Efeitos de Aloe barbadensis (aloe vera) em células mesangiais humanas cultivadas em meio com alta concentração de glicose: possíveis mecanismos regulatórios no sistema renina-angiotensina
Data
2015-01-31
Autores
Arita, Lilian Saemi 
Orientadores
Casarini, Dulce Elena
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A nefropatia diabética está relacionada com a hiperglicemia descontrolada ou crônica, e é caracterizada por hipertrofia dos glomérulos, hipoperfusão, espessamento da membrana basal glomerular e hiperfiltração glomerular. O aumento da atividade do Sistema Renina Angiotensina (SRA) intra-renal contribui para a nefropatia diabética. Bloqueadores do SRA tem conferido proteção renal para pacientes diabéticos. Apesar da melhoria nas condições dos pacientes diabéticos através desse bloqueio, nenhum tratamento tem atingido uma modulação eficiente até o momento, justificando que mais estudos continuem a ser realizados em nível molecular e genético para encontrar melhores maneiras de impedir os danos causados pelo SRA no rim diabético. Estudos pré-clínicos e clínicos mostram o efeito hipoglicemiante do gel de Aloe barbadensis (Aloe vera). A investigação de seus compostos com inúmeras propriedades medicinais e a sua importância no tratamento de diabetes, torna-se inevitavelmente instigante. Considerando que não exsitem dados na literatura sobre o papel da A. Vera e/ou de seus compostos purificados sobre os componentes no SRA de células mesangiais, o objetivo deste estudo foi avaliar os efeitos de componentes isolados e purificados de A. vera sobre células mesangiais humanas cultivadas em meio com alta concentração de glicose, em especial sobre os componentes o SRA. Primeiramente, foram realizados experimentos in vivo com a fração butanólica (FB) e o extrato bruto (EB) de A. vera em comparação aos efeitos de insulina e glibenclamida, nos quais verificamos a ação hipoglicemiante da FB nos ratos diabéticos. A FB de A.vera foi purificada por Cromatografia Líquida de Alta Eficiência (HPLC), e então, selecionamos dois componentes com tempos de retenção próximos ao componente conhecido Aloe emodina (AE), nomeados de P9 e P9A. De acordo com dados da literatura, AE tem demonstrado efeitos anti-diabéticos em diferentes modelos in vitro. Avaliamos a ação dos componentes P9 e P9A AE e insulina comparados com um grupo controle sem tratamento nas atividades da renina e enzima conversora de angiotensina (ECA), bem como na expressão proteica dos componentes do SRA tais como a renina, ECA, receptor AT1 (AT1R), receptor AT2 (AT2R) e receptor Mas. P9 e P9A atuam em diferentes níveis da cascata, mas vale destacar os seus efeitos inibitórios sobre a atividade da renina das células cultivadas em meio com alta concentração de glicose, de aproximadamente 98% e de 50%, respectivamente, quando comparados ao CT. Além disso, ambos diminuíram a expressão proteica da renina e o P9A também diminuiu a expressão proteica da ECA. No entanto, a alta capacidade inibitória de P9 não está relacionada com a diminuição da expressão proteica da renina. Além disso, a fração P9 aumentou a expressão de AT2R e, em contrapartida, a fração P9A aumentou a expressão de AT1R sugerindo maior efeito protetor do P9 contra os efeitos deletérios do diabetes. Nossos resultados demonstraram a presença de AE na fração P9, e um possível novo composto na fração P9A. Tanto a AE que utilizamos como controle como a detectada na fração P9, tem ações diretas sobre o SRA nunca antes estudado, evidenciando uma inibição da renina, lembrando uma similaridade com o alisquireno. Ambas as frações purificadas de A.vera, inibem a atividade da renina, sendo possíveis potenciais compostos para o tratamento do diabetes e hipertensão.
Diabetic nephropathy is associated with uncontrolled or chronic hyperglycemia and characterized by glomerular hypertrophy and hyperfiltration, hypoperfusion and thickening of the glomerular basement membrane. Activation of the intrarenal ReninAngiotensin System (RAS) contributes to diabetic nephropathy. RAS blockers has been shown to provide renal protection for diabetic patients. Despite RAS blockage improves diabetic patients’ conditions, there is no treatment capable to efficiently suppress the RAS yet. Thus, further studies have to be conducted at the molecular and genetic levels to find better ways to prevent the damage caused by the RAS in diabetic kidney. Clinical and preclinical studies show hypoglycemic effect of Aloe barbadensis (Aloe vera) in gel. Investigating A. vera compounds, due to their numerous medicinal properties and importance in the treatment of diabetes, has being inevitably attractive. Whereas there in no findings in the literature about the role of A. vera and and its purified compounds in the RAS components of mesangial cells, the aim of this study was to evaluate the effects of isolated and purified compounds of A. vera in human mesangial cells cultured in high glucose concentration, especially in RAS componentes. First, we performed in vivo experiments using butanol fraction (FB) and crude extract (EB) of A. vera, comparing with insulin and glibenclamide effects, in which we find the hypoglycemic action of FB in diabetic rats. The FB fraction was purified by High Performance Liquid Chromatography (HPLC) and then we selected two components with retention times similar to the known componente, Aloe emodin (AE), which we called P9 and P9A. According to the literature, AE has demonstrated anti-diabetic effects in different in vitro models. We evaluated the action of P9, P9A, AE and insulin compared with control group in the activities of renin and angiotensin-I converting enzyme (ACE), and in the protein expression of RAS components such as renin, ACE, AT1 (AT1R), AT2 (AT2R) and Mas receptors. P9 and P9A act at different levels of angiotensin formation, but it is worth highlighting its inhibitory effects on renin activity of the cells in cultured in a high concentration of glucose, approximately 98% and 50%, respectively in comparison to control. Inhibition of P9 treatment around 98% of renin activity could be due to the reduced expression of the enzyme with lower availability. However, in treatments with P9A and insulin, the expressions were much lower than in the group treated with P9 and their actions as inhibitors were not so efficient, indicating the possibility of high inhibitory capacity of P9 is not being related to decreased protein expression of renin. Moreover, P9 fraction increased AT2R expression, indicating a protective effect of this treatment against the deleterious effects of diabetes. In treatment with P9A fraction, despite its efficiency as renin inhibitor, it has increased AT1R expression, showing that its effect is not so protector. Our results demonstrated the presence of AE in P9 fraction and a possible new compound in P9A fraction. Both AE which were used as control as the one detected in P9 fraction, have direct actions on the RAS, which was never studied before, indicating an inhibition of renin, seeing a similarity with aliskiren. Both purified fractions of A. vera, inhibit renin activity, being possible potencial compounds for the treatment of diabetes and hypertension.
Diabetic nephropathy is associated with uncontrolled or chronic hyperglycemia and characterized by glomerular hypertrophy and hyperfiltration, hypoperfusion and thickening of the glomerular basement membrane. Activation of the intrarenal ReninAngiotensin System (RAS) contributes to diabetic nephropathy. RAS blockers has been shown to provide renal protection for diabetic patients. Despite RAS blockage improves diabetic patients’ conditions, there is no treatment capable to efficiently suppress the RAS yet. Thus, further studies have to be conducted at the molecular and genetic levels to find better ways to prevent the damage caused by the RAS in diabetic kidney. Clinical and preclinical studies show hypoglycemic effect of Aloe barbadensis (Aloe vera) in gel. Investigating A. vera compounds, due to their numerous medicinal properties and importance in the treatment of diabetes, has being inevitably attractive. Whereas there in no findings in the literature about the role of A. vera and and its purified compounds in the RAS components of mesangial cells, the aim of this study was to evaluate the effects of isolated and purified compounds of A. vera in human mesangial cells cultured in high glucose concentration, especially in RAS componentes. First, we performed in vivo experiments using butanol fraction (FB) and crude extract (EB) of A. vera, comparing with insulin and glibenclamide effects, in which we find the hypoglycemic action of FB in diabetic rats. The FB fraction was purified by High Performance Liquid Chromatography (HPLC) and then we selected two components with retention times similar to the known componente, Aloe emodin (AE), which we called P9 and P9A. According to the literature, AE has demonstrated anti-diabetic effects in different in vitro models. We evaluated the action of P9, P9A, AE and insulin compared with control group in the activities of renin and angiotensin-I converting enzyme (ACE), and in the protein expression of RAS components such as renin, ACE, AT1 (AT1R), AT2 (AT2R) and Mas receptors. P9 and P9A act at different levels of angiotensin formation, but it is worth highlighting its inhibitory effects on renin activity of the cells in cultured in a high concentration of glucose, approximately 98% and 50%, respectively in comparison to control. Inhibition of P9 treatment around 98% of renin activity could be due to the reduced expression of the enzyme with lower availability. However, in treatments with P9A and insulin, the expressions were much lower than in the group treated with P9 and their actions as inhibitors were not so efficient, indicating the possibility of high inhibitory capacity of P9 is not being related to decreased protein expression of renin. Moreover, P9 fraction increased AT2R expression, indicating a protective effect of this treatment against the deleterious effects of diabetes. In treatment with P9A fraction, despite its efficiency as renin inhibitor, it has increased AT1R expression, showing that its effect is not so protector. Our results demonstrated the presence of AE in P9 fraction and a possible new compound in P9A fraction. Both AE which were used as control as the one detected in P9 fraction, have direct actions on the RAS, which was never studied before, indicating an inhibition of renin, seeing a similarity with aliskiren. Both purified fractions of A. vera, inhibit renin activity, being possible potencial compounds for the treatment of diabetes and hypertension.
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Citação
ARITA, Lilian Saemi. Efeitos de Aloe barbadensis (aloe vera) em células mesangiais humanas cultivadas em meio com alta concentração de glicose: possíveis mecanismos regulatórios no sistema renina-angiotensina. 2015. Tese (Doutorado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.