RESUMO
Las proteínas NPC1L1, ABCG5 y ABCG8 participan en la absorción intestinal de colesterol. Ezetimiba inhibe este proceso bloqueando a NPC1L1, sin embargo, su efecto sobre ABCG5 y ABCG8 aún no está claro. Así, el objetivo del presente trabajo fue evaluar en ratones C57BL/6 con hipercolesterolemia inducida por dieta y tratados con ezetimiba, la expresión de NPC1L1, ABCG5 y ABCG8 mediante PCR en tiempo real y Western blot, en 3 grupos de animales: 1, dieta hipercolesterolémica D12336; 2, dieta D12336 más 5 mg/kg/día de ezetimiba; 3, dieta control. El nivel sérico de colesterol total fue significativamente diferente entre los grupos estudiados (control: 1,85 +/- 0,49 mmol/L; dieta D12336: 3,11 +/- 0,73 mmol/L; ezetimiba: 2,11 +/- 0,50 mmol/L, P = 0,001). La expresión génica de NPC1L1 aumentó 5,4 veces en el grupo que recibió la dieta D12336 (P = 0,003). Por otro lado, la expresión génica de ABCG5 y ABCG8 no fue diferente en el grupo con hipercolesterolemia (P = 0,239 y P = 0,201, respectivamente). Después del tratamiento con ezetimiba, la expresión génica de ABCG5 se incrementó 15,6 veces (P = 0.038). No hubo diferencias significativas en la expresión génica de NPC1L1 (P = 0,134) y ABCG8 (P = 0,067). En relación a la expresión proteica, la dieta D12336 incrementó los niveles de expresión de NPC1L1 (P = 0,022) y ABCG5 (P = 0,008); el tratamiento con ezetimiba incrementó los niveles de NPC1L1 (P = 0,048) y redujo los niveles de ABCG5 (P = 0,036) y ABCG8 (P = 0,016). En conclusión, nuestros resultados sugieren que tanto la dieta hipercolesterolémica como el tratamiento con ezetimiba, en un modelo experimental, afectan los niveles de expresión de NPC1L1, ABCG5 y ABCG8, sugiriendo que ABCG5 y ABCG8 están involucrados en la respuesta hipolipemiante a este fármaco. No obstante, el mecanismo mediante el cual se explica esta interacción requiere de un futuro estudio.
Proteins NPC1L1, ABCG5 and ABCG8 are involved in the intestinal absorption of cholesterol. Ezetimibe inhibits this process by blocking NPC1L1, however, its effect on ABCG5 and ABCG8 is not yet clear. Thus, the objective of this study was to evaluate in C57BL / 6 mice with diet-induced hypercholesterolemia treated with ezetimibe, the expression of NPC1L1, ABCG5 and ABCG8 by real time PCR and Western blot, in 3 groups of animals: 1, diet hypercholesterolemic D12336, 2, D12336 diet plus 5 mg/kg/ day of ezetimibe, 3, diet control. The serum level of total cholesterol was significantly different between groups (control: 1.85 +/- 0.49 mmol / L; diet D12336: 3.11 +/- 0.73 mmol / L; ezetimibe: 2.11 +/- 0.50 mmol / L, P = 0.001). NPC1L1 gene expression increased 5.4-fold in the group receiving the diet D12336 (P = 0.003). Furthermore, the gene expression of ABCG5 and ABCG8 was not different in the group with hypercholesterolemia (P = 0.239 and P = 0.201, respectively). After treatment with ezetimibe, ABCG5 gene expression was increased 15.6 times (P = 0.038). No significant differences in gene expression of NPC1L1 (P = 0.134) and ABCG8 (P = 0.067). Regarding protein expression, the D12336 diet increased the levels of expression of NPC1L1 (P = 0.022) and ABCG5 (P = 0.008), treatment with ezetimibe increased the levels of NPC1L1 (P = 0.048) and reduced levels of ABCG5 (P = 0.036) and ABCG8 (P = 0.016). In conclusion, our results suggest that both hypercholesterolemic diet as treatment with ezetimibe, in an experimental model, affect the expression levels of NPC1L1, ABCG5 and ABCG8, suggesting that ABCG5 and ABCG8 are involved in lipid-lowering response to this drug. However, the mechanism by which this interaction is explained requires further study.
Assuntos
Animais , Ratos , Anticolesterolemiantes/administração & dosagem , Azetidinas/administração & dosagem , Hipercolesterolemia/tratamento farmacológico , Lipoproteínas/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Transportadores de Cassetes de Ligação de ATP/fisiologia , Western Blotting , Colesterol na Dieta , Modelos Animais de Doenças , Expressão Gênica , Lipoproteínas/genética , Proteínas de Membrana Transportadoras/genética , Reação em Cadeia da Polimerase em Tempo Real , Transportadores de Cassetes de Ligação de ATP/genéticaRESUMO
In this study we evaluated the possible association between five single nucleotide polymorphisms in ABCG5 (rs6720173) and ABCG8 (rs11887534, rs4148211, rs4148217 and rs6544718) genes and ezetimibe response in Chilean hypercholesterolemic subjects. A total of 60 non-related hypercholesterolemic subjects, aged 18 to 65 years old were included in this study. These subjects were treated with ezetimibe (10mg/day) during one month. The ABCG5 and ABCG8 genotypes were assessed by PCR-RFLP. The genotype distribution of the ABCG5/ABCG8 polymorphisms was in Hardy-Weinberg equilibrium. Our results showed that the investigated polymorphisms were not associated with the response to ezetimibe. Nevertheless, the T allele of rs6544718 polymorphism was related to higher baseline levels of LDL-cholesterol (p<0.001). In addition, the G allele for the rs4148211 polymorphism was associated with greater baseline concentrations of triglycerides (P=0.019). This allele was also associated with lower concentrations of HDL-cholesterol (P=0.027), after ezetimibe treatment. Our results suggest that the studied polymorphisms do not affect the therapeutic response to ezetimibe in the evaluated subjects.
En este estudio se evaluó la posible asociación entre cinco polimorfismos de nucleótido único en los genes ABCG5 (rs6720173) y ABCG8 (rs11887534, rs4148211, rs4148217 y rs6544718) y la respuesta a ezetimiba en pacientes hipercolesterolémicos chilenos. Un total de 60 individuos hipercolesterolemicos, no relacionados, con edades entre 18 y 65 años fueron incluidos. Estos sujetos fueron tratados con ezetimiba (10mg/día) durante un mes. Los genotipos de ABCG5 y ABCG8 fueron evaluados por PCR-RFLP. La distribución de genotipos de los polimorfismos de ABCG5/ABCG8 se encontraba en equilibrio de Hardy-Weinberg. Nuestros resultados mostraron que los polimorfismos estudiados no se asociaron con la respuesta a la ezetimiba. Sin embargo, el alelo T del polimorfismo rs6544718 fue relacionado con niveles basales elevados de LDL-colesterol (p <0,001). Además, el alelo G para el polimorfismo rs4148211 se asoció con una mayor concentración basal de triglicéridos (p = 0,019). Este alelo también se asoció con concentraciones más bajas de HDL-colesterol (p = 0,027), después del tratamiento con ezetimiba. Nuestros resultados sugieren que los polimorfismos estudiados no afectan a la respuesta terapéutica a la ezetimiba en los sujetos evaluados.
Assuntos
Feminino , Pessoa de Meia-Idade , Azetidinas/farmacologia , Hipercolesterolemia/genética , Polimorfismo Genético , Transportadores de Cassetes de Ligação de ATP/genética , Anticolesterolemiantes/farmacologia , Variação Genética , HDL-Colesterol , HDL-Colesterol/sangue , Hipercolesterolemia/tratamento farmacológico , LDL-Colesterol , LDL-Colesterol/sangue , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase/métodos , Triglicerídeos/sangueRESUMO
Este trabalho teve como objetivo estudar o polimorfismo Gln604Glu do gene ABCG5 e a sua correlação com os parâmetros lipídicos, em uma população de voluntários residentes na cidade de São Roque (SP), em 2009. Foram determinados os perfis lipídicos de 150 voluntários por meio de métodos enzimáticos colorimétricos (Advia-1650-Bayer) e em seguida a realização de análise do polimorfismo Gln604Glu do gene ABCG5 pela técnica de reação em cadeia da polimerase seguida de análise de enzima de restrição. Os resultados mostraram que 45% dos indivíduos apresentaram hipercolesterolemia, 31% exibiram hipertriacilglicerolemia e 11% hipoalfalipoproteinemia. A análise do polimorfismo Gln604Glu do gene ABCG5 demonstrou que 55% dos indivíduos eram heterozigotos, 37% homozigotos normais e 8% homozigotos mutantes. Não foi observada diferença significativa entre os perfis lipídicos dos homozigotos selvagens, homozigotos mutantes e heterozigotos, não existindo correlação entre os perfis lipídicos e o polimorfismo. Os resultados sugerem que na população estudada, a presença do polimorfismo não pode ser considerada como um biomarcador para dislipidemia. Sendo necessários estudos mais amplos que avaliem a relação entre o perfil genético do indivíduo e a resposta ao tratamento preconizado.
The aim of the present work is to study the Gln604Glu polymorphism of the ABCG5 gene and its correlation with the lipid profile in a volunteer population resident in the city of São Roque in the state of São Paulo (2009). For the analysis of the lipid profile and the Gln604Glu polymorphism, we used the techniques of colorimetric analysis, Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP), respectively. The results showed that 45% of the individuals presented with hypercholesterolemia, 31% with hypertriacylglyce-rolemia and 11% with hypoalphalipoproteinemia. The analysis of the Gln604Glu polymorphism showed that 55% of the individuals were heterozygous, 37% normal heterozygous and 8% mutant heterozygous. No significant difference was observed between the lipid profiles of the wild homozygous, mutant homozygous and heterozygous, there being no correlation between the lipid profile and polymorphisms. The results suggest that the presence of polymorphism cannot be used as a dyslipidemia biomarker, in the studied population. Broader studies are required that can evaluate the relationship between the genetic profile of the individual and the response to the advocated treatment.