Study of molecular and cellular responses in healthy elderly subjects performing a resistance training program

Resumen

Aging is associated with an immunosenescence process, which manifests as a chronic sterile low-grade inflammation state (inflammaging). This inflammaging has been related to the biological aging process and aging-related diseases. The accumulation of cellular damage as damage-associated molecular patterns (DAMPs) with age appear to be the key to activate Nod-like receptors (NLRs) and star the inflammatory response through NLRP3 inflammasome signaling. The autophagic process, responsible for the elimination of damaged molecules and organelles, is closely linked to the inflammatory response, so that the deterioration of autophagic capacity experienced with age leads to an excessive accumulation of DAMPs in the cell cytosol, stimulating the inflammatory response. An important source of DAMPs in aging is the production of reactive oxygen species (ROS) in the mitochondria, especially when they suffer alterations in their membrane potential and mitophagy is inhibited, such that damaged mitochondria accumulate. Moreover, the apoptosis process, also closely linked to inflammation, increase with age, especially in the thymus, increasing the alteration of the inflammatory response, as well as the quantity and quality of immune cells, and in the muscle, favoring the loos of muscular tissue, named sarcopenia. On the other hand, during aging there is an increase in misfolded or unfolded proteins, generating endoplasmic reticulum stress (ERS). The age-relate ERS appears to be due to a failure of the folding systems - chaperones - as well as a decline in the main mediators of the unfolded protein response (UPR). Furthermore, senescent cells secrete high levels of inflammatory mediators resulting in what is known as the somatic cell senescence-associated secretory phenotype (SASP). Along with an increase in cfDNA release, these SASP mediators include cytokines, proteases, growth factors or chemokines, as well as different types of microRNAs, such as the senescence- and inflammation-related mitochondrial miR-146a-5p. It is widely demonstrated that regular exercise provides benefits throughout the body. Specifically, it has been speculated that resistance exercise can, not only increase strength and stop the loss of muscle mass in aging, but also have great anti-inflammatory effects, either directly modifying the inflammatory response or indirectly through the regulation of autophagy, and mitophagy, apoptosis and UPR cellular processes or by modulating the age-associated biomarkers miR-146a-5p and cfDNA release into the circulation. Although in last decades it has been suggested that these systemic effects may be due to the release of myokines by the muscle, recent evidence seems to indicate that other cell types participate in the exercise-mediated release of charged molecules in extracellular vesicles, as exosomes. However, the evidence of the capacity of the resistance training in modifying the mentioned molecular and cellular processes in the elderly are very little or, in some cases, nonexistent. Therefore, the objective of the present Doctoral Thesis was to evaluate the effects of 8-week resistance exercise on NLRP3 inflammasome response, autophagy capacity and mitophagy initiation, apoptosis activation, UPR signaling, and miR-146a-5p, cfDNA and exosome release in healthy elderly individuals. Thirty-eight women and men were assigned to a strength training group (TG), who performed an 8-week resistance training program, or to a control group (CG), who continued with their daily routine. Before and after the intervention, peripheral blood mononuclear cells (PBMCs) were isolated. On the one hand, protein expression of beclin-1, Atg12, Atg16 and LAMP-2 increased following the training program, while expression of p62/SQSTM1 and phosphorylation of ULK-1 at Ser757 were significantly lower. Resistance exercise also induced a decrease in NLRP3 expression and in the caspase-1/procaspase-1 ratio. Expression of Bcl-2 and Bcl-xL, as well as the Bad/BcL-2, ratio were reduced, and there was a significant decrease in the protein content of caspase-3. On the other hand, the phosphorylation of PERK and IRE1, as well as ATF4, and XBP1 protein expression, significantly increased following the training, while expression of BiP, AFT6 and CHOP remain without changes. Additionally, the intervention also induced an increase in PGC-1a and Mfn1 protein levels, while no changes were found in Drp1 expression. Finally, the resistance protocol was not able to activate PINK1/Parkin and Bnip3/Nix pathways. Finally, results showed no changes in plasma miR-146a-5p and cfDNA levels with training. Furthermore, the levels of exosome markers (Flot-1, CD9, and CD81) as well as exosome-carried proteins (CD14 and VDAC1) remained unchanged, whereas an attenuated CD63 response was found in trained group compared to the controls. The results indicate that the 8-week resistance exercise carried out is capable of decreasing the inflammatory response mediated by the NLRP3 inflammation, and that it confirms a lower expression of the pro-inflammatory exosomal marker CD63 in the group trained after exercise. However, no effect was observed on the expression of other exosomal markers, as well as on the release into plasma of the biomarkers miR-146a and cfDNA. In addition, resistance exercise is capable of promoting autophagy, without influencing mitophagy, as well as the processes of mitochondrial biogenesis and fusion and UPR, decreasing the apoptosis process in elderly PBMCs. Although further research is needed to present decisive outcomes, the results of the present Doctoral Thesis suggest that the well-established interrelationship between inflammation, autophagy and apoptosis, as well as the increase of the mitochondrial network and the activation of the UPR could be the underlying mechanisms involved. on the exercise-mediated systemic beneficial effects on the elderly and reinforces the idea that regular physical activity is one of the most powerful anti-aging treatments that exists, being the key in the healthy aging process.