy and distal mucous fistula. They were divided into two groups according to euthanasia that was performed 2 or 4 weeks after the intervention. Each group was divided into three subgroups according to the enema applied daily: saline alone, SCF at 1 g/kg/day, or SCF at 2 g/kg/day. Colitis was diagnosed by the histological analysis adopting the previous validate scale. The tissue expression of both proteins was identified by immunohistochemical technique. The content of proteins was quantified by computer-assisted image analysis. RESULTS: The inflammatory score was high in colonic segments without fecal transit, and enemas with SCF reduced the inflammatory score in these segments, mainly in those animals submitted to intervention with SCF in greater concentration and for a longer period of intervention. There was an increase in tissue content of claudin-3 and occludin, related to SCF concentration. The tissue content of both proteins was not related to the intervention time. CONCLUSION: Enemas with SCF reduced the inflammation and increased the tissue content of claudin-3 and occludin in colonic mucosa without fecal stream.

Enemas with mesalazine increase the tissue contents of mucins in the colonic mucosa devoid of fecal stream

Abstract Purpose: To evaluate the inflammatory reaction and measure the content of mucins, in the colonic mucosa without fecal stream submit to intervention with mesalazine. Methods: Twenty-four rats were submitted to a left colostomy and a distal mucous fistula and divided into two groups according to euthanasia to be performed two or four weeks. Each group was divided into two subgroups according daily application of enemas containing saline or mesalazine at 1.0 g/kg/day. Colitis was diagnosed by histological analysis and the inflammatory reaction by validated score. Acidic mucins and neutral mucins were determined with the alcian-blue and periodic acid of Schiff techniques, respectively. Sulfomucin and sialomucin were identified by high iron diamine-alcian blue technique. The tissue contents of mucins were quantified by computer-assisted image analysis. Mann-Whitney test was used to analyze the results establishing the level of significance of 5%. Results: Enemas with mesalazine in colonic segments without fecal stream decreased the inflammation score and increased the tissue content of all subtypes of mucins. The increase of tissue content of neutral, acid and sulfomucin was related to the time of intervention. Conclusion: Mesalazine enemas reduce the inflammatory process and preserve the content of mucins in colonic mucosa devoid of fecal stream.

Eficacia del tinidazol en la profilaxis terapéutica de la colitis amebiana en pacientes con leucemia aguda de novo que reciben quimioterapia intensiva / Efficacy of tinidazole in the therapeutic prophylaxis of amoebic colitis in patients with de novo acute leukemia receiving intensive chemotherapy

Resumen Introducción: En México la seroprevalencia de la Entamoeba histolytica es del 8.4%. La amebiasis intestinal en pacientes con leucemia aguda de novo posterior al inicio de quimioterapia (QT), en el Servicio de Hematología del CMN 20 de Noviembre, es del 12%, aún si muestran test coprológico negativo basal. Objetivo: Averiguar si la administración de tinidazol, en pacientes con leucemia aguda y coprológico negativo, al principio de la QT, disminuye la incidencia de colitis amebiana durante la inducción a la remisión. Método: Prospectivo y no comparativo. Enfermos con diagnóstico de leucemia aguda de novo que inician QT de inducción y coprológico inicial. Se indicó tinidazol, 2 g/día durante 5 días en la primera semana de comenzada QT. Se vigilaron hasta que la inducción concluyó y se inició la recuperación hematopoyética. Resultados: 38 pacientes, 15 mujeres y 23 hombres con edad media de 44 años (16-72). Con leucemia aguda linfoblástica 19, con mieloblástica 16 y con promielocítica 3. Casos sin y con amebiasis intestinal, 35 y 3, respectivamente. Los pacientes con amebiasis solo recibieron tinidazol durante 3 días y se dio después de 2 días de empezada la QT. Conclusión: El tinidazol, en pacientes con leucemia aguda de novo que inician QT de inducción, es efectivo en la prevención de la amebiasis intestinal, durante la etapa de inducción, si se administra a 2 g/día, durante cinco días, a partir del día 1 de la QT.

Abstract Introduction: In Mexico, seroprevalence of Entamoeba histolytica is 8.4%. The intestinal amebiasis in patients with acute leukemia of novo, after the start of chemotherapy (CT) in the Hematology Service of the CMN 20 de Noviembre is 12%, even if patients show a negative baseline coprological test. Objective: To find out if the administration of tinidazole, in patients with acute leukemia and negative coprological test, at the beginning of the CT, decreases the incidence of amoebic colitis during the induction to remission. Method: Prospective and not comparative study. Patients with de novo diagnosis of acute leukemia who initiate induction and initial coprological CT. Tinidazole was indicated, 2 g/day for 5 days in the first week of CT started. They were monitored until the induction was concluded and hematopoietic recovery started. Results: 38 patients, 15 women and 23 men with a mean age of 44 years (16-72), with acute lymphoblastic leukemia 19, myeloblastic 16 and promyelocytic 3. Cases without and with intestinal amebiasis were 35 and 3, respectively. Patients with amebiasis only received tinidazole for 3 days and it was given 2 days after the CT started. Conclusion: Tinidazole, in patients with acute de novo leukemia who initiate induction CT, is effective in the prevention of intestinal amebiasis, during the induction stage, if administered at 2 g/day, for five days, starting on day 1 of the CT.

L-carnitine has a protective effect on the colonic mucosa during abdominopelvic radiotherapy in rats

ABSTRACT PURPOSE: To evaluate histopathologically the radioprotective effect of L-carnitine on the colonic mucosa in rats undergoing abdominopelvic irradiation. METHODS: Thirty-two rats were randomly assigned to four experimental groups: intraperitoneal administration of normal saline (group 1) or L-carnitine (300 mL/kg; group 2), followed in groups 3 and 4, respectively, by one dose of abdominopelvic radiation (20 Gy) 30 min later. Rats were sacrificed 5 days after radiation, and their descending colons were resected for histopathological evaluation of the presence and severity of damage. RESULTS: Average damage scores did not differ significantly between groups 1 and 2 (0.13 ± 0.35 and 0.25 ± 0.46, respectively); the group 3 score was highest (10.25 ± 0.71), and the group 4 score (3.63 ± 1.41) was significantly lower than that of group 3 (both p = 0.0001). Pre-radiation L-carnitine administration significantly reduced mucosal thinning, crypt distortion, reactive atypia, inflammation, cryptitis, and reactive lymph-node hyperplasia (all p < 0.01). CONCLUSIONS: L-carnitine had a radioprotective effect on rat colonic mucosa. L-carnitine use should be explored for patients with gastrointestinal cancer, who have reduced serum L-carnitine levels.

The effect of progesterone in the prevention of the chemically induced experimental colitis in rats / Efeito da progesterona na prevenção de colite experimental induzida quimicamente em ratos

PURPOSE: To study the effects of progesterone on an experimental colitis model. METHODS: Wistar albino rats were treated subcutaneously with 2mg/kg once a day during seven days Colitis was induced by intrarectal administration of 5mg trinitrobenzene sulfonic acid (TNBS). Disease activities, macroscopic and microscopic scores were evaluated. To determine the response provoked by progesterone we measured Colonic malondialdehyde (MDA), TNF alfa, IL-6 and Nitric oxide (NO) levels in addition to the MPO (Myeloperoxidase) and caspase-3 activities. RESULTS: Progesterone ameliorated significantly the macroscopic and microscopic scores. TNBS-induced colitis significantly increased the colonic MDA levels and caspase-3 activities in group 2 in comparison to the control group. The results of the study revealed a decline in MDA, NO, IL6 and TNF-α levels in the colon tissue and in blood due to progesterone therapy in group 3 when compared to the group 2, a significant improvement. Progesterone treatment was associated with decreased MDA, MPO, TNF alfa and caspase-3 activity. CONCLUSION: Progesterone therapy decreased oxidative damage in the colonic mucosa.

OBJETIVO: Investigar os efeitos da progesterona em um modelo de colite experimental. MÉTODOS: Ratos albinos Wistar foram tratados subcutaneamente com 2mg/kg por dia durante sete dias. A colite foi induzida por administração intrarretal de 5mg ácido sulfônico trinitrobenzeno (TNBS). Foram avaliadas as atividades da doença, escores macroscópicos e microscópicos Para determinar a resposta provocada pela progesterona foi medida no cólon os níveis de malondialdeído (MDA), TNF alfa, IL-6 e óxido nítrico (NO), além da atividade da MPO (Myeloperoxidase) e caspase-3. RESULTADOS: A progesterone melhorou significantemente os escores macroscópicos e microscópicos. A colite induzida pelo TNBS significantemente aumentou os níveis colônicos de MDA e a atividade da caspase-3 no grupo 2 em comparação com o grupo controle. Os resultados do estudo revelaram um declínio nos níveis de MDA, NO, IL6 e TNF-α no tecido colônico e no sangue devido à terapia com a progesterona no grupo 3 quando comparado ao grupo 2. O tratamento com a progesterona foi associado com decréscimo do MDA, MPO, TNF alfa e atividade da caspase-3. CONCLUSÃO: A terapia com progesterona decresce o dano oxidativo na mucosa do cólon.

Reduction in oxidative stress levels in the colonic mucosa without fecal stream after the application of enemas containing aqueous Ilex paraguariensis extract / Redução dos níveis de estresse oxidativo na mucosa cólica sem trânsito intestinal após aplicação de enemas contendo extrato aquoso de Ilex paraguariensis

PURPOSE: To evaluate the antioxidant effects of enemas containing aqueous extract of Ilex paraguariensis, comparing segments with and without fecal stream and correlating the segments with the duration of intervention. METHODS: Twenty-six Wistar rats were subjected to a diversion of the fecal stream in the left colon by a proximal colostomy and distal mucosal fistula. The rats were distributed randomly into two experimental groups of 13 animals each based on the time of sacrifice after surgical procedure (two or four weeks). Each group was then divided into two experimental subgroups that received either second daily enemas containing 0.9 percent saline solution or aqueous extract of Ilex paraguariensis at 0.2g/100g. Colitis was diagnosed by histopathological analysis and the detection of oxidative tissue damage by measuring the levels of malondialdehyde. The Mann-Whitney test was used to compare the tissue levels of malondialdehyde between colon segments with and without fecal stream in each experimental group, and the Kruskal-Wallis test was used to verify the variance between the levels of oxidative stress according the duration of the irrigation; both tests determined significance at 5 percent (p<0.05). RESULTS: The levels of malondialdehyde in the animals subjected to intervention in the colon with saline with and without fecal stream after two and four weeks of irrigation were 0.05±0.006 and 0.06±0.006, and 0.05± 0.03 and 0.08 ±0.02, respectively. The malondialdehyde levels in the animals irrigated with Ilex paraguariensis with and without fecal stream after two and four weeks of irrigation were 0.010±0.002 and 0.02±0.004, and 0.03±0.007 and 0.04±0.01, respectively. After two and four weeks of intervention, the levels of malondialdehyde were lower in the animals irrigated with Ilex paraguariensis regardless of the time of irrigation (p=0.0001 and p=0.002, respectively). CONCLUSION: The daily rectal application of enemas containing aqueous extract of Ilex paraguariensis decreases oxidative tissue damage in the colon without fecal stream regardless of the time of irrigation.

OBJETIVO: Avaliar os efeitos antioxidantes de clisteres contendo extrato aquoso de Ilex paraguariensis comparando segmentos com e sem trânsito fecal e correlacionando com o tempo de intervenção. MÉTODOS: Vinte e seis ratos Wistar machos foram submetidos à derivação do trânsito no cólon esquerdo pela confecção de colostomia proximal e fístula mucosa distal. Foram divididos de forma randomizada em dois grupos experimentais de 13 animais, de acordo com o sacrifício ser realizado após duas ou quatro semanas do procedimento cirúrgico. Cada grupo foi dividido em dois subgrupos experimentais segundo a aplicação diária ter sido realizada com enemas diários contendo solução fisiológica a 0,9 por cento ou extrato aquoso de Ilex paraguariensis na concentração de 0,2 g/100 ml. O diagnóstico de colite foi estabelecido por análise histológica e os níveis de estresse oxidativo pela dosagem tecidual de malondialdeído Para comparar os níveis de malondialdeído entre os cólons com e sem trânsito fecal em cada grupo experimental adotou-se o teste de Mann-Withney, e o teste de Kruskal-Wallis para verificar a variação dos níveis de estresse oxidativo segundo o tempo de irrigação, estabelecendo-se para ambos o nível de significância de 5 por cento (p<0,05). RESULTADOS: Os níveis de malondialdeído nos animais submetidos à intervenção com soro fisiológico nos cólons providos e desprovidos de trânsito fecal após duas e quatro semanas de irrigação foram de 0,05±0,006, 0,06±0,006 e 0,05±0,03, 0,08±0,02 respectivamente. Os níveis de malondialdeído nos animais irrigados com Ilex paraguariensis, nos cólons providos e desprovidos de trânsito, após duas e quatro semanas de irrigação foram de 0,010±0,002, 0,02±0,004 e 0,03±0,007, 0,04±0,01, respectivamente. Após duas e quatro semanas de intervenção os níveis de malondialdeído foram menores nos animais irrigados com Ilex paraguariensis, independente do tempo de irrigação (p=0,0001 e p=0,002, respectivamente). CONCLUSÃO: A aplicação diária de extrato aquoso de Ilex paraguariensis reduz os níveis de dano oxidativo tecidual nos segmentos cólicos sem trânsito fecal, independente do tempo de irrigação.

Comparison of prophylactic and therapeutic use of short-chain fatty acid enemas in diversion colitis: a study in Wistar rats

OBJECTIVES: To study the effect of short-chain fatty-acids on atrophy and inflammation of excluded colonic segments before and after the development of diversion colitis. INTRODUCTION: Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, possibly evolving with mucous and blood discharge. The most favored hypotheses to explain its development is short-chain fatty-acid deficiency in the colon lumen. METHODS: Wistar rats were submitted to colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after post-operative day 40). The mucosal thickness of the excluded colon was measured histologically. The inflammatory reaction of the mucosal lamina propria and the lymphoid tissue response were quantified through established scores. RESULTS: There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 showed no mucosal thickness recovery and significant increases in the numbers of lymphocytes (p = 0.0006) and eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). CONCLUSION: Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy.

Prophylactic Effect of Lactobacillus GG in Animal Colitis and Its Effect on Cytokine Secretion and Mucin Gene Expressions / 대한소화기학회지

BACKGROUND/AIMS: Lactobacillus rhamnosus GG (LGG) has been used in acute colitis treatment. However, it is unclear whether the LGG prevents chronic colitis. The aim of this study was to examine the prophylactic effect of LGG on animal colitis, cytokine secretion, and mucin gene expression. METHODS: BALB/c mice (n=64) were exposed to 5% dextran sulfate sodium (DSS) for 7 days followed by 10 days recovery period and repeatedly exposed for 4 days. Then, the mice were devided into three group; group of oral LGG adminstration throughout the recovery and repeated colitis period; PBS group of PBS administration; control group. Colon length, histologic score, tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10) levels, mucin gene expressions were determined at each period. RESULTS: In acute colitis period, the LGG group showed higher levels of disease activity index (DAI), histologic score, TNF-alpha, IL-10, but shorter colon length, lower levels of mucin gene expressions than the control group. However, in repeated colitis period, the LGG group showed markedly lower levels of DAI and IL-10 but significantly longer colon length than PBS group (p<0.05). There was no difference in the mucin gene expression. CONCLUSIONS: These results suggest that LGG prevents chronic murine colitis. It may be associated with cytokine modulation and competitive inhibition of pathogenic bacteria. However, it may not be related with gene expression.