Pterostilbene targets hallmarks of aging in the gene expression landscape in blood of healthy rats

Scope: Polyphenols from the phytoestrogen group, including pterostilbene (PTS), are known for their antioxidant, anti-inflammatory and anti-cancer effects. In recent studies, phytoestrogens attenuate age-related diseases, however their pro-longevity effects in healthy models in mammals remain unknown. As longevity studies demonstrate age-related transcriptomic signatures in human blood, we propose that phytoestrogen-supplemented diet may induce changes in gene expression that ultimately confer pro-longevity benefits. Methods and Results: In the present study, we conducted RNA sequencing to determine transcriptome-wide changes in gene expression in whole blood of healthy rats consuming diets supplemented with phytoestrogens. Ortholog cell deconvolution was applied to analyze the omics data. We discovered that PTS leads to changes in the gene expression landscape and PTS-target genes are associated with functions counteracting hallmarks of aging, including genomic instability, epigenetic alterations, compromised autophagy, mitochondrial dysfunction, deregulated nutrient sensing, altered intercellular interaction, and loss of proteostasis. These functions bridge together under anti-inflammatory effects through multiple pathways, including immunometabolism, where changes in cellular metabolism (e.g., ribosome biogenesis) impact the immune system. Conclusion: Our findings provide a rationale for pre-clinical and clinical longevity studies and encourage investigations on PTS in maintaining cellular homeostasis, decelerating the process of aging, and improving conditions with chronic inflammation. Overall design: A total of 24 male Fischer 344 rats at 4 weeks of age were obtained from Charles River (Indianapolis, IN, USA) and housed 2 rats per cage in a temperature-controlled (24°C) room with a 12-h light/dark cycle, and ad libitum access to water and a chow diet [i.e., choline-su?cient amino acid-defined (CSAA)]. After a week of acclimation, rats were randomly divided into 4 groups, each containing 6 animals. Using the biological and analytical variability from previous data, it could be determined that 6 rats per group (n = 6) were required to detect a 50% difference with the variation of the endpoint 25% (i.e., gene expression measured by qRT-PCR) and with 80% power at a nominal significance of P < 0.05. The 4 groups were as follows: Group 1 - fed the CSAA diet; Group 2: fed a CSAA diet supplemented with resveratrol (CSAA+RSV, 1.2 g/kg of diet); Group 3 – fed a CSAA diet supplemented with pterostilbene (CSAA+PTS, 1.34 g/kg of diet); Group 4 – fed a CSAA diet supplemented with chlorogenic acid (CSAA+CGA, 1.9 g/kg of diet). All diets were pelleted at Dyets Inc. (Bethlehem, PA, USA). The compounds (BIOTANG Inc., Lexington, MA, USA) were used at equal molar concentrations. Following a 20-day feeding period, blood samples were collected by retro-orbital plexus sampling from anesthetized rats, where the retro-orbital plexus was penetrated with a capillary tube to promote blood flow from the capillaries behind the eye. The amount of collected blood did not exceed 10% of circulating blood volume based on body weight. Blood samples were collected from each rat into spray coated K2EDTA tubes (BD & Co,. Franklin Lakes, NJ, USA) and stored in -80°C until RNA was extracted. Rats were treated strictly following the animal use protocol #1112000342 approved by the Institutional Animal Care and Use Committee at the involved institutions. Mouse RiboPure™-Blood RNA Isolation Kit (Invitrogen) was used to extract RNA from rat whole blood samples as recommended by the manufacturer.