5-Azacytydine and resveratrol reverse senescence and ageing of adipose stem cells via modulation of mitochondrial dynamics and autophagy.

Obesity and endocrine disorders have become prevalent issues in the field of both human and veterinary medicine. Equine metabolic syndrome is a complex disorder involving alternation in metabolism and chronic systemic inflammation. It has been shown that unfavourable microenvironment of inflamed adipose tissue negatively affects adipose stem cell population (ASC) residing within, markedly limiting their therapeutic potential. ASCs are characterized by increased senescence apoptosis, excessive accumulation of reactive oxygen species (ROS), mitochondria deterioration and "autophagic flux." The aim of the present study was to evaluate whether treatment of ASCs with a combination of 5-azacytydine (AZA) and resveratrol (RES) would reverse aged phenotype of these cells. For this reason, we performed the following analyzes: molecular biology (RT-PCR), microscopic (immunofluorescence, TEM) and flow cytometry (JC-1, ROS, Ki67). We evaluated the mitochondrial status, dynamics and clearance as well as autophagic pathways. Furthermore, we investigated epigenetic alternations in treated cells by measuring the expression of TET genes and analysis of DNA methylation status. We have demonstrated that AZA/RES treatment of ASCs is able to rejuvenate these cells by modulating mitochondrial dynamics, in particular by promoting mitochondrial fusion over fission. After AZA/RES treatment, ASCs were characterized by increased proliferation rate, decreased apoptosis and senescence and lower ROS accumulation. Our findings offer a novel approach and potential targets for the beneficial effects of AZA/RES in ameliorating stem cell dysfunctions.