Introduction

Extracellular vesicles (EVs) are released by different cellular types during normal development as well as in pathogenic conditions [1]. Exosomes are a subtype of EVs secreted into the extracellular space through fusion of multivesicular bodies with the plasma membrane [2]. Membrane-specific proteins, for example tetraspanins, such as CD63, and acetylcholinesterase (AChE) have been considered as exosome markers [3, 4].

Several evidences have suggested that exosomes play an important role in intercellular communication by delivering proteins, mRNA, and miRNAs among different cells [2, 5, 6]. Increasing attention has been given to its ability to transfer toxic proteins, such as a mutant superoxide dismutase 1 (SOD1) in amyotrophic lateral sclerosis [7] and amyloid-β (Aβ) peptide in Alzheimer disease (AD) [8]. Although the exosomes have been linked to age-related pathological conditions, including neurodegenerative diseases [8], the involvement of EVs in aging process has been rarely addressed. Lehmann et al. [9] showed increased exosome releases in senescence model in vitro using human fibroblasts and prostate tumor cells. In this context, the use of exosomes for prognostic and/or diagnostic in neurodegenerative diseases has been widely proposed; Fiandaca et al. [8] suggested that Aβ levels in circulating exosomes are a potential biomarker of AD progression. However, to our knowledge, there are no reports evaluating Aβ levels in circulating exosomes in aging process.

Exosomes have been shown to transfer antioxidant enzymes, namely SOD and catalase, between oligodendrocytes and neurons [10]. Interestingly, it was recognized that circulating exosomes are able to produce reactive species during pathological conditions, specifically septic shock [11, 12]. Thus, it is reasonable to hypothesize that exosomes could be involved with the aging-related oxidative stress [13, 14].

Exercise has been associated with increased longevity [15] and reduced risks of aging-related diseases, such as cardiovascular diseases [16], cognitive impairment [17] and AD [18]. Frühbeis et al. [19] observed an increased release of small EVs into circulation immediately after acute exercise in physically active healthy men. Despite these findings, to our knowledge, there are no reports evaluating the exercise impact on circulating exosomal profile in aging process. Although it is widely recognized that regular exercise improves aging-related oxidative stress in several organs and tissues [20, 21], there is a lack of studies reporting the relationship between exosomal oxidative status and exercise effects during aging process.

Our working hypothesis was that the aging process and exercise could alter circulating EVs components. Therefore, our aim was to evaluate transitory and delayed exercise effects on serum exosome quantification through CD63 content and AChE activity in young adult and aged Wistar rats. We also intended to study EVs oxidative status parameters, specifically reactive species levels and SOD, and the Aβ content as well.

Methods

Animals

Male Wistar rats of different ages, 3-, 21-, and 26-months, were used (n = 4–6). The choice of age groups was based on some previous works performed by our group demonstrating that this exercise protocol can modulate inflammatory, epigenetic and oxidative parameters in brain areas of young adult (3-month old) and aged (20- to 21-month-old) rats [22,23,24,25]. Additionally, the oldest age was chosen based on results describing increased Aβ1–42 levels in brains of 26-month-old healthy rats [26]. The animals were provided by the Centro de Reprodução Animal de Laboratório (CREAL) and maintained under standard conditions (12-h light/dark, 22 ± 2 °C) with food and water ad libitum. The Local Ethics Committee (CEUA—Comissão de Ética no Uso de Animais—UFRGS; nr.23464) approved all animal procedures and experimental conditions.

Treadmill exercise

The animals were divided in sedentary (SED) or exercised groups (EXE). Exercise training consisted of running sessions on a motorized rodent treadmill (AVS Projetos, São Paulo, Brazil) using a moderate daily treadmill protocol (20 min running session each day for 2 weeks). All animals ran at 60% of their maximal oxygen uptake \(VO_2\), which was measured indirectly prior to training [23, 24]. Previously, we demonstrated the neuroprotective effects of this treadmill exercise protocol (20 min/day during 2 weeks), since it was able to reduce in vitro ischemic damage in hippocampal slices of Wistar rats [27]. Moreover, recently, it was described neuroprotective effects of this exercise protocol in aversive memory performance and biochemical parameters in cerebral areas of Wistar rats [23,24,25]. Animals in the SED group were daily placed on the treadmill for 5 min without any stimulus to run. Gentle tapping on their back encouraged animals that initially refused to run. Neither electric shock nor physical prodding was used in this study, and the treadmill exercise was performed between 2:00 and 5:00 PM.

Sample preparation

In order to verify the transitory and delayed exercise effects, rats were decapitated 1 and 18 h after the last training session of treadmill exercise. 1 and 18 h after the last exercise training session correspond to the afternoon and early morning, respectively, allowing to compare two time points of the circadian rhythm. The trunk blood was collected, centrifuged (10,000 rpm) at room temperature for 10 min and the serum resultant was stored at −80 °C.

Circulating extracellular vesicles isolation

The isolation was performed using a commercial kit based on vesicles precipitation (miRCURY™ Exosome Isolation Kit, Exiqon, Denmark) following the manufacturer’s instructions. Firstly, 0.6 mL serum was centrifuged for 5 min at 10,000g to remove cell debris and the supernatant was transferred into a new vial. In the new vial, it was added 200 μL of precipitation buffer, mixed by vortexing and incubated for 60 min at 4 °C. After the incubation, the samples were centrifuged for 30 min at 1500g at room temperature and the supernatant was completely discarded. The resultant pellets were resuspended in 270 μL of buffer using a vortex shaker for 5–15 min at room temperature and then stored at −20 °C. Although we have used a commercial kit called Exosome Isolation Kit, it is impossible to exclude the possibility of co-isolation of EVs containing some degree of microvesicles and other blood components such as lipoproteins of a similar size. Davidson et al. [28] affirmed that there is no perfect method to isolate and purify pure exosomes.

In addition, the size of serum-derived nanoparticles was assessed using nanosight nanoparticle characterization system LM10-HS (Nanosight Ltd., Amesbury, UK) that is considered one of the best methods for EVs characterization [28]. NTA software indicated that the nanoparticle sizes ranged from 90 to 300 nm. The size distribution of circulating EVs from rats here described had a similar profile those obtained with human being [29].

The total protein content of circulating exosomes was measured by the Coomassie blue method using bovine serum albumin as standard [30].

CD63 levels

The specificity of our isolation procedure was characterized at the protein level by the presence of CD63, a membrane protein for exosomes. The CD63 levels were measured using a specific kit (ExoELISA kit, System Biosciences) following the manufacturer’s instructions. In a 96-well plate, 50 µl of prepared protein standards or sample was added and incubated overnight at 4 °C. After incubation, 50 µl of the specific primary antibody CD63 was added and incubated at room temperature for 1 h followed by incubation with secondary antibody. Finally, 50 µl of super-sensitive TMB ELISA substrate was added and the absorbance was read at 450 nm. The CD63 levels were calculated by comparison to the standard curve per mg of total protein and expressed as percentage of control; young adult SED group was taken as 100%.

Acetylcholinesterase (AChE) activity

The AChE activity was evaluated in samples by slight modifications in the colorimetric method described by Ellman et al. [31] using acetylthiocholine iodide (Sigma, USA) as a substrate. The hydrolysis rate of acetylthiocholine iodide was measured at 412 nm through the release of the thiol compound that reacts with 5, 5-dithiobis-(2-nitrobenzoic acid) DTNB producing the color-forming compound TNB. The AChE activity was normalized for total protein content.

Reactive species levels

Reactive species levels were quantified using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) as a probe [29]. In a 96-well plate, 15 μl of sample was incubated with DCFH-DA (100 mM) at 37 °C for 30 min. The oxidized fluorescent derivative (DCF) formation was monitored at excitation (488 nm) and emission (525) wavelengths using fluorescence spectrophotometer. All procedures were performed in the dark, while the blanks containing DCFH-DA (no exosomes) were processed for auto fluorescence measurement [32]. Reactive species content was measured using a DCF standard curve and normalized for total protein.

Superoxide dismutase (SOD) activity

The superoxide dismutase activity was evaluated in samples using a specific kit following the manufacturer’s instructions (RANSOD kit, Randox Labs, USA). This method employs xanthine and xanthine oxidase to generate superoxide anion radical that reacts with 2-(4-iodophenyl)-3-(4-nitrophenol)-5 phenyl tetrazolium chloride (I.N.T.) to form a red formazan dye, which was measured at 37 °C using a spectrophotometer reader (505 nm). The inhibition of chromogen production was proportional to the enzyme activity in the sample. The units of SOD activity were normalized for total protein.

Western blot evaluation

Western blot was performed to evaluate the SOD1 content of the EV extract (n = 4 animals per group). Proteins (20 μg) were separated by 14% polyacrylamide gel electrophoresis (SDS-PAGE, 1.5 mm, 130 V) and transferred to PVDF membranes (Millipore). The membrane was blocked with 5% nonfat dry milk in TBS-T and then blotted with primary antibodies anti-SOD1 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. After, the membrane was incubated with a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. A standard molecular weight marker (RPN 800 rainbow full range Bio-Rad, CA, USA) was used as a reference to determine the molecular weights of the bands. Ponceau method was used for normalization [33].

Aβ1–42 peptide levels

The Aβ1–42 peptide levels were measured using a specific kit (Sensolyte ELISA kit-AnaSpec, Fremont, CA) following the manufacturer’s instructions. In a 96-well plate, 100 µl of standards or samples and 50 µl of the diluted detection antibody solution were added; the plate was incubated overnight at 4 °C. After incubation, 100 μl of TMB color substrate solution was added in each well followed by addition of 50 μl of the Stop Solution. The plate was read at 450 nm using a microplate absorbance reader. Aβ1–42 peptide levels were quantified using a standard curve and normalized for total protein.

Statistical analysis

The results were expressed as percentage of control (mean ± SD); young adult SED group was taken as 100%. Age, exercise, and time point factors were analyzed using two-way analysis of variance (ANOVA) with followed by Tukey post hoc test when appropriate (age × exercise; time points × age). Test t was used to compare the SOD1 content between groups. The accepted level of significance was p < 0.05 in all tests (Fig. 1).

Fig. 1
figure 1

Timeline of exercise protocol and sample collection. Animals were subjected to a treadmill exercise protocol 20 min/day during 14 days and samples were obtained 1 h (afternoon) and 18 h (morning) after the last exercise session

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Results

Aging effects on circulating extracellular vesicles profile

To confirm the presence of exosomes in the EV extract and to quantify age and exercise-related changes in exosome levels, we measured CD63 levels, a standard exosomes marker. Two-way ANOVA showed an age effect on CD63 levels (F (2,34) = 11.788; p < 0.001) since this parameter was decreased in 21- and 26-month-old rats compared to young adult groups in the late afternoon (Fig. 2a, Tukey’s post hoc, p < 0.05). However, this age-dependent response disappeared in the early morning (Fig. 2b, 18-h groups; two-way ANOVA, F (2,33) = 0.741; p = 0.486).

Fig. 2
figure 2

Aging and exercise effects on CD63 levels in circulating exosomes from 3-, 21-, and 26-month-old Wistar rats. a 1-h groups, 3-month-old SED and EXE (n = 6); 21-month-old SED (n = 5) and EXE (n = 6); 26-month-old SED and EXE (n = 6). b 18-h groups, 3-month-old SED and EXE (n = 6); 21-month-old SED and EXE (n = 6); 26-month-old SED and EXE (n = 5). The columns represent the mean ± SD (% of control). Two-way ANOVA followed by Tukey’s test. #Values significantly different from 3-month-old groups; *Values significantly different from its respective sedentary

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There were increased exosomal AChE activities in both aged groups, 21- and 26-month-old rats, compared to young adult ones (Fig. 3a, b, respectively, F (2,29) = 3.661; p = 0.040; F (2,27) = 4.420; p = 0.024; two-way ANOVA).

Fig. 3
figure 3

Aging and exercise effects on AChE activity in circulating exosomes from 3-, 21-, and 26-month-old Wistar rats. a 1-h groups, 3-month-old SED and EXE (n = 6); 21-month-old SED (n = 4) and EXE (n = 5); 26-month-old SED (n = 4) and EXE (n = 5). b 18-h groups, 3-month-old SED (n = 5) and EXE (n = 6); 21-month-old SED and EXE (n = 4); 26-month-old SED (n = 4) and EXE (n = 5). The columns represent the mean ± SD (% of control). Two-way ANOVA followed by Tukey’s test. #Values significantly different from 3-month-old groups; *Values significantly different from its respective sedentary

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In addition, a significant age effect on reactive species levels from circulating EVs (Fig. 4a, b) was observed in both time of day (two-way ANOVA, F (2,26) = 10.371; p = 0.001 and F (2,32) = 22.889, p < 0.001, respectively, 1- and 18-h groups). Aged animals (21- and 26-month-old) showed higher reactive species levels compared to young adult ones (Tukey post hoc, p < 0.05). There was an age effect on SOD activity (two-way ANOVA, F (2,29) = 8.948; p = 0.001), with decreased SOD activity in EV extracts from 26-month-old animals compared with 3-month-old animals (Fig. 5b). The decrease in SOD activity was only observed at the 18-h time point (morning, Fig. 5b), and not at the 1-h time point (afternoon, Fig. 5a) (F (2,33) = 1.491; p = 0.224). In order to investigate if SOD1 content, an established cargo of mammalian exosomes [34], was related with reduced SOD activity in aging process, western blot was performed. There was no significant difference in the SOD1 content between 3- and 26-month-old groups (Test t, p = 0.07; Fig. 6).

Fig. 4
figure 4

Aging and exercise effects on reactive species levels in circulating EVs from 3-, 21-, and 26-month-old Wistar rats. a 1-h groups, 3-month-old SED and EXE (n = 4); 21-month-old SED (n = 4) and EXE (n = 6); 26-month-old SED (n = 4) and EXE (n = 5). b 18-h groups, 3-month-old SED and EXE (n = 6); 21-month-old SED (n = 6) and EXE (n = 5); 26-month-old SED (n = 4) and EXE (n = 6). The columns represent the mean ± SD (% of control). Two-way ANOVA followed by Tukey’s test. #Values significantly different from 3-month-old groups; *Values significantly different from its respective sedentary

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Fig. 5
figure 5

Aging and exercise effects on SOD activity in circulating EVs from 3-, 21-, and 26-month-old Wistar rats. a 1-h groups, 3-month-old SED and EXE (n = 6); 21-month-old SED (n = 5) and EXE (n = 6); 26-month-old SED (n = 5) and EXE (n = 6). b 18-h groups, 3-month-old SED (n = 4) and EXE (n = 6); 21-month-old SED and EXE (n = 5); 26-month-old SED (n = 4) and EXE (n = 6). The columns represent the mean ± SD (% of control). Two-way ANOVA followed by Tukey’s test. #Values significantly different from 3-month-old groups

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Fig. 6
figure 6

Aging and exercise effects on SOD1 content in circulating exosomes from 3- and 26-month-old Wistar rats (n = 4). The columns represent the mean ± SD (Test t, p = 0.07)

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Finally, the Aβ1–42 peptide levels remained unchanged in all groups evaluated (two-way ANOVA; Fig. 7a, F (5,31) = 2.072; p = 0.101 and Fig. 7b, F (5,31) = 1.727; p = 0.164).

Fig. 7
figure 7

Aging and exercise effects on Aβ1-42 levels in circulating EVs from 3-, 21-, and 26- month-old Wistar rats. a 1-h groups, 3-month-old SED and EXE (n = 6); 21-month-old SED and EXE (n = 5); 26-month-old SED and EXE (n = 5). b 18-h groups, 3-month-old SED (n = 5) and EXE (n = 6); 21-month-old SED (n = 6) and EXE (n = 5); 26-month-old SED (n = 4) and EXE (n = 6). The columns represent the mean ± SD (% of control). Two-way ANOVA followed by Tukey’s test

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Exercise effects on circulating extracellular vesicles

It is important to consider that samples were obtained 1- and 18-h after the last training session of treadmill exercise to verify the transitory and delayed exercise effects, respectively; they were obtained at afternoon and early morning period.

Two-way ANOVA indicated a significant main effect of exercise; since aerobic exercise increased almost 10% CD63 levels at all ages (Fig. 2b; two-way ANOVA, F (1,33) = 4.292; p = 0.048).

An exercise effect on AChE activity (Fig. 3b; F (1,27) = 10.659, p = 0.004), as well an interaction between age and exercise factors (F (2,27) = 5.245, p = 0.014) were showed by two-way ANOVA. Moreover, a decreased AChE activity was observed in aged rats 18-h after exercise indicating a delayed effect (Fig. 3b).

In addition, there was a delayed exercise effect on DCF levels (two-way ANOVA, F (1,32) = 16.470, p < 0.001), given that our exercise protocol was able to decrease reactive species levels 18 h after the last session at all ages (Tukey post hoc, p < 0.05; Fig. 4b).

Effects of time points on circulating extracellular vesicles profile: comparison between early morning and afternoon

Two-way ANOVA showed an effect of time points only in CD63 levels (F (1,33) = 6.563, p = 0.016; Fig. 8); since higher CD63 levels were observed in young SED group in the late afternoon (1-h group). However, there was no interaction between time points and age factors (F (1,33) = 2.228, p = 0.126; Fig. 8).

Fig. 8
figure 8

Time point effects on CD63 levels in circulating extracellular vesicles profile from 3-, 21-, and 26-month-old sedentary Wistar rats. 3-month-old groups 1 h (n = 6), 18 h (n = 5); 21-month-old groups 1 and 18 h (n = 5); 26-month-old groups 1 h (n = 5), 18 h (n = 4). The columns represent the mean ± SD (% of control). Two-way ANOVA followed by Tukey’s test. *Values significantly different from 18-h group

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Discussion

Aging effects on circulating extracellular vesicles

The current study adds evidence for the role of EVs in the aging process. To our knowledge, this is the first study describing the aging process and exercise effects on circulating EVs. Interesting findings regarding peripheral exosome markers, such as CD63 levels and AChE activity, were here demonstrated in aged samples; therefore, it is possible to suggest that the aging process alters the circulating EVs profile.

Although the EVs isolation has been performed using a significantly reproducible method based on vesicles precipitation and the Nanosight™ technology was used to characterize EVs, it is impossible to exclude that other blood particles, such as lipoproteins that can share the same size range, could contaminate the isolated vesicles. However, the quantification of CD63, a specific exosome marker, was performed. Taken that aged groups had decreased CD63 levels compared to young adult rats, we can suppose that there is a decrease in circulating exosomes in aging process.

Other interesting result that emerges from this work is that AChE activity of circulating exosomes/EVs increases with age. Although some studies have used AChE activity to evaluate the exosome levels [3, 35, 36], these studies did not compare exosomes from different ages for example young and old samples. A marked decrease of central and peripheral cholinergic system, including AChE activity alterations, seems to have a central role in neurodegenerative processes and cognitive dysfunction in healthy aging process [37, 38]. Therefore, although AChE has been used as EVs marker [3], our result indicates that this enzyme is not adequate to compare EVs among different ages. Considering that substantial evidence has demonstrated an intrinsic ability of the exosomes to cross the blood–brain barrier (BBB) [39, 40], it is possible to infer that circulating exosomes can also reflect neural-secreted exosome cargo, such as AChE.

A clear temporal profile was observed in CD63 content, since aged groups have decreased exosomal CD63 levels only in the late afternoon, at the end of their habitual rest period. Taken that exosomes seem to be involved in the waste removal [41] and biomolecule clearance systems are strongly stimulated by sleep [42], it is possible to infer that our result can be related to failure of cellular waste disposal processes leading to accumulation of pathogenic proteins in aging process [43, 44].

Although Aβ levels in circulating exosomes as a potential AD progression biomarker have been suggested, this was the first work evaluating peripheral EVs Aβ levels in the healthy aging. An interesting idea regarding exosomal Aβ in different biological compartments can be proposed, since aged monkeys showed a decreased CSF exosomes Aβ levels associated to its accumulation in brain tissue [45], while in the present work there were no changes on circulating EVs Aβ content. Therefore, we suggest that this work was unable to validate peripheral EVs Aβ as a good marker of aging.

In this study, for the first time, it was observed an increased reactive species levels in circulating EVs of 21- and 26-month-old rats compared to young adult ones. At this moment, it is impossible to affirm if reactive species are generated as an intrinsic production, since studies using platelet-derived exosomes from septic shock patients suggested a reactive species generation by NADPH oxidase and nitric oxide synthase enzymes [11,12 46]. We are unable to exclude the involvement of oxidative modification of other blood microparticles isolated with exosomes, such as lipoproteins. In addition, exosomes can travel substantial distances; consequently, high reactive species content in circulating EVs could reflect its tissue levels. Therefore, we can suppose that higher reactive species levels observed in circulating exosomes from aged rats can contribute to the general oxidative stress in aging process.

As observed in CD63 levels, the impact of aging on SOD activity in EVs has a clear temporal profile, because aged rats had lesser activities compared to young adult ones in the afternoon period. Once SOD may scavenge superoxide radicals [47, 48] and exosome was related to an exchange of SOD between different cells [10], our result supports the idea that lesser protective molecules exchange contributes to an impaired cellular oxidative status in older tissues. In addition, we can suggest that the decreased SOD activity observed in 26-month-old rats is independent of SOD1 isoenzyme content. Accordantly, it is possible to infer that aging could induce SOD aggregation similarly to those observed in amyotrophic lateral sclerosis. 20% of inherited cases of ALS were linked to mutations SOD1 with high susceptibility to misfold [49]. Takeuchi et al. [50] demonstrated that secretion and intercellular transmission of chaperones can be mediated by exosomes. Chaperones usually support the proper folding of proteins preventing their aggregation [51, 52]. A number of microRNAs (miRNAs) have been identified in exosomes. In addition, SOD can be a target of miRNAs, for example, miRNA-23a-3p is able to increase [53], while miRNA-25 and miRNA-182 decreased SOD activity [54, 55]. Taken together, it is possible to suggest that unidentified compounds, such as chaperones and/or miRNAs, can interact with SOD modifying its activity.

Exercise effects on circulating extracellular vesicles

The remarkable data obtained in this study are that two-way ANOVA indicated a significant main effect of exercise, since aerobic exercise increased almost 10% CD63 levels at all ages. Therefore, we can suggest that treadmill exercise increases released exosome levels into the circulation taken that CD63 is a specific exosome-associated protein. Evidences that EVs are altered at long term of exercise are limited. Our results suggest a delayed exercise effect (18 h after the last session) without any acute effect, nevertheless Frühbeis et al. [19] demonstrated that acute exercise is able to raise small EVs levels in the blood of physically active healthy men. At this point, it is impossible to determine the source of circulating exosomes induced by exercise. Safdar et al. [56] suggested a release of exosomes from muscle, because an acute decrease on ALIX levels, another exosomal marker, in skeletal muscle following a bout of endurance exercise was observed.

Considering that the exercise seems to be able to increase the EVs release into the circulation and few cardioprotective strategies have been related to increased number of EVs in the blood [28], our results led us to the hypothesis that exercise-related release of EVs into the circulation could be associated with cardioprotective exercise effects. Vicencio et al. [57] demonstrated that plasma exosomes are able to protect the myocardium from ischemia–reperfusion injury. In accordance, increased release of EVs from the heart after preconditioning stimuli was related to cardioprotection [58].

The link between exercise and oxidative status has been widely documented [20, 21, 59]. Here, it was investigated the moderate daily aerobic exercise effects on circulating EVs, specifically reactive species content, in different developmental stages. The exercise protocol used was able to decrease EVs reactive species content in all tested ages; however, SOD activity, an antioxidant enzyme, remained unchanged. Therefore, it is possible to infer that these declines are related to a reduced reactive species generation instead of altering the EVs antioxidant system. Given that the exosomes have a capacity to cross biological barriers, a lesser reactive species content observed in circulating exosomes might reflect the effect induced by exercise on reactive species tissular production. In this context, if peripheral-derived reactive species can be transferred to CNS by the exosomes [39, 40], we can speculate that neuroprotective effects of our exercise protocol could be associated at least in part with its impact on reactive species levels observed in circulating exosomes.

In addition, exercise effects on cardio metabolic variables in patients with type 2 diabetes can be related to its impact on EVs oxidative status. Jansen et al. [60] in an elegant study using a diabetes cellular model demonstrated that an increased reactive species in EVs (100–1000 nm), including exosomes, is involved in the pathophysiology of cardiovascular complications, specifically atherosclerotic lesions. Therefore, it is possible to infer that the impact of exercise on reactive species in EVs might be related at least in part to its protective effect described in type 2 diabetes. In view of the fact that exosomes are considered as mediators of intercellular communication, regulating cell signaling, it is possible to suggest that the exercise-induced changes on exosome profile can be responsible for its wide range of beneficial effects on the body, specially, preventing or treating oxidative stress-related diseases [20, 21, 59].

A remarkable finding that emerges from this work is the exercise protocol ability to reverse the effects of aging process on EVs AChE activity. Interestingly, this effect was detected at 18 h after the last exercise session, showing a delayed effect of exercise, without any acute effect (1-h time point).

Accumulating evidences have demonstrated that the use of AChE inhibitors (AChEIs) as a therapeutic strategy is able to improve cognition in neurodegenerative disorders, such as AD [61, 62]. In this context, we can suggest a beneficial treadmill exercise cognitive effects can be at least in part related to its impact on AChE activity. Accordantly, other studies have demonstrated that exercise training is able to reduce hippocampal and cortical AChE activities impacting on cognitive functions [63, 64].

Effects of time points on circulating extracellular vesicles profile

Our results can indicate the influence of the time of the day on circulating exosome levels, since higher CD63 levels were observed in samples obtained in the late afternoon, end of the rest period, in SED group. Accordantly, it has been described that EVs vary with the time of day [58]. Although, variable time points, specifically “Zeitgeber Time” (ZT), must be evaluated to study properly the circadian rhythm, we cannot disregard the impact of the time points on CD 63 levels, as above cited, an exosome mark. This result can represent a daily rhythm of exosomes content with elevated levels at the end of the rest period. Our data give insights and might open new avenues for further studies regarding the modulation of EVs content by time of day.”

Conclusion

In this study, we provide evidence that the healthy aging process and treadmill exercise are able to modify the components of EVs. An important finding that emerges of this work is a disruption on circulating exosomes, evaluated by CD63 levels, in aged rats. Besides, increased reactive species levels were found in EVs from aged rats, which can contribute to the age-related oxidative status. Moreover, the exercise is able to impact peripheral EVs that could be related to its protective effects including in normal aging. Interestingly, the data here obtained contribute to understand and provide exciting lines of investigation about the exosomes role on healthy aging process and exercise effects.