Luis A. Salazar

Leptin G-2548A promoter polymorphism is associated with increased plasma leptin and BMI in Brazilian women

Variants in leptin gene (LEP) have been implicated in the pathogenesis of obesity. The relationship between LEP G-2548A polymorphism and obesity-related traits was evaluated in a sample of Brazilian women (n = 228) who were randomly selected from two clinical centers in Sao Paulo city. Blood samples were collected for DNA extraction, plasma leptin and serum lipids measurements. LEP G-2548A genotypes were identified by a PCR- RFLP strategy using the endonuclease Alw44I. LEP G-2548A was associated with obesity after adjustment for covariates (age, hypertension, coronary artery disease, smoking and physical activity). Women carrying G allele had a four times higher risk of obesity than the A allele carriers (OR: 4.11, CI95%: 1.06-15.90, p = 0.041). G allele was also related to increased plasma leptin (p = 0.024) and body mass index (p = 0.027). Hypertension, hyperglycemia, dyslipidemia and coronary artery disease were associated with obesity. However LEP G-2548A polymorphism was not related to these variables. All together these data suggest that LEP G-2548A polymorphism has an important role in regulating plasma leptin levels and body mass index in women.

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.

Anti-atherogenic and anti-angiogenic activities of polyphenols from propolis.

Propolis is a polyphenol-rich resinous substance extensively used to improve health and prevent diseases. The effects of polyphenols from different sources of propolis on atherosclerotic lesions and inflammatory and angiogenic factors were investigated in LDL receptor gene (LDLr-/-) knockout mice. The animals received a cholesterol-enriched diet to induce the initial atherosclerotic lesions (IALs) or advanced atherosclerotic lesions (AALs). The IAL or AAL animals were divided into three groups, each receiving polyphenols from either the green, red or brown propolis (250 mg/kg per day) by gavage. After 4 weeks of polyphenol treatment, the animals were sacrificed and their blood was collected for lipid profile analysis. The atheromatous lesions at the aortic root were also analyzed for gene expression of inflammatory and angiogenic factors by quantitative real-time polymerase chain reaction and immunohistochemistry. All three polyphenol extracts improved the lipid profile and decreased the atherosclerotic lesion area in IAL animals. However, only polyphenols from the red propolis induced favorable changes in the lipid profiles and reduced the lesion areas in AAL mice. In IAL groups, VCAM, MCP-1, FGF, PDGF, VEGF, PECAM and MMP-9 gene expression was down-regulated, while the metalloproteinase inhibitor TIMP-1 gene was up-regulated by all polyphenol extracts. In contrast, for advanced lesions, only the polyphenols from red propolis induced the down-regulation of CD36 and the up-regulation of HO-1 and TIMP-1 when compared to polyphenols from the other two types of propolis. In conclusion, polyphenols from propolis, particularly red propolis, are able to reduce atherosclerotic lesions through mechanisms including the modulation of inflammatory and angiogenic factors.

The antifungal effect of six commercial extracts of Chilean propolis on Candida spp

Propolis has been used in traditional medicine for many centuries because of its beneficial health properties, including its antimicrobial capacity. Prosthesis stomatitis affects a significant percentage of users of removable dentures; Candida albicans is the most common fungal species associated with the development of this pathology. Thus, the objectives of this study were: a. To evaluate the antifungal activity of six commercial propolis extracts against Candida spp. that was isolated from the oral cavity of removable dentures users, and b. To determine chemical characteristics of the propolis extracts evaluated. Among the results, we note that these concentrations of polyphenols varied between 9 ± 0.3 and 85 ± 2.1 mg mL-1. Chromatographic analysis was able to detect 35 compounds, among which were caffeic acid, myricetin, quercetin, kaempferol, apigenin, pinocembrin, galangin, and caffeic acid phenyl ester (CAPE). All strains tested were inhibited by the liquid extracts of propolis. The MID ranged between 1:40 and 1:1280, and the MIC for C. albicans ranged from 197 µg mL-1 to 441 µg mL-1. From the results obtained in this investigation, we can conclude that all propolis extracts evaluated are capable of inhibiting the development of Candida spp. However, they show significant differences in the concentration of polyphenols present and in antifungal activity. El propoleo ha sido utilizado por la medicina tradicional desde hace muchos siglos debido a sus propiedades beneficas para la salud, entre las que destaca su capacidad antimicrobiana. La estomatitis subprotesica, afecta a un porcentaje importante de usuarios de protesis dental removible, siendo Candida albicans la especie fungica mas comun asociada al desarrollo de esta patologia. Asi, los objetivos de este estudio fueron: a) evaluar la actividad antifungica de seis extractos de propoleos comerciales sobre cepas de Candida spp. aisladas de la cavidad oral de usuarios de protesis dental removible, y b) determinar algunas caracteristicas quimicas de los extractos de propoleos utilizados. Entre los resultados obtenidos, podemos senalar que estos mostraron concentraciones de polifenoles que variaron entre 9 ± 0,3 y 85 ± 2,1 mg/mL. El analisis cromatografico permitio detectar 35 compuestos, entre los cuales se logro identificar la presencia de acido cafeico, miricetina, quercetina, kaempferol, apigenina, pinocembrina, galangina y acido cafeico fenil ester (CAPE). Todas las cepas de Candida spp. evaluadas fueron inhibidas por los seis extractos liquidos de propoleos, observandose que la DIM vario entre 1/40 y 1/1280, y la CIM para C. albicans vario entre 197 µg/mL y 441 µg/mL. A partir de los resultados obtenidos en esta investigacion podemos concluir que todos los propoleos evaluados son capaces de inhibir el desarrollo de Candida spp. , sin embargo, estos muestran importantes diferencias en la concentracion de los polifenoles presentes y en la actividad antifungica.

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.

No association between common Gly972Arg variant of the insulin receptor substrate-1 and polycystic ovary syndrome in Southern Chilean women

BACKGROUND Previously studies have indicated that the insulin receptor substrate-1 (IRS-1) Gly972Arg (G972R) polymorphism is associated with polycystic ovary syndrome (PCOS). We examined the possible association between G972R common variant of the IRS-1 gene and PCOS in Southern Chilean women with PCOS and controls. METHODS A total of 50 women with PCOS (29.1+/-8.1 yr) and 75 healthy women (29.3+/-9.3 yr) were included. Serum lipids, glucose and uric acid concentrations were determined by enzymatic-colorimetric methods. The G972R variant of the IRS-1 gene was detected by PCR-RFLP. RESULTS Women with PCOS exhibited a higher concentrations of total testosterone, glucose, insulin, total cholesterol, triglycerides, LDL-C and uric acid, and lower HDL-C concentrations than controls (P<0.05). The presence of G972R polymorphism in PCOS and control women was not significantly different (16% vs. 6.6%, P=0.276). The OR for PCOS associated to 972R variant was 2.67 (95% CI: 0.82-8.69, P=NS). Moreover, neither association between G972R genotypes and metabolic parameters were observed in PCOS women or controls. CONCLUSION Our data do not support an association between G972R variant of the IRS-1 with PCOS or its metabolic parameters in Southern Chilean women.

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.

Polymorphisms of the NOS3 gene in Southern Chilean subjects with coronary artery disease and controls

BACKGROUND Nitric oxide (NO) from the endothelium, produced by oxidation of l-arginine to l-citruline for the action at the endothelial nitric oxide synthase (eNOS) is considered an important atheroprotective factor. The 894G>T, -786T>C and 4a/4b polymorphic variants of the NOS3 gene have been implicated in the development of coronary artery disease (CAD). We investigated the association between occurrence of CAD documented by angiography and the 894G>T, -786T>C and 4a/4b polymorphisms of the NOS3 gene in Southern Chilean individuals. METHODS A total of 112 unrelated patients with diagnosis of CAD confirmed by angiography and 112 controls were included in this study. The 894G>T and -786T>C single nucleotide polymorphisms were analyzed by PCR-RFLP, and 4a/4b polymorphism just for PCR. RESULTS The genotype distribution and the relative allelic frequencies for the 3 variants investigated were not significantly different between CAD and control subjects (p=NS). Moreover, the odds ratio for CAD associated with the 894T (OR=1.22, 95% CI 0.76-1.95), -786C (OR=1.16, 95% CI 0.75-1.80) and 4a (OR=0.97, 95% CI 0.48-1.95) variants failed to reach statistical significance. CONCLUSION These findings suggest that the 894G>T, -786T>C and 4a/4b polymorphisms of the NOS3 were not associated with CAD in the studied subjects.

Association between CAPN10 UCSNP-43 gene polymorphism and polycystic ovary syndrome in Chilean women

BACKGROUND Studies have suggested that the Calpain-10 gene polymorphisms may play a role in polycystic ovary syndrome (PCOS) susceptibility. Thus, the aim of the present study was to evaluate the possible association between three single nucleotide polymorphisms (SNPs) in the calpain-10 gene (UCSNPs -43, -19, and -63) and polycystic ovary syndrome (PCOS) in Chilean women. METHODS Fifty women with PCOS (28.8+/-8.2 y) and 70 healthy women (28.6+/-8.6 y) were included in this study. Serum lipids, hormonal status, fasting glucose, oral glucose tolerance test (OGTT), insulin, HOMAIR, and uric acid levels were determined by conventional methods. The calpain-10 gene variants were detected by PCR and PCR-RFLP, respectively. RESULTS The presence of uncommon allele (A) for the UCSNP-43 was associated with increased risk of PCOS (odds ratio=1.93, 95% CI: 1.11-3.34). The UCSNP-63 (OR=1.11, 95% CI: 0.59-2.11) and UCSNP-19 (OR=0.93, 95% CI: 0.55-1.57) were not associated to PCOS. However, the PCOS women carrying the CC genotype for UCSNP-63 exhibited higher values of total cholesterol and LDL-C (P<0.05). Similarly, control women carrying the CC genotype showed higher serum levels of triglycerides, HDL-C and uric acid (P<0.05). CONCLUSION Our data suggest the contribution of CAPN10 UCSNP-43 gene polymorphism to PCOS in Chilean women. However, further studies with larger samples are necessary to confirm this observation.