Alejandro Cuevas

Modulation of Immune Function by Polyphenols: Possible Contribution of Epigenetic Factors

Several biological activities have been described for polyphenolic compounds, including a modulator effect on the immune system. The effects of these biologically active compounds on the immune system are associated to processes as differentiation and activation of immune cells. Among the mechanisms associated to immune regulation are epigenetic modifications as DNA methylation of regulatory sequences, histone modifications and posttranscriptional repression by microRNAs that influences the gene expression of key players involved in the immune response. Considering that polyphenols are able to regulate the immune function and has been also demonstrated an effect on epigenetic mechanisms, it is possible to hypothesize that there exists a mediator role of epigenetic mechanisms in the modulation of the immune response by polyphenols.

Identification of pharmacogenetic predictors of lipid-lowering response to atorvastatin in Chilean subjects with hypercholesterolemia.

BACKGROUND Statins are normally the first-line therapy for hypercholesterolemia (HC); however, the lipid-lowering response shows high interindividual variation. We investigated the effect of four polymorphisms in CYP3A4, CYP3A5 and ABCB1 genes on response to atorvastatin and CYP3A4 activity in Chilean subjects with HC. METHODS A total of 142 hypercholesterolemic individuals underwent atorvastatin therapy (10mg/day/1month). Serum lipid levels before and after treatment were measured. Genetic variants in CYP3A4 (-290A>G, rs2740574), CYP3A5 (6986A>G, rs776746) and ABCB1 (2677G>A/T, rs2032582 and 3435C>T, rs1045642) were analyzed by PCR-RFLP. CYP3A4 enzyme activity in urine samples was assessed through determination of 6β-hydroxycortisol/cortisol free ratio (6βOHC/FC). RESULTS After 4weeks of therapy, a significant reduction in total cholesterol (TC) and LDL-c was observed (P<0.001). The G allele for -290A>G polymorphism was related to higher percentage of variation in TC and LDL-c (P<0.001). Moreover, same allele was associated with higher HDL-c variation (P=0.017). In addition, CYP3A4 enzyme activity was lower in subjects carrying this polymorphism (P=0.009). No differences were observed for CYP3A5 and ABCB1 variants. CONCLUSION Our results suggest that presence of G allele for -290A>G polymorphism determines a better response to atorvastatin, being also associated with lower CYP3A4 activity in vivo, causing an increased atorvastatin activity.

Identification of microRNAs involved in the modulation of pro-angiogenic factors in atherosclerosis by a polyphenol-rich extract from propolis.

New vessel formation plays a critical role in the progression and vulnerability of atherosclerotic lesions. It has been shown that polyphenols from propolis attenuate the progression of atherosclerosis and also exert inhibitory effects on angiogenic factors. However, the mechanisms underlying these effects are not completely understood. Thus, this study aimed to identify microRNAs (miRNAs) involved in the modulation of pro-angiogenic factors in the atherosclerotic plaques of LDL receptor gene knockout mice treated with a polyphenol-rich extract of Chilean propolis. The progression of the atherosclerotic lesions was significantly attenuated in treated mice compared with control mice. Using microarray analysis and a bioinformatic approach, we identified 29 differentially expressed miRNAs. Many of these miRNAs were involved in biological processes associated with angiogenesis, such as the cell cycle, cell migration, cell growth and proliferation. Among them, three miRNAs (miR-181a, miR-106a and miR-20b) were over-expressed and inversely related to the expression of Vegfa (vascular endothelial growth factor A) and Hif1a (hypoxia inducible factor 1 alpha). In addition, VEGF-A protein expression was attenuated in histological sections obtained from the aortic sinuses of treated mice. VEGFA is a key pro-angiogenic factor in atherosclerotic plaques, and Hif1a, which is expressed in the necrotic nucleus of the atheroma, is its main inducer. We found a correlation between the over-expression of miR-181a, miR-106a and miR-20b and their target genes, Hif1a and Vegfa, which is consistent with attenuation of the atherosclerotic lesion. In conclusion, our data analysis provides evidence that the anti-angiogenic effects of polyphenols from Chilean propolis can be modulated by miRNAs, in particular miR-181a, miR-106a and miR-20b.

Chemical and botanical characterization of Chilean propolis and biological activity on cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus

Propolis is a non-toxic natural substance with multiple pharmacological properties including anti-cancer, antioxidant, fungicidal, antibacterial, antiviral, and anti-inflammatory among others. The aim of this study was to determine the chemical and botanical characterization of Chilean propolis samples and to evaluate their biological activity against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. Twenty propolis samples were obtained from beekeeping producers from the central and southern regions of Chile. The botanical profile was determined by palynological analysis. Total phenolic contents were determined using colorimetric assays. Reverse phase HPLC and HPLC-MS were used to determine the chemical composition. The minimum inhibitory concentration (MIC) was determined on S. mutans and S. sobrinus. All propolis samples were dominated by structures from native plant species. The characterization by HPLC/MS, evidenced the presence of quercetin, myricetin, kaempferol, rutine, pinocembrin, coumaric acid, caffeic acid and caffeic acid phenethyl ester, that have already been described in these propolis with conventional HPLC. Although all propolis samples inhibited the mutans streptococci growth, it was observed a wide spectrum of action (MIC 0.90 to 8.22 μg mL−1). Given that results it becomes increasingly evident the need of standardization procedures, where we combine both the determination of botanical and the chemical characterization of the extracts. Research conducted to date, describes a promising effectiveness of propolis in the prevention of caries and other diseases of the oral cavity, making it necessary to develop studies to identify and understand the therapeutic targets or mechanisms of molecular action of the various compounds present on them.

ERK1/2 and HIF1α Are Involved in Antiangiogenic Effect of Polyphenols-Enriched Fraction from Chilean Propolis

Propolis has been shown to modulate the angiogenesis in both in vitro and in vivo models. Thus, we aimed to evaluate the antiangiogenic properties of an ethanolic extract of Chilean propolis (EEP) and Pinocembrin (Pn). Migration, formation of capillary-like structures of endothelial cells, and sprouting from rat aortic rings were used to assess the antiangiogenic properties of EEP or Pn. In addition, microRNAs and VEGFA mRNA expression were studied by qPCR. ERK1/2 phosphorylation and HIF1α stabilization were assessed by western blot. EEP or Pn attenuated the migration, the capillary-like tube formation, and the sprouting in the in vitro assays. In addition, the activation of HIF1α and ERK1/2 and the VEGFA mRNA expression was significantly inhibited in a dose-dependent manner. In summary, these results suggest that HIF1α and ERK1/2 phosphorylation could be involved in the antiangiogenic effect of Chilean propolis, but more studies are needed to corroborate these findings.

Efecto del Propóleos Chileno sobre el Metabolismo de Glucosa en Ratones Diabéticos

En el presente estudio se evaluo el efecto del propoleos sobre el metabolismo de la glucosa en ratones C57/BL-6 con diabetes mellitus tipo 2 inducida por dieta alta en grasa. Se midieron los cambios en las concentraciones sericas de lipidos, glucosa e insulina, y el efecto sobre la captacion de 2-deoxi-[2,6-3H]-D-glucosa, sintesis de [14C]-glicogeno y descarboxilacion de [U-14C]-D-glucosa inducida por insulina en musculo aislado. Los resultados muestran que en ratones diabeticos, el tratamiento con propoleos (150 mg/kg/dia) reduce los niveles de insulina e indice HOMA (P<0.05). Tambien disminuyo la obesidad abdominal de estos animales (P<0.05). Por otro lado, no modifico las concentraciones plasmaticas de glucosa, colesterol total y trigliceridos. Se observo tambien que la captacion de 2-deoxi-[2,6-3H]-D-glucosa, sintesis de [14C]-glicogeno y descarboxilacion de [U-14C]-D-glucosa inducida por insulina en musculo soleo de ratones tratados con propoleos fue significativamente superior al grupo control (P<0.05). En resumen, nuestros datos confirman que el propoleos es capaz de modular el metabolismo de glucosa en ratones C57/BL-6 con diabetes mellitus tipo 2 inducida por dieta alta en grasa. Los datos obtenidos constituyen un importante antecedente que avala el posible uso del propoleos como fuente de polifenoles con actividad antidiabetogenica.

Polyphenols from Chilean Propolis and Pinocembrin Reduce MMP-9 Gene Expression and Activity in Activated Macrophages

Polyphenols from diverse sources have shown anti-inflammatory activity. In the context of atherosclerosis, macrophages play important roles including matrix metalloproteinases synthesis involved in degradation of matrix extracellular components affecting the atherosclerotic plaque stability. We prepared a propolis extract and pinocembrin in ethanol solution. Propolis extract was chemically characterized using LC-MS. The effect of treatments on gene expression and proteolytic activity was measured in vitro using murine macrophages activated with LPS. Cellular toxicity associated with both treatments and the vehicle was determined using MTT and apoptosis/necrosis detection assays. MMP-9 gene expression and proteolytic activity were measured using qPCR and zymography, respectively. Thirty-two compounds were identified in the propolis extract, including pinocembrin among its major components. Treatment with either ethanolic extract of propolis or pinocembrin inhibits MMP-9 gene expression in a dose-dependent manner. Similarly, an inhibitory effect was observed in proteolytic activity. However, the effect showed by ethanolic extract of propolis was higher than the effect of pinocembrin, suggesting that MMP-9 inhibition results from a joint contribution between the components of the extract. These data suggest a potential role of polyphenols from Chilean propolis in the control of extracellular matrix degradation in atherosclerotic plaques.

Frequency of Common Variants in Genes Involved in Lipid-Lowering Response to Statins in Chilean Subjects with Hypercholesterolemia

Polimorfismos de los genes CYP3A4, CYP3A5 y ABCB1 se han asociado a variaciones en la respuesta a farmacos hipolipemiantes, como las estatinas; principales medicamentos utilizados para disminuir los niveles plasmaticos de colesterol (CT). Sin embargo, la frecuencia de estas variantes geneticas puede variar entre las poblaciones. Asi, el objetivo de este trabajo fue evaluar la frecuencia de tres polimorfismos de los genes CYP3A4, CYP3A5 y ABCB1, relacionados previamente a la respuesta a estatinas, en individuos chilenos hipercolesterolemicos (HC) y controles. Se analizaron 135 sujetos con diagnostico de hipercolesterolemia primaria (CT 240 mg/dL) y 120 controles (CT 200 mg/dL) pertenecientes a la Region de La Araucania (Chile). La genotipificacion de las variantes geneticas se efectuo mediante la tecnica de reaccion en cadena de la polimerasa seguido de restriccion enzimatica (PCR-RFLP). La distribucion de genotipos para la variante 3435C>T del gen ABCB1 en los individuos HC (CC: 49%, CT: 37%, TT: 14%) y controles (CC: 41%, CT: 48%, TT: 11%) fue semejante (P = 0,186). De forma similar, la distribucion de genotipos para el polimorfismo -290A>G del gen CYP3A4 en los pacientes HC (AA: 73%, AG: 27%, GG: 0%) y controles (AA: 71%, AG: 29%, GG: 0%) fue equivalente (P = 0,863). Del mismo modo, la distribucion de genotipos para la variante 6986A>G del gen CYP3A5 en el grupo HC (AA: 4%, AG: 41%, GG: 55%) y grupo control (AA: 4%, AG: 47%, GG: 49%) fue similar (P = 0,594). En resumen, nuestro estudio demuestra que las frecuencias de los polimorfismos 3435C>T (ABCB1), -290A>G (CYP3A4) y 6986A>G (CYP3A5) no difieren entre individuos HC y controles, y son comparables a las frecuencias encontradas en poblaciones asiaticas. Su efecto sobre el tratamiento con estatinas en la poblacion chilena debe ser investigado.

Asociación del polimorfismo rs2241766 del gen de la adiponectina y enfermedad arterial coronaria en individuos del sur de Chile

La adiponectina, hormona peptidica secretada por los adipocitos, actua inhibiendo la formacion de la placa ateromatosa en casi todas sus etapas, ya sea modulando la respuesta inflamatoria, inhibiendo la expresion de moleculas de adhesion, la activacion de monocitos, la formacion de celulas espumosas y la migracion y proliferacion de celulas de musculo liso. En el presente estudio, fue evaluada la frecuencia del polimorfismo 45TMJ (rs2241766) del gen que codifica para la hormona adiponectina (ADIPOQ), junto con su posible contribucion al desarrollo de enfermedad coronaria en individuos del sur de Chile. Metodos: Fueron evaluados 416 individuos adultos (30 a 68 anos); 200 casos confirmados por angiografia y 216 controles. La genotipificacion del polimorfismo 451>G del gen ADIPOQ se realizo mediante la tecnica de PCR-RFLP. Resultados: El analisis de los resultados demuestra que la presencia del alelo G del polimorfismo 45T>G del gen ADIPOQ duplica el riesgo de padecer enfermedad coronaria en la poblacion estudiada (OR= 2.06; I.C.95%: 1.36-3.14). Conclusion: Nuestros datos revelaron la existencia de una interesante asociacion entre el polimorfismo 45T>G del gen ADIPOQ y enfermedad arterial coronaria en los sujetos analizados. Es importante destacar, que este hallazgo constituye el primer antecedente en poblacion chilena.

Bacterial Community Profile of the Gut Microbiota Differs between Hypercholesterolemic Subjects and Controls

The role of gut microbiota in the development of metabolic illnesses has been abundantly demonstrated. Recent studies suggest that gut microbiota alterations may also be related to the development of hypercholesterolemia. Therefore, we aimed to assess differences in the gut bacterial community profiles between hypercholesterolemic subjects and controls. Thirty cases diagnosed with hypercholesterolemia and 27 normocholesterolemic controls were included. A fasting whole blood sample was obtained to determine the lipid profile. In parallel, stool samples were collected and total DNA was isolated to assess the bacterial community profiles by denaturing gradient gel electrophoresis (DGGE). In addition, the Richness, Shannon-Weaver, and Simpson indexes were used to evaluate the richness and diversity of bacterial communities. As expected, serum concentrations of total cholesterol, triglycerides, and LDL-cholesterol were significantly higher in the cases compared with controls. Moreover, DGGE analysis showed a lower richness and diversity of bacterial communities in hypercholesterolemic subjects. In conclusion, our results showed differences in the profiles of bacterial communities between hypercholesterolemic subjects and controls, suggesting a possible role of the gut microbiota in the development of hypercholesterolemia.