Jornal de Distúrbios Nutricionais e Terapia

Abstrato

Nutrição Clínica 2019: Os efeitos do chá de kombucha na integridade intestinal em camundongos - Elaheh Mahmoudi - Universidade de Ciências Médicas de Alborz

Elaheh Mahmoudi

Objetivo(s): O estresse oxidativo está implicado na patogênese da hiperpisia, um fator de risco para morbidade e mortalidade cardiovascular. Vários estudos em humanos mostraram que os carotenoides do tomate podem afetar vários aspectos da saúde humana. Durante esta apresentação, o autor abordará duas questões: a) equilibrar a resposta das células da pele à irradiação UV e b) a redução do sinal vital elevado. a) Vários estudos em humanos mostraram que os carotenoides do tomate podem reduzir os danos induzidos por UV, reduzindo o eritema e melhorando o equilíbrio entre a produção e a degradação do colágeno. Nós hipotetizamos que uma mistura de carotenoides do tomate junto com polifenóis pode produzir melhor proteção da pele do que a esperada da soma de sua atividade. Verdadeiramente, entendemos que misturas de complexo de nutrientes do tomate (contendo licopeno) com extrato de alecrim (contendo o ácido carnósico polifenólico) reduziram sinergicamente os marcadores inflamatórios e induziram a atividade antioxidante nas células da pele, principalmente para diminuir a Metaloproteinase da Matriz (MMPs) e, portanto, podem diminuir a degradação do colágeno e retardar o envelhecimento da pele. b) A hiperpiésia pode ser um fator de risco para morbidade e mortalidade cardiovascular. Realizamos uma análise dose-resposta para descobrir a dose efetiva ideal de um suplemento de complexo nutricional de tomate na manutenção de sinais normais entre indivíduos hipertensos. Materiais e métodos:

O intestino tipo peneira foi persuadido em duas coleções de jovens e adultos usando sulfato de sódio de dextrano em bebida por sete dias. Anteriormente, o fKT era administrado aos camundongos pretensiosos por colite e relacionado aos animais normais e não tratados com colite da mesma idade.

Preparação do chá de Kombucha (KT)

Chá preto (Golestan, Teerã, Irã) foi adicionado à água fervente (1,2% p/v), misturado e deixado em infusão por 10 min. O chá foi então filtrado por uma peneira estéril, e sacarose (10%) foi dissolvida dentro do chá. Para organizar o KT, 200 ml do chá resfriado foram inoculados com 3% p/v de fungo de chá mais 10% v/v de líquido KT que foi previamente fermentado. A coleta foi então deixada para fermentar por incubação a 28 °C por 14 dias. Para formar o KT filtrado (fKT), o chá fermentado resultante foi centrifugado a 5000 rpm por 20 min e filtrado empregando um filtro de celulose de 0,45 µm equipado com uma bomba de ar.

Grupos experimentais e desenho do estudo

Male NMR mice were purchased from the Pasteur Institute Experimental Animal Center, Tehran, Iran. All animals were housed for one week before the experiments began in light- and temperature-regulated rooms at the traditional animal department of Alborz University of Medical Sciences.  All experimentations were permitted by the native ethical group (reference No Abzums.Rec.1395.51) and performed consistent with Animal Care and Use Protocol of Alborz University of Medical Sciences.

The animals were divided into two groups of young and old. Each group was then further subdivided into two groups (8 in each) including normal and colitis-induced. because the figure indicates, each of colitis-induced old or young groups was further subdivided into two subgroups, including colitis-induced with no treatment and colitis-induced treated with fKT. The study was performed in three phases. within the initiative , DSS-induced colitis was found out in young (2 months) and old (16 months) mice during a period of 21 days during which weight loss and therefore the clinical score were evaluated and compared with the age-matched healthy animals. within the second phase, the effect of fKT administration on survival analysis and therefore the clinical score were evaluated during a period of 21 days. After completion of phase I clinical trial and II of the study, molecular and histological evaluations were performed on young and old healthy controls, DSS-induced colitis, and DSS-induced colitis treated with fKT animals in phase III clinical trial . Considering the deathrate and clinical signs that occurred within the animals with colitis, animals at this phase of the study were sacrificed on day 14 after the start of the study.

Colitis induction

Colitis was induced on day 0 using administration of drinking water containing 3.5% (w/v) dextran sodium sulfate salt (DSS) (40000 kDa, MP Biomedical, Eschwege, Germany) per mouse per day. The animals and clinical signs of disease were daily monitored, indicated by weight loss, occurrence of blood in the stool or around the rectum, and diarrhea until day seven after the colitis induction. Weight loss was determined by comparing the body weight for each mouse to the baseline body weight and expressed as a percentage of weight loss. Other symptoms were scored according to the previously suggested system by Siegmund et al. Briefly, the different signs for stool consistency were scored as follows: score 0, well-formed pellets; score 2, pasty and semi-formed stools that did not adhere to the anus; score 4, liquid stools that did adhere to the anus. The different signs for bleeding were scored as follows: score 0, no blood measured using the Hemoccult system (Beckman Coulter); score 2, positive Hemoccult; score 4, gross bleeding. Animals with borderline scores were given a one-half score.

Histological and histopathological analysis

To perform histological evaluation of the colon, the animals were sacrificed under ether anesthesia after the last treatment with beverage or fKT. Colon was initially flushed with 1x ice-cold phosphate buffered saline (PBS) to get rid of feces completely. Tissue samples of the colon were then removed, fixed in 10% buffered formalin, and processed for paraffin sectioning. Sections of about 5 μm thickness were taken and stained with Hematoxylin and Eosin (H&E). The stained sections were examined with an Olympus cX41 microscope and photographed using an Olympus D330 camera . Damage score ranged from 0 to 4 scale was judged based on: inflammation represented by number and extent of leukocyte infiltration, epithelial defects represented by the severity of injury to the somatic cell layer, crypt atrophy estimated visually for the percent of atrophy within the crypts, edema, polymorphonuclear cells(PMNs) infiltration, and mucosal disruption.

Immunofluorescence studies of ZO-1 and ZO-2 expression

Sections of 5 µm paraffin-embedded colon tissues were prepared from each sample and then dewaxed, hydrated, and incubated in a protein block solution. Subsequently, the sections were incubated with the primary rabbit monoclonal ZO-1 or ZO-2 antibody (diluted 1:100 in 0.01 mol /L PBS; Zo-1: ab214228, Zo-2: ab2273, UK) followed by incubation with a goat anti-rabbit Alexa flour 488 (ab150077, Abcam, Cambridge shire, UK). The images were captured using a DeltaPix fluorescent microscope (Smorum, Denmark) and evaluated independently by two expert pathologists.

Analysis of gene expression by real-time PCR

Total RNA was extracted from ~50 mg of frozen colon tissue using guanidine/phenol solution (reagent lysis Qiazol-USA) consistent with the manufacturer’s instruction. the standard and quantity of RNA concentrations were monitored employing a NanoDrop 2000c (Eppendorf, Germany). Then, 1 μg RNA was reversely transcribed to DNA using Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit (Munich, Germany), consistent with the manufacturer’s instructions. The relative expression of mRNA for GAPDH, ZO-1, and ZO-2 decided by preparing reaction mixer with PCR Master Mix (2X) (Amplicon-Denmark) and gene-specific primers with diluted cDNA and final volume made up to 10 μl with nuclease-free water. Quantification and analysis were administered in ABI real-time PCR. The sequences of primers, designed by Integrated DNA Technologies, were forward 5′-TGTCCCACTTGAATCCCC-3′ and reverse 5′-TGTTTCCTCCATTGCTGTG-3′ for ZO-1 and forward 5′-CTCCCTCTTCACATCTGCTTC-3′ and reverse R: 5′-CTGTTACTTGCTTTGGTCTGG-3′ for ZO-2. The efficiencies for primers utilized in the study varied between 95% and 105%. Primer pairs were validated to make sure the right size of the PCR product and therefore the absence of primer dimers. The GAPDH gene was chosen as an indoor control against which mRNA expression of the target gene was normalized. The resultant organic phenomenon level was presented as 2-ΔCt, during which ΔCt was the difference between Ct values of the target gene and GAPDH.

Statistical analysis

Statistical analysis was performed using Graph Pad Prism 7.01. Data are presented as means± SD. ANOVA was wont to indicate any significant difference between the groups. Survival rates were illustrated using Kaplan–Meier plots and compared using the log-rank test. Value of P was considered statistically significant when it had been but 0.05.

Results:

Characteristics and clinical course of DSS-induced colitis

A colite induzida por DSS em camundongos é o modelo animal comum para lidar com a patogênese da colite e avaliar abordagens terapêuticas. Este modelo foi descoberto em nosso laboratório e monitorado por um período de 21 dias (Fase I). Para isso, camundongos NMR machos receberam 3,5% de DSS em bebida por sete dias. Os animais foram verificados diariamente durante a quantidade do estudo para taxa de sobrevivência, perda de peso e sinais clínicos de colite, incluindo sangramento e diarreia, e comparados com animais saudáveis ​​da mesma idade. A análise de sobrevivência dos animais jovens tratados com DSS demonstrou que 66% e 33% dos animais estavam vivos nos dias 7 e 14, respectivamente, e todos estavam mortos no dia 2. Apenas no caso dos animais velhos tratados com DSS, a análise de sobrevivência revelou que 66% e 50% dos animais estavam vivos nos dias 7 e 14, respectivamente, e todos estavam mortos no dia 21. Houve uma grande perda de peso dentro dos grupos jovens e velhos tratados com DSS em comparação com os animais saudáveis ​​da mesma idade. Os camundongos jovens tratados com DSS perderam aproximadamente 10% e 46% do seu peso nos dias 7 e 14, respectivamente. Os camundongos velhos tratados com DSS perderam aproximadamente 13,5% e 15% do seu peso nos dias 7 e 14, respectivamente. Quanto aos sinais de distúrbios digestivos, sangramento e diarreia foram observados em camundongos jovens e velhos tratados com DSS no dia 2 e três após a administração de DSS, respectivamente. Esses resultados demonstram que camundongos jovens tratados com DSS apresentam sinais clínicos mais graves e menor taxa de sobrevivência do que camundongos velhos tratados com DSS.

Observações histológicas

A análise histológica de seções de tecido coradas com H&E (Figura 6) de todos os camundongos jovens e velhos desafiados com DSS mostrou aumento da infiltração de PMNs, perda críptica, defeito epitelial, ruptura da mucosa, apoptose, edema e afinamento da mucosa em comparação com os camundongos saudáveis ​​da mesma idade. O tratamento com fKT diminuiu a extensão da lesão, embora não tenha causado uma reversão completa ao estado saudável pelo regime administrado durante este estudo. Notavelmente, camundongos velhos e saudáveis ​​tiveram mais infiltração de PMNs, perda críptica e edema do que os animais jovens e saudáveis. Conforme ilustrado na Figura 6c, a espessura da mucosa foi diminuída pela administração de DSS porque a pontuação clínica aumentou tanto em jovens quanto em velhos com colite induzida por DSS. ^{Este trabalho foi} parcialmente apresentado na 24ª  Conferência Internacional sobre Nutrição Clínica , de 4 a 6 de março de 2019, realizada em Barcelona, ​​Espanha.