Efeito da mistura de triterpenos alfa e beta-amirina, isolada de Protium heptaphyllum, sobre a adipogênese in vitro

Abstract

The obesity is defined by World Health Organization as the abnormal and excessive accumulation of body fat, which causes health damage. This condition is associated with several comorbidities, such as type 2 diabetes mellitus, cardiovascular diseases, dyslipidemias and cancer. The global epidemic of obesity has led to several studies on its physiology and the search for new treatments for this disease. The 3T3-L1 fibroblast cell line is widely used for the study of adipocyte differentiation in vitro, since it is a stable cell culture that resembles adipose tissue itself. Studies with this lineage have shown that several triterpenes could inhibit the differentiation of these cells, such as ursolic acid and oleanolic acid. The mixture of α,β-amyrin triterpenes is the main constituent from resin of Protium heptaphyllum (“almecegueira”). Like P. heptaphyllum resin, the α,β-amyrin mixture showed antiobesity action in mice. The aim of this work was to evaluate the effect of α,β-amyrin on the adipogenesis of 3T3-L1 cells by staining with Oil Red O and analysis of protein expression (Western Blot) and gene expression (qPCR) of important transcription factors involved in this process. MTT assay showed that α,β-amyrin didn’t cause cell viability reduction at concentrations of 3.125; 6.25; 12.5; 25; 50 and 100 μg/mL on undifferentiated cells; however, it causes viability reduction in 61.5% and 87% in concentrations of 200 and 400 μg/mL, respectively. On differentiated cells, concentrations of 100, 200 and 400 μg/mL caused a reduction in cell viability in 21.9; 83.2 and 94.7%, respectively. Results of Oil Red O show that α,β-amyrin at concentrations of 6.25; 12.5; 25 and 50 μg/mL reduced the accumulation of lipid in 3T3-L1 cells in 34, 44, 68 and 75%, respectively, when compared to the control group (p<0.05). Concentrations of 12.5; 25 and 50 μg/mL were chosen for the other experiments. Concentrations of 25 and 50 μg/mL α,β-amyrin significantly reduced (p<0.05) the protein expression of PPARγ in 54 and 54.8%, respectively. Concentrations of 12.5; 25 and 50 μg/ mL reduced (p<0.05) the protein expression of C/EBPα in 50.9; 50.1 and 62%, respectively, and the concentration of 50 μg/mL reduced the expression of SREBP-1 in 41.1%. The α,β-amyrin doesn’t modify C/EBPβ protein expression and it doesn’t participate in AMPKα phosphorylation at any of the concentrations tested. The α,β-amyrin at concentrations 12.5; 25 and 50 μg/mL significantly reduced (p<0.05) the PPARγ2 gene expression by 1.7, 1.3 and 1.9-fold, respectively. Gene expression of C/EBPα was reduced (p<0.05) in 1.9; 2.1 and 7.3-fold at the concentrations tested and there was no change in the C/EBPβ and C/EBPδ gene expression. Thus, α,β-amyrin reduces the differentiation of 3T3-L1 cells by down-regulation expression of key transcription factors of adipogenesis. Through these results, this mixture of triterpenes becomes a possible candidate for the development of new substances for the control of adipogenesis.