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Previous issue date: 2016-08-30 === Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) === Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) === A urolit?ase afeta aproximadamente 10% da popula??o mundial e est? associada fortemente a presen?a de cristais de oxalato de c?lcio (OxCa). Atualmente n?o existe nenhum composto eficiente que pode ser utilizado para prevenir esta doen?a. No entanto, alguns polissacar?deos sulfatados (PS) de algas marinhas marrons demonstraram capacidade de inibir a forma??o de cristais de OxCa e alterar a morfologia destes in vitro. Os PS da alga vermelha Gracilaria birdiae t?m atividades biol?gicas importantes, mas at? o presente momento n?o foi avaliada como inibidora da forma??o de cristais de OxCa. O presente estudo teve como objetivo obter PS e verificar seu efeito na forma??o do OxCa. Para tal, extratos ricos em polissacar?deos foram obtidos utilizando extra??o alcalina, sonica??o, digest?o proteol?tica, seguida por precipita??o com etanol. Esses extratos ricos em PS (EB) foram separados em 5 fra??es (F-0.25; F-0.5; F-0.75; F-1.0 e F-1.25) atrav?s de cromatografia de troca i?nica DEAE-celulose. A eletroforese em gel de agarose, infra vermelho e an?lises qu?micas mostraram que essas fra??es cont?m o mesmo pool de polissacar?deos sulfatados ricos em galactose. F-0.25; F-0.5; F-0.75; F-1.0 foram capazes de inibir etapas importantes da forma??o dos cristais de OxCa, como a nuclea??o e agrega??o, no entanto a F-0.25 promoveu a maior inibi??o da nuclea??o em 76,92% e da agrega??o em 68,57%. As fra??es F-0.25 e F-0.5 foram capazes de inibir em 6 e 7 vezes, respectivamente, o n?mero de cristais OxCa monohidratados (COM) formados in vitro, enquanto que as fra??es F-0.75 e F-1.0, reduziram apenas em 1,5 vezes. A morfologia dos cristais de OxCa foi afetada principalmente pelas amostras, EB, F-0.25; F-0.5 e F-0.75, pois promoveram as varia??es mais destoantes em rela??o ao controle. A an?lise do potencial zeta (?) dos cristais formados na presen?a das amostras, constatou um aumento de cargas negativas em suas superf?cies. Atrav?s das imagens obtidas por microscopia de fluoresc?ncia, foi constatado que os PS est?o distribu?dos de forma homog?nea nos cristais dihidratados (COD) e de forma perif?rica nos COM. Os dados obtidos atrav?s da microscopia eletr?nica de varredura(MEV) e espectroscopia de energia dispersiva (EDS) revelaram grandes varia??es na distribui??o de ?tomos de oxig?nio e c?lcio na superf?cie dos cristais na presen?a das F-0.25 e F-0.5. A c?lulas renais HEK-293, MDCK e 786-0 tiveram baixa toxicidade na presen?a do extrato bruto e das fra??es F-0.25; F-0.5; F-0.75. Estas amostras protegeram c?lulas renais MDCK e verificou-se um efeito reparador contra danos causados pelo H2O2 e OxCa. Por fim, as c?lulas MDCK submetidas ao tratamento pr?vio e simult?neo com a F-0.25 e a F-0.5 na presen?a de H2O2 e OxCa, reduziram a atividade das enzimas super?xido dismutase (SOD) e catalase (CAT). Com isto, verifica-se que PS da G. birdiae podem ser potenciais alvos farmacol?gicos do ponto de vista preventivo, terap?utico e reparador de danos causados pela urolit?ase, por?m ? importante que outros estudos sejam realizados in vivo, bem como, sejam realizados experimentos para elucidar os mecanismos precisos de a??o, pelos quais estes PS protegem as c?lulas contra danos por H2O2. === The urolithiasis disease affects approximately 10% of the world's population and is strongly associated calcium oxalate crystals (CaOx). Until now, there is not an efficient compound that can be used to prevent this disease. However, some sulfated polysaccharides (SP) from brown seaweeds exhibited an ability to inhibit the formation of CaOx crystals and change the morphology in vitro. SP from the red seaweed Gracilaria birdiae has important biological activities, but they have not been evaluated as inhibitor of CaOx crystals formation. This study aimed to obtain SP from this seaweed and evaluate their effect on CaOx crystals formation. Thus, sulfated polysaccharide-rich extract (EC) was obtained using alkaline extraction, sonication, proteolytic digestion followed by ethanol precipitation. This extract was fractionated into five fractions (F-0.25; F-0.5; F-0.75; F-1.0; F-1.25) by DEAE-cellulose ion-exchange chromatography. Agarose gel electrophoresis, infrared and chemical analysis showed that these fractions contain the same pool of sulfated galactose-rich polysaccharides. F-0.25; F-0.5; F, 0.75; F-1.0 were able to inhibit important formation steps of the CaOx crystals, as nucleation and aggregation, we highlight F-0.25 that promoted the highest inhibition of nucleation in 76.92% and aggregation in 68.57%. The F-0.25 and F-0.5 fractions were able to reduce approximately 6 and 7 times, respectively, the number of these CaOx monohydrated crystals (COM) formed in vitro, whereas F-0.75 and F-1.0 fractions reduced this number only 1.5 times. The morphology of CaOX crystals was mainly affected by the samples EC, F-0.25; F-0.5 and F-0.75, they promoted the most discordant variations in the control. The analysis of the zeta potential (?) of the crystals formed in the presence of the samples was found an increase of negative charges on their surfaces. Through the images obtained by fluorescence microscopy revealed that the PS are distributed homogeneously in the dehydrated crystals (COD) and peripherally in COM. The data obtained by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) revealed wide variations in the distribution of oxygen and calcium atoms on the surface of the crystals in the presence of F-0.25 and F-0.5. The kidney cells HEK-293, MDCK and 786-0 showed low toxicity in the presence of EC and fractions F-0.25; F-0.5; F, 0.75. These samples protected kidney MDCK cells against damage caused by H2O2 and CaOx. Finally, the MDCK cells treated beforehand and concomitant with both F-0.25 and F-0.5 in the presence of H2O2 and CaOx, reduced the activity of superoxide dismutase and catalase enzymes. Overall, the data showed that PS G. birdiae may be potential pharmacological targets for preventive, therapeutic and repairing damage caused by urolithiasis. However, it is important that to make set in vivo experiments, as well as, experiments to elucidate the precise mechanism of action by which these PS protect the H2O2 damage on cells.
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