Avaliação de extratos vegetais de Azadirachta indica A. Juss como inibidores reversíveis do ciclo celular de fibroblastos bovinos

Abstract

Errors in epigenetic reprogramming in reconstructed embryos by technique Somatic Cell Nuclear Transfer (SCNT) reduce your success rate. The synchronization of the receiver oocyte cycle with the donor cell was described as one of the main factors that contribute to an appropriate reprogramming. Extracts of Azadirachta indica A. Juss (Neem) have demonstrated potential to inhibit the cell cycle of cancerigens cells and growth of tumors, which led to the hypothesis that it may be effective also in nucleus donor somatic cell for cloning. This study aimed to evaluate the effects of Neem extracts on cell cycle of bovine fibroblasts and verify if these treatments reduce the viability of cells and change the expression of stress and apoptosis genes. Were tested four concentrations (50, 100, 200 and 300 μg/mL) of the aqueous, ethanolic, and hexane extract, by exposure to 12 or 24 hours, using a positive control (inhibition serum starvation) and a control with 0 μg/ml . The inhibition and reversibility of cycle were analyzed by Flow Cytometry, the expression of genes HSP70.1A, HSBP1, HSP27.P1, BAX, BCL-2 and WNT5A evaluated by real-time PCR, and cell viability by Trypan Blue. The results were subjected to analysis of variance and means were compared by the Student Newman Keuls test, with values P<0.05 considered significant. The treatments of 100 and 200 μg/ml of the ethanol extract, for 24 hours of exposure, were able to synchronize the cell cycle in the G0/G1 (88,59 ± 0,26% and 88,36 ± 0,32%, respectively), compared to the control with 0 μg/ml (81.89 ± 0,43%), being the only ones that were reversible, returning to cycle within 12 hours after the removal of extract. Just these treatments, therefore, cell viability and gene expression analysis was performed. For the group of fibroblasts treated with 200 μg/mL was reduced the viability (50,16 ± 2,76%) compared to the others, which were similar (P>0.05) between them. For both treatments ran under expression of most genes, with the exception of HSP27 to 100 μg/mL and HSP70 to 200 μg/mL, when they have been compared to the control with 0 μg/mL. In conclusion, among the treatments tested, the best was the ethanol extract, 100 μg/mL, for 24 hours of exposure. Therefore, this treatment have potential to be tested in the production of embryos by SCNT technique and helping to improve their efficiency.