Effect of diacerein and vitamin E on dystrophic fibers of mdx mice

Orientador: Elaine MinatelTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A distrofia muscular de Duchenne (DMD) é considerada a mais comum e devastadora das distrofias musculares humanas. Inflamação crônica e estresse oxidativo são características marcantes da doença. A Diacereína é uma antraquinona usada no tratamento de osteoartrite, que exibe propriedades anti-inflamatórias e a Vitamina E apresenta um papel importante como potente antioxidante e na preservação da integridade da membrana celular. Com base nas ações desses medicamentos, o objetivo do presente estudo foi avaliar os efeitos da Diacereína e da Vitamina E na mionecrose, no estresse oxidativo e na resposta inflamatória dos músculos diafragma, esternomastóideo e tibial anterior de camundongos mdx. Neste estudo, foram utilizados camundongos das linhagens C57BL/10 e camundongos mdx. Os camundongos mdx foram divididos em 5 grupos: mdx (tratado com solução salina); mdxP (tratado com Prednisona - 5mg/kg; controle positivo); mdxD (tratado com Diacereína ¿ 20mg/kg); mdxVE (tratado com Vitamina E ¿ 40mg/kg) e mdxVED (tratado com Vitamina E e Diacereina). Os camundongos C57BL/10 não receberam nenhum tratamento e foram utilizados como controle. Todos os animais foram submetidos à análise de medida de força. Amostras de sangue foram utilizadas para determinação de creatina quinase (CK) e para a quantificação de IL-1? e IL-6. Os músculos diafragma (DIA), esternomastóideo (STN) e tibial anterior (TA) foram retirados e submetidos à técnicas histológicas (HE; IgG; F4/80; DHE e lipofuscina), Western blotting (TNF-?, NF-?B, 4-HNE, SOD2, Catalase, GR) e para determinação da atividade da GSH. Os resultados demonstraram que os camundongos mdx tratados com Diacereína, Vitamina E e/ou com associação desses dois medicamentos apresentaram redução da perda da força, do dano muscular, da resposta inflamatória exacerbada e dos danos oxidativos. Em comparação com o tratamento com Prednisona, os resultados demonstraram que o tratamento com Diacereína e Vitamina E melhoraram o fenótipo distrófico dos músculos analisados, semelhante ao observado no tratamento com o glicocorticoide. Em conjunto, os resultados demonstraram os efeitos benéficos do tratamento com Diacereína e Vitamina E, administrados isoladamente ou em associação, sobre o processo de regeneração muscular, resposta inflamatória e estresse oxidativo nos músculos DIA, STN e TA de camundongos mdxAbstract: Duchenne muscular dystrophy (DMD) is considered the most common and devastating of human muscular dystrophies. Chronic inflammation and oxidative stress are striking features of disease. Diacerein is an anthraquinone used in the osteoarthritis treatment, which exhibits anti-inflammatory and antioxidant properties and the Vitamin E presents an important role as a potent antioxidant and in preserving the integrity of the cell membrane. The aim of the present study was evaluated the effects of Diacerein and Vitamin E against myonecrosis, oxidative stress and inflammatory response in the diaphragm, sternomastoid and tibialis anterior muscles of mdx mice. In this study, C57BL/10 and mdx mice were used. The mdx mice were randomly divided into 5 groups: mdx (treated with saline); mdxP (treated with Prednisone ¿ 5mg/kg; positive control); mdxD (treated with Diacerein ¿ 20mg/kg); mdxVE (treated with Vitamin E ¿ 40mg/kg) and mdxVED (treated with Vitamin E and Diacerein). The C57BL/10 mice received no treatment and were used as a control group. All animals were submitted to force measurement analysis. Blood samples were used for the determination of creatine kinase (CK) and for the quantification of IL-1? and IL-6 levels. The diaphragm (DIA), sternomastoid (STN) and tibialis anterior (TA) muscles were removed and submitted to histological techniques (HE, IgG, F4/80, DHE and lipofuscin), Western blotting (TNF-?, NF-?B, 4-HNE, SOD2, Catalase, GR) and for the determination of GSH activity. The results showed that mdx mice treated with Diacerein, Vitamin E and/or association of these two drugs presented a reduction in strength loss, muscle damage, exacerbated inflammatory response and oxidative damage. Compared with Prednisone treatment, the results demonstrated that treatment with Diacerein and Vitamin E improved the dystrophic phenotype of the muscles analyzed, similar to that observed with glucocorticoid treatment. Taken together, the results demonstrate the beneficial effects of treatment with Diacerein and Vitamin E, administered alone or in combination, in the process of muscle regeneration, inflammatory response and oxidative stress in the DIA, STN and TA muscles of mdx miceDoutoradoAnatomiaDoutor em Biologia Celular e Estrutural2014/01970-6147862/2014-0FAPESPCNPQCAPE

The Immunomodulation To Diabetes Control: New Proposals For The Reversion Of This Disease.

The diabetes mellitus is a metabolic disorder, characterized by the hyperglycemia with deficiency in the use of carbohydrates, fats and proteins, resultant of the impairment in secretion and/or insulin action. Severely, the type 1 diabetes provokes the compromise of several organs, causing different disorders and until death of patient. In this way, the literature has shown the general treatments for the type 1 diabetes and currently the focus in immunotherapy and/or immunomodulation, to control of this hyperglycemic condition. The use of new therapies is necessary due to the high increase of incidence of this disease around the world. Recent studies showed an increase of 40% in the cases since 1997. This disease affects different organs, including the glandular tissues, mainly the pancreas. Despite all therapies for diabetes control, the damages occurred remain irreversible. Thus, in addition to general treatments, the use of immunotherapy may open new perspectives for treatment of this disease. Within this aspect, the anti-CD3 monoclonal antibodies may be effective, mainly by protect and maintain the pancreatic acinar cells. Thus, these treatments based in the immunomodulation can be an option for diabetes control and to reverse the damage caused by this disease.9210-21

Dipeptidyl peptidase IV inhibitor improves the salivary gland histology of spontaneously diabetic mice

Objectives: The incretin-based therapy might be effective in patients possessing certain levels of preserved pancreatic beta-cells. However, doubts still exist regarding the efficacy of this atment in the recovery of tissues damaged by type 1 diabetes. Thus, the objective of this study was to evaluate the treatment with MK0431 in salivary glands of spontaneously diabetic mice, focusing mainly on the possible therapeutic and hypoglycaemic effects of this dipeptidyl peptidase IV inhibitor in the recovery of these salivary tissues. Methods and results: Twenty mice were divided into two groups of 10 animals each: group I (NOD diabetic/untreated) and group II (NOD diabetic MK0431/treated). The group II was treated during 4 weeks with MK0431 mixed in the food. The group I was maintained in the same way without receiving, however, any treatment. Glucose levels were monitored during treatment and salivary glands samples were collected at the end of treatment for the histological examination under both transmitted and polarized light microscopy. High glucose levels were observed in untreated animals, while in animals with treatment, reduction of these levels was observed. Tissue restructuring was also observed in animals submitted to therapy with MK0431, mainly in relation to the attempt to extracellular matrix reorganization. Conclusions: According to results, the treatment with this dipeptidyl peptidase IV inhibitor contributed to the general homeostasis of the organism and to the reestablishment of both epithelial and stromal compartments which were damaged by the hyperglycaemic condition, demonstrating that the incretin-based therapy may be an important complementary treatment for the type 1 diabetic condition. © 2012 Elsevier Ltd

Inflammatory response morphology in dystrophic diaphragm muscle.

<p>Diaphragm (DIA) cross-sections showing inflammatory area (outline) in C57BL/10 (A), saline-treated <i>mdx</i> mice (B), prednisone-treated <i>mdx</i> mice (C) and diacerhein-treated <i>mdx</i> mice (D). The graphs (E) show the inflammatory area (%) in the DIA muscle of C57BL/10 mice (Ctrl), saline-treated <i>mdx</i> mice (<i>mdx</i>), prednisone-treated <i>mdx</i> mice (<i>mdx</i>P) and diacerhein-treated <i>mdx</i> mice (<i>mdx</i>D). DIA cross-sections showing F4/80 staining (white arrow) in Ctrl (F), <i>mdx</i> (G), <i>mdx</i>P (H) and <i>mdx</i>D (I). In (J), graphs show the F4/80 staining (%) in the DIA muscle of Ctrl, <i>mdx</i>, <i>mdx</i>P and <i>mdx</i>P groups. ^a<i>P</i> ≤ 0.05 compared with Ctrl mice, ^b<i>P</i> ≤ 0.05 compared with <i>mdx</i> mice, ^c<i>P</i> ≤ 0.05 compared with prednisone-treated <i>mdx</i> mice (one-way ANOVA with Tukey’s post-hoc test).</p

Oxidative stress in dystrophic diaphragm muscle.

<p>Diaphragm (DIA) cross-sections showing dihydroethidium (DHE) fluorescence in C57BL/10 (A), saline-treated <i>mdx</i> mice (B), prednisone-treated <i>mdx</i> mice (C) and diacerhein-treated <i>mdx</i> mice (D). The graphs (E) show the DHE staining area (%) in C57BL/10 mice (Ctrl), saline-treated <i>mdx</i> mice (<i>mdx</i>), prednisone-treated <i>mdx</i> mice (<i>mdx</i>P) and diacerhein-treated <i>mdx</i> mice (<i>mdx</i>D). DIA cross-sections showing autofluorescent lipofuscin granules (white arrow) in Ctrl (F), <i>mdx</i> (G), <i>mdx</i>P (H) and <i>mdx</i>D (I). The graphs (J) show the number of lipofuscin granules x 10⁻⁴/mm³ in Ctrl, <i>mdx</i>, <i>mdx</i>P and <i>mdx</i>D groups. In (K), western blotting analysis of 4-hydroxynonenal (4-HNE)-protein adducts. Bands corresponding to protein (top row), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; used as loading control) (bottom row), are shown. The graphs show protein levels in the crude extracts of DIA muscle from Ctrl, <i>mdx</i>, <i>mdx</i>P and <i>mdx</i>D groups. The intensities of each band were quantified and normalized to those of the corresponding Ctrl (in order to obtain relative values). All values expressed as mean ± standard deviation (SD). ^a<i>P</i> ≤ 0.05 compared with Ctrl mice, ^b<i>P</i> ≤ 0.05 compared with <i>mdx</i> mice, ^c<i>P</i> ≤ 0.05 compared with prednisone-treated <i>mdx</i> mice (one-way ANOVA with Tukey’s post-hoc test).</p

Degeneration/regeneration process in dystrophic diaphragm muscle.

<p>Diaphragm (DIA) cross-sections showing IgG staining (white arrow) in C57BL/10 (A), saline-treated <i>mdx</i> mice (B), prednisone-treated <i>mdx</i> mice (C) and diacerhein-treated <i>mdx</i> mice (D). The graphs (E) show the IgG staining (%) in the DIA muscle of C57BL/10 mice (Ctrl), saline-treated <i>mdx</i> mice (<i>mdx</i>), prednisone-treated <i>mdx</i> mice (<i>mdx</i>P) and diacerhein-treated <i>mdx</i> mice (<i>mdx</i>D). The graphs (F) show the creatine kinase (CK) levels in Ctrl, <i>mdx</i>, <i>mdx</i>P and <i>mdx</i>D. CK values expressed as mean ± standard deviation (SD). DIA cross-sections showing fibers which central nuclei (black arrow head) and peripheral nuclei (black arrow) in Ctrl (G), <i>mdx</i> (H), <i>mdx</i>P (I) and <i>mdx</i>D (J). The graphs show the nuclei central fibers (K) and peripheral nuclei fibers (L) in the DIA muscle of Ctrl, <i>mdx</i>, <i>mdx</i>P and <i>mdx</i>D groups. ^a<i>P</i> ≤ 0.05 compared with Ctrl mice, ^b<i>P</i> ≤ 0.05 compared with <i>mdx</i> mice (one-way ANOVA with Tukey’s post-hoc test).</p

Dystrophic phenotype improvement in the diaphragm muscle of <i>mdx</i> mice by diacerhein

<div><p>Chronic inflammation and oxidative stress are striking features of Duchenne muscular dystrophy disease. Diacerhein is an anthraquinone, which exhibits anti-inflammatory and antioxidant properties. Based on their actions, the present study evaluated the effects of diacerhein against myonecrosis, oxidative stress and inflammatory response in the diaphragm muscle of <i>mdx</i> mice and compared these results to current treatment widely used in DMD patients, with a main focus on the impact of prednisone. The results demonstrated that diacerhein treatment prevented muscle damage indicated by a decrease in the IgG uptake by muscle fibers, lower CK levels in serum, reduction of fibers with central nuclei with a concomitant increase in fibers with peripheral nuclei. It also had an effect on the inflammatory process, decreasing the inflammatory area, macrophage staining and TNF-α and IL-1β content. Regarding oxidative stress, diacerhein treatment was effective in reducing the ROS and lipid peroxidation in the diaphragm muscle from <i>mdx</i> mice. Compared to prednisone treatment, our findings demonstrated that diacerhein treatment improved the dystrophic phenotype in the diaphragm muscle of <i>mdx</i> mice similar to that of glucocorticoid therapy. In this respect, this work suggests that diacerhein has a potential use as an alternative drug in dystrophinopathy treatment and recommends that its anti-inflammatory and antioxidants properties in the dystrophic muscle should be better understood.</p></div

Factor and inflammatory cytokines involved in dystrophic process.

<p>Western blotting analysis of tumor necrosis factor alpha (A, TNF-α), and nuclear factor-kappa (B, NF-κB). The blots of the proteins (top row) and of glyceraldehyde-3-phosphate dehydrogenase (loading control, bottom row), are shown. The graphs show protein levels in the crude extracts of diaphragm (DIA) muscle from C57BL/10 mice (Ctrl), saline-treated <i>mdx</i> mice (<i>mdx</i>), prednisone-treated <i>mdx</i> mice (<i>mdx</i>P) and diacerhein-treated <i>mdx</i> mice (<i>mdx</i>D). The intensities of each band were quantified and normalized to those of the corresponding Ctrl. Relative values are expressed as mean ± standard deviation (SD). Detection of IL-1β (C) and IL-6 (D) levels in serum of Ctrl, <i>mdx</i>, <i>mdx</i>P and <i>mdx</i>D groups. ^a<i>P</i> ≤ 0.05 compared with Ctrl mice, ^b<i>P</i> ≤ 0.05 compared with <i>mdx</i> mice (one-way ANOVA with Tukey’s post-hoc test).</p

Body weights and forelimb muscle strength in control and dystrophic mice.

<p>Body weights and forelimb muscle strength in control and dystrophic mice.</p

Coenzyme Q10 supplementation acts as antioxidant on dystrophic muscle cells

Increased oxidative stress is a frequent feature in Duchenne muscular dystrophy (DMD). High reactive oxygen species (ROS) levels, associated with altered enzyme antioxidant activity, have been reported in dystrophic patients and mdx mice, an experimental model of DMD. In this study, we investigated the effects of coenzyme Q10 (CoQ10) on oxidative stress marker levels and calcium concentration in primary cultures of dystrophic muscle cells from mdx mice. Primary cultures of skeletal muscle cells from C57BL/10 and mdx mice were treated with coenzyme Q10 (5 μM) for 24 h. The untreated mdx and C57BL/10 muscle cells were used as controls. The MTT and live/dead cell assays showed that CoQ10 presented no cytotoxic effect on normal and dystrophic muscle cells. Intracellular calcium concentration, H2O2 production, 4-HNE, and SOD-2 levels were higher in mdx muscle cells. No significant difference in the catalase, GPx, and Gr levels was found between experimental groups. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H2O2, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. Our findings also suggest that the decrease of oxidative stress reduces the need for upregulation of antioxidant pathways, such as SOD and GSH24611751185CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo001Não temNão temThis study was financed in part by the Coordenação de Pessoal de Nivel Superior Brasil, (CAPES)–Finance Code 001, Fundação de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), CNPq and FAEPEX. L.H.R.M., A.R.F., and R.D.M. were the recipient of a FAPESP fellowship. D.S.M, T.A.H., and C.C.L are the recipient of a CAPES fellowshi