Biogerontology (2023) 24:753–769
https://doi.org/10.1007/s10522-023-10042-1
RESEARCH ARTICLE
Long‑term detraining reverses the improvement of lifelong
exercise on skeletal muscle ferroptosis and inflammation
in aging rats: fiber‑type dependence of the Keap1/Nrf2
pathway
Zhuang‑Zhi Wang · Hai‑Chen Xu · Huan‑Xia Zhou · Chen‑Kai Zhang · Bo‑Ming Li · Jia‑Han He ·
Pin‑Shi Ni · Xiao‑Ming Yu · Yun‑Qing Liu · Fang‑Hui Li
Received: 11 December 2022 / Accepted: 27 May 2023 / Published online: 8 June 2023
© The Author(s), under exclusive licence to Springer Nature B.V. 2023
Abstract We investigated the effects of lifelong
aerobic exercise and 8 months of detraining after
10 months of aerobic training on circulation, skel-
etal muscle oxidative stress, and inflammation in
aging rats. Sprague–Dawley rats were randomly
assigned to the control (CON), detraining (DET),
and lifelong aerobic training (LAT) groups. The
DET and LAT groups began aerobic treadmill exer-
cise at the age of 8 months and stopped training at
the 18th and 26th month, respectively; all rats were
sacrificed when aged 26 months. Compared with
CON, LAT remarkably decreased serum and aged
skeletal muscle 4-hydroxynonenal (4-HNE) and
Supplementary Information The online version
contains supplementary material available at https://​doi.​
org/​10.​1007/​s10522-​023-​10042-1.
Z.-Z. Wang · C.-K. Zhang · B.-M. Li · J.-H. He · P.-S. Ni ·
F.-H. Li (*)
School of Sport Sciences, Nanjing Normal University,
Nanjing 210023, China
e-mail: 12356@njnu.edu.cn
H.-C. Xu · H.-X. Zhou · X.-M. Yu
Department of Rehabilitation, Shanghai Seventh People’s
Hospital, Shanghai University of Traditional Chinese
Medicine, Shanghai 200137, China
Y.-Q. Liu
Changzhou Sports Hospital, Changzhou 213022, China
F.-H. Li
School of Sport Sciences, Zhaoqing University,
Zhaoqing 222023, China
8-hydroxy-2-deoxyguanosine (8-OHdG) levels.
Superoxide dismutase 2(SOD2) levels were higher
in the LAT group than in the CON group in skel-
etal muscle. However, DET remarkably decreased
SOD2 protein expression and content in the skeletal
muscle and increased malondialdehyde (MDA) level
compared with LAT. Compared with LAT, DET
remarkably downregulated adiponectin and upregu-
lated tumor necrosis factor alpha (TNF-α) expres-
sion, while phosphoinositide 3-kinase (PI3K), protein
kinase B (AKT), and 70-kDa ribosomal protein S6
kinase (P70S6K) protein expression decreased, and
that of FoxO1 and muscle atrophy F-box (MAFbX)
proteins increased in the quadriceps femoris. Adi-
ponectin and TNF-α expression in the soleus mus-
cle did not change between groups, whereas that of
AKT, mammalian target of rapamycin (mTOR), and
P70S6K was lower in the soleus in the DET group
than in that in the LAT group. Compared with that
in the LAT group, sestrin1 (SES1) and nuclear fac-
tor erythroid 2–related factor 2 (Nrf2) protein expres-
sion in the DET group was lower, whereas Keap1
mRNA expression was remarkably upregulated in
the quadriceps femoris. Interestingly, the protein and
mRNA levels of SES1, Nrf2, and Keap1 in soleus
muscle did not differ between groups. LAT remark-
ably upregulated ferritin heavy polypeptide 1(FTH),
glutathione peroxidase 4(GPX4), and solute carrier
family 7member 11 (SLC7A11) protein expression in
the quadriceps femoris and soleus muscles, compared
with CON. However, compared with LAT, DET
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downregulated FTH, GPX4, and SLC7A11 protein
expression in the quadriceps femoris and soleus mus-
cles. Long-term detraining during the aging phase
reverses the improvement effect of lifelong exercise
on oxidative stress, inflammation, ferroptosis, and
muscle atrophy in aging skeletal muscle. The quadri-
ceps femoris is more evident than the soleus, which
may be related to the different changes in the Keap1/
Nrf2 pathway in different skeletal muscles.
Keywords Detraining · Aged skeletal muscle ·
Oxidation stress · Inflammatory · Nrf2 pathway
Introduction
Aging is a process of systemic tissue and organ func-
tion loss in which abnormal changes in body compo-
sition occur (Ponti et al. 2020), chronic inflammation,
and oxidative stress (Liguori et al. 2018; Furman et al.
2019), which is a major contributor to aging-related
chronic diseases, such as cardiovascular disease, type
2 diabetes, sarcopenia, and even disability. Regular
physical training is an effective measure for main-
taining physical health, resisting oxidative stress and
chronic diseases, and promoting healthy aging (Nal-
bant et al. 2009; Mora and Valencia 2018). Lifelong
endurance exercise is closely associated with a longer
life span and a delay in the onset of chronic diseases
(Ruegsegger and Booth 2018). Further, lifelong aero-
bic exercise with intensity of 60%-75%VO2max four
to seven times a week can improve body composi-
tion (Carrick-Ranson G et al. 2014), downregulate the
expression of circulating inflammatory factors, such
as high-sensitivity C-reactive protein (hsCRP) (Mik-
kelsen et al. 2013), increase superoxide dismutase 2
(SOD2) capacity (Barranco-Ruiz et al. 2017), and
reduce skeletal muscle oxidative stress (Belaya et al.
2018), ultimately maintaining skeletal muscle meta-
bolic function (Liang et al. 2021; Baek et al. 2022).
Considering that the benefits of exercise are
dependent on adherence to training programs, because
of subjective or objective reasons, some people, such
as professional athletes and those with disabilities
caused by accidents, may not be able to achieve life-
long or maintain high-intensity physical exercise
(Mujika and Padilla 2000a, b). Short- or long-term
training stimulation is insufficient or if training is
stopped; this process is termed the detraining stage,
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and the adaptive effect caused by training will be par-
tially or completely lost (Mujika and Padilla 2000a,
b). Compared with lifelong exercise, detraining leads
to an increase in body obesity and the content of cir-
culating serum inflammatory factors in rats (Kilic-
Erkek et al. 2016). Moreover, the degree of exercise
benefit loss caused by detraining is affected by the
degree of aging (Toraman 2005). Compared with
those of young people, the muscle mass and physi-
cal performance of older people decrease to a greater
extent after detraining (Ivey et al. 2000; Bickel et al.
2011), which has been hypothesized to be partially
caused by an increase in body inflammation and oxi-
dative stress levels because of aging itself (Pyo et al.
2020). In addition, the decline in exercise adaptation
after detraining may be related to the type and dura-
tion of exercise and detraining. After 9 and 24 weeks
of resistance training with intensity of 75–80% 1RM,
6 and 12 weeks of detraining caused only a slight
decline in skeletal muscle mass and physical perfor-
mance among older people, whereas 31 and 52 weeks
of detraining led to a complete loss of exercise adap-
tation or even lower than the pre-training level (Taaffe
and Marcus 1997; Lemmer et al. 2000; Ivey et al.
2000; Toraman 2005). Moreover, compared with
resistance exercise, aerobic exercise with the same
detraining duration results in a higher loss of exercise
adaptation (Lo et al. 2011). In rats, detraining over
4–6 weeks led to the regression of 8 weeks of endur-
ance training with intensity of 50–60% V ­ O2max for
physical performance adaptation to the baseline level
or even more (Bocalini et al. 2010; Lee et al. 2017;
Sertie et al. 2019). Moreover, 2–4 weeks of detraining
in rats led to the loss of exercise benefits of reducing
the level of peroxidation and inflammation caused by
8–12 weeks of aerobic exercise with a running speed
of 26 m·min−1 (Bradic J et al 2018; Dinari Ghozhdi
et al. 2021). Together, these studies indicate that the
loss of exercise adaptation gradually increases with
the extension of detraining duration. However, the
exercise and detraining times in previous studies
were relatively short (most studies lasted < 4 months),
and whether a longer detraining program would lead
to a greater loss of exercise adaptation is yet to be
determined.
The skeletal muscle plays an important role in
maintaining metabolic homeostasis and is an impor-
tant energy metabolism and endocrine organ. How-
ever, skeletal muscles in the aging body are more
Biogerontology (2023) 24:753–769
prone to oxidative stress and inflammation, which
accelerate aging skeletal muscle atrophy and meta-
bolic dysfunction (Szentesi et al. 2019). Ferroptosis
is a cell death process caused by oxidative stress-
induced lipid peroxidation, which can also accel-
erate skeletal muscle atrophy during aging (Chen
et al. 2022a, b; Alves et al. 2022). In addition. the
Keap1/Nrf2 pathway as an important oxidative stress
defense mechanism in various tissues (Bellezza et al.
2018), and activation of the nuclear factor eryth-
roid 2–related factor 2 (Nrf2) pathway can inhibit
the occurrence of ferroptosis (Anandhan et al. 2020;
Chen et al. 2022a, b). Moreover, aerobic exercise can
activate the Nrf2 pathway and improve skeletal mus-
cle quality and exercise performance in aged rats (Yan
et al. 2022). However, it remains unclear whether
long-term detraining inhibits the activation of the
Keap1/Nrf2 pathway in aging skeletal muscles and
causes an increase in oxidative stress and ferroptosis,
ultimately leading to a decline in exercise efficiency.
Based on these observations, the present study
examined the differences in the effects of lifelong
aerobic training (LAT) and 10-month aerobic train-
ing, following 8 months of training cessation (i.e., not
exercising during the aging phase (Ma et al. 2020))
on circulating serum, skeletal muscle oxidative stress
and ferroptosis, and the Keap1/Nrf2 pathway in natu-
rally aging rats.
Materials and methods
Animals
Thirty-six Sprague–Dawley female (SD) rats
aged ~ 8 months were purchased from the Guang-
dong Medical Animal Experimental Center (Animal
license number: SCXK [Guangdong] 2013-0002),
where free feed and supplies were increased accord-
ing to weight gain and natural light; the bedding
was changed 2–3 times per week, the temperature
was 20–23 °C, and relative humidity was 50–70%.
Cages were ventilated and dried, and the Guang-
dong Medical Animal Experimental Center provided
national standard rodent routine feed and bedding
material. Animal experiments were conducted in
accordance with the Animal Management Regula-
tions proposed by the Ministry of Health of China
and approved by the Ethics Committee of Guangdong
755
Medical Animal Experimental Center. All the
experiments were approved by the animal experi-
ment ethical checklist of Nanjing Normal University
(IACUU-1903006).
Experimental design
SD rats were adaptively fed for two weeks, followed
by adaptive treadmill exercise for one week. The
animals were randomly divided into three groups
(n = 12 rats/group): control (CON), detraining (DET),
and LAT. The animal experimental platform [model
FD000043] was purchased from Guangzhou Feidi
Biological Technology Co., Ltd.
The formal training period was 18:30–22:00 from
Monday to Friday, and training was stopped on Sat-
urdays and Sundays. The maximal oxygen uptake
­(VO2max) of the 24 rats in the DET and LAT groups
was determined using a breath analysis exercise test.
The exercise program for formal training intensity
and time changes was based on the work of Li (2018)
and Ramez (2019). Before and after formal train-
ing, the rats were allowed to warm up and relax by
running at a speed of 10 m·min−1 (intensity 30–40%
­VO2max) for 1 min. The DET and LAT groups were
trained together at 17 m·min−1 (75–80% V ­ O2max)
until the 18th month. The DET group stopped train-
ing, and the LAT group continued training until the
26th month; the total exercise time was 45 min/day,
five days/week. A schematic of the experimental
design is shown in Fig. 1. To eliminate noise interfer-
ence from animal experiments, rats in the CON group
were placed next to the animal running platform with-
out exercise. Of note, two rats from the CON group
(owing to severe eye infection) and one from the DET
group died from exercise-unrelated causes, and one
rat from the LAT group died owing to non-adherence
to the training protocol.
Body composition
The body composition of the rats was determined
using dual-energy X-ray 48 h after the last train-
ing cycle (to avoid acute effects). Before testing, the
rats were anesthetized by intraperitoneal injection of
10% chloral hydrate (3 ml·kg−1) and placed prone
on an XR-36Norland dual-energy X-ray absorption
platform (Unigamma X-ray Plus, Cerro Maggiore,
Milano, Italy). Rats were scanned in small animal
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Fig. 1  Schematic diagram of experimental design
mode and observed for body weight (BW), lean mass,
and fat mass. Finally, the relative index was corrected
for BW.
Sampling
All rats were anesthetized with 10% chloral hydrate
at a dose of 4 ml·kg−1. Blood was collected from the
abdominal aorta, the rats were euthanized by decapi-
tation under anesthesia, and 1.5 ml of blood mixed
with sodium heparin was stored in 5-ml EP tubes for
each rat. The blood was incubated at 23 °C ± 2 °C for
30 min and then incubated at 4 °C for 1 h followed by
centrifugation for 15 min at 3500 r·min−1 to separate
serum and plasma; the remaining serum and plasma
were divided into freezing tubes and stored in liquid
nitrogen. The gastrocnemius, quadriceps femoris,
extensor digitorum longus, and soleus muscles were
rapidly removed and weighed, and the ratio of skel-
etal muscle mass to BW of each rat was used as the
skeletal muscle index. All tissues were wrapped in
aluminum foil and stored in a freezer at − 80 °C for
future experiments.
Biochemical tests
Malondialdehyde (MDA, #A003-1) content and
SOD2 (#A001-2-1) activity were determined using
a colorimetric method. An enzyme-linked immuno-
sorbent assay was used to determine trans-4-hydroxy-
2-nonenal (4-HNE, #H268), 8-hydroxy deoxyguano-
sine (8-OHdG, #H165), and insulin-like growth factor
1 (IGF-1, #H041) levels. All kits used were purchased
from the Nanjing Jiancheng Bioengineering Institute
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(Nanjing, China). All procedures were performed in
strict accordance with the manufacturer’s instruc-
tions. Each index was measured three times, and the
mean values were calculated.
Western blotting
In present study, we randomly selected 3 rats from
each group for western blotting experiment. Pro-
teins were extracted from the quadriceps femo-
ris (the vastus lateralis in the quadriceps femoris
was extracted in the present study) and soleus tis-
sues via standard methods using RIPA lysis buffer
(150 mM sodium chloride, 1.0% (v/v) Triton X-100,
0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate [SDS], 50 mM Tris, pH = 8.0) containing
protease (Complete Protease Inhibitor Cocktail Tab-
lets, Roche) and phosphatase inhibitors (PhosSTOP
Phosphatase Inhibitor Cocktail Tablets, Roche).
After centrifugation, the supernatant was used for
subsequent western blotting using 12% SDS-PAGE
with a 5% stacking gel on a Bio-Rad electropho-
resis system (Hercules, CA, USA) and then trans-
ferred to a nitrocellulose membrane. The mem-
branes were blocked and incubated overnight at
4 °C with primary antibodies. The membrane was
later incubated with horseradish peroxidase-conju-
gated anti-rabbit IgG (1:10000 dilution; AF7021,
Affinity Biosciences) secondary antibody, incubated
for 1.5 h at approximately 20 °C, and then washed
with TBST. All the antibodies used are listed in
Table 1. All antibodies purchased and used in the
present study have been validated in other studies,
and the reference list is provided in Supplementary
Biogerontology (2023) 24:753–769 757

Table1  All antibody used for western blot analysis level was calculated as follows: relative expression
Proteins Dilution ratio Manufacturer brand Art.No. level = ratio of the target protein gray value/β-actin
(article gray value.
number)
RNA isolation and reverse transcription‑quantitative
SOD2 1:1000 Biosso bs-1080R
PCR (RT‑qPCR)
Adiponectin 1:1000 Biosso bs-0471R
TNF-α 1:1000 Affinity AF7014
Total RNA was isolated from the quadriceps femo-
PI3K 1:1000 Affinity AF6241
AKT 1:1000 Affinity AF6261
ris (the vastus lateralis in the quadriceps femoris
mTOR 1:1000 Affinity AF6308
was extracted in the present study) and soleus tissues
FoxO1 1:1000 Affinity AF3461
using TRIzol reagent (R401-01, Vazyme, Nanjing,
P70S6K 1:1000 Affinity AF6226
China) according to the manufacturer’s instructions.
MAFbX 1:1000 Affinity DF7075
Briefly, total RNA (1 μg) was reverse-transcribed into
SES1 1:1000 Biosso bs-8325R
cDNA using the HiScript II first-strand cDNA syn-
Keap1 1:1000 Biosso bs-4900R
thesis kit (R211-02, Vazyme). The cDNA was then
Nrf2 1:1000 Servicebio GB113808
mixed with ChamQ SYBR qPCR reagent (Q331-02,
FTH 1:1000 ABmart T55648F
Vazyme) and amplified using a StepOnePlus real-
GPX4 1:1000 ABmart T56959F
time PCR system (Rockford Thermoelectric Science,
SLC7a11 1:1000 ABmart T57046F
IL, USA). The relative mRNA abundance was nor-
β-actin 1:1000 Biosso bs-0061R
malized to the expression of the housekeeping gene
glyceraldehyde phosphate dehydrogenase (GAPDH).
The PCR primer sequences used in this study are
listed in Table 2.
Information. The bands were visualized using an
enhanced chemiluminescence detection kit (Amer- Statistical analysis
sham Pharmacia Biotech, Little Chalfont, UK).
Chemiluminescent signals were captured using the GraphPad Prism software (version 7.0) was used for
Bio-Rad ChemiDoc XRS system. The resulting data analysis and mapping. The data are expressed as
images were quantified using the ImageJ software mean ± standard error of the mean (SEM). Training
(NIH, Bethesda, MD, USA). The relative expression intervention differences among the CON, DET, and

Table 2  Primer sequences for quantitative real-time PCR

Gene name Sequence 5′-3′ (forward) Sequence 5′-3′ (reverse)

CAT​ CTA​TTG​CCG​TCC​GAT​TCT​CCA​CAG​ CAC​AAG​GTC​CCA​GTT​ACC​ATC​TTC​AG

SOD1 CCA​GTT​GTG​GTG​TCA​GGA​CAG​ATT​AC GGA​CCG​CCA​TGT​TTC​TTA​GAG​TGA​G
ACSL4 ATC​ATG​CGG​TGC​TGG​AAC​AGT​TAC​ TGG​ACA​ATT​CTT​CAG​TGC​GGC​TTC​
GPX4 CCG​ATA​CGC​CGA​GTG​TGG​TTTAC​ TAG​AGA​TAG​CAC​GGC​AGG​TCC​TTC​
SES1 ATG​AAG​TGA​GGT​GGG​AGG​GTCTG​ GGG​ACA​TCT​CTT​GGA​CAG​CAT​AAG​C
SES2 AGC​CTA​CAG​CCT​CAC​CTA​TAA​CAC​C GTA​CAT​TCT​GCG​GGT​CGT​CTT​CTC​
SES3 TCG​TGG​TCC​CTG​GAG​AGA​CTTTC​ CAG​CGT​GGT​AGT​GTC​AAC​ATC​CTC​
Keap1 AGT​GCT​CAA​CCG​CTT​GCT​GTATG​ GCG​ATG​CTT​CAT​GGA​GGC​TACG​
Nrf2 TTA​AGC​AGC​ATA​CAG​CAG​GAC​ATG​G GAC​TGA​CTA​ATG​GCA​GCA​GAG​GAA​G
FPN TCC​CAG​ATC​GCA​GAA​CCC​TTCC​ CTG​AGA​ACA​GAC​CAG​TCC​GAA​CAA​G
FTH GAA​CCA​GCG​AGG​TGG​ACG​AATC​ CCC​AGG​GTG​TGC​TTG​TCA​AAGAG​
FTL GAT​CGG​GAT​GAC​GTG​GCT​TTGG​ TAC​GGA​GGT​TGG​TCA​GGT​GGTTG​
HO-1 GGA​ACT​TTC​AGA​AGG​GTC​AGG​TGT​C GCT​GTG​TGG​CTG​GTG​TGT​AAGG​
GAPDH TGC​CGC​CTG​GAG​AAA​CCT​GC TGA​GAG​CAA​TGC​CAG​CCC​CA

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LAT groups were assessed using one-way analysis of
variance (ANOVA) followed by Fisher’s LSD post-
hoc test. For all calculations, p < 0.05 indicated a sta-
tistically significant difference.
Results
Changes in body composition and relative weight of
rat skeletal muscle
Compared with the CON and LAT groups, the DET
group showed significantly higher fat-to-lean mass
(p < 0.05, Fig. 2a). Moreover, the DET group exhib-
ited a lower lean mass to BW % ratio than did the
CON group (p < 0.01, Fig. 2f). The LAT group
exhibited significantly higher relative weights of
the quadriceps femoris (p < 0.05) and gastrocne-
mius (p < 0.05) than did the CON group (Fig. 2g, j).
The DET group showed significantly lower relative
weights of the quadriceps femoris (p < 0.01), gas-
trocnemius (p < 0.01), soleus (p < 0.05), and extensor
digitorum longus (p < 0.05) muscles than did the LAT
group (Fig. 2g–j).
Fig. 2  Body composition and Relative weight of skeletal
muscle in each group of rats. Fat to lean mass (a), gastrocne-
mius weight (b), soleus weight (c), extensor digitorum longus
weight (d), quadriceps femoris weight (e), Lean mass to BW
(f), Relative gastrocnemius weight (g), Relative soleus weight
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Changes in protein synthesis and degradation‑related
protein expression
Compared with that in the CON group, the expres-
sion of phosphoinositide 3-kinase (PI3K) (p < 0.05),
protein kinase B (AKT) (p < 0.01), mammalian target
of rapamycin (mTOR) (p < 0.05), and 70-kDa ribo-
somal protein S6 kinase (P70S6K) (p < 0.05) pro-
teins were significantly upregulated in the quadriceps
femoris of the LAT group (Fig. 3f–h, n). However,
compared with that in the LAT group, the expres-
sion of PI3K (p < 0.05), AKT (p < 0.01), and P70S6K
(p < 0.05) proteins was significantly downregulated
in the quadriceps femoris of the DET group, and
that of FoxO1 (p < 0.05) and muscle atrophy F-box
(MAFbX) (p < 0.05) was significantly upregulated
(Fig. 3f, g, l–n). Compared with that in the CON
group, the expression of AKT (p < 0.01) and P70S6K
(p < 0.01) proteins was significantly upregulated
in the soleus muscle in the LAT group (Fig. 3d, k).
However, compared with that in the LAT group, the
expression of AKT (p < 0.05), mTOR (p < 0.05), and
P70S6K (p < 0.05) proteins was significantly down-
regulated in the soleus muscle in the DET group
(Fig. 3d, e, k). Interestingly, there were no significant
(h), Relative extensor digitorum longns weight (i), Relative
quadriceps femoris weight (j). All data were analyzed by one-
way ANOVA followed by Fisher’s LSD post-hoc test. Data are
presented as means ± SEM. *Represents p < 0.05; **Repre-
sents p < 0.01
Biogerontology (2023) 24:753–769
Fig. 3  Protein synthesis and degradation-related protein
expression in each group of rats. Western blotting bands of
protein synthesis and degradation-related protein expression
in soleus (a); Western blotting bands of protein synthesis and
degradation-related protein expression in quadriceps femo-
ris (b). Phosphatidylinositide 3-kinases, PI3K in soleus (c);
Protein kinases B, AKT in soleus (d); Mammalian Target of
Rapamycin, mTOR in soleus (e); PI3K in quadriceps femo-
differences in the expression of FoxO1 and MAFbX
proteins in the soleus between the groups (Fig. 3i, j).
Changes in the expression of skeletal muscle
inflammation‑related proteins
As shown in Fig. 4, compared to the CON group,
LAT significantly upregulated the protein expression
of adiponectin (p < 0.01) in the quadriceps femoris
(Fig. 4e). However, compared to the LAT group, DET
significantly downregulated the protein expression of
adiponectin (p < 0.05) and upregulated that of tumor
necrosis factor alpha (TNF-α; p < 0.05) in the quadri-
ceps femoris (Fig. 4e, f). Interestingly, there were no
significant differences in the expression of adiponec-
tin and TNF-α proteins in the soleus between the
groups (Fig. 4c, d). IGF-1 levels were significantly
759
ris (f); AKT in quadriceps femoris (g); mTOR in quadriceps
femoris (h); Fork head Box 01, FoxO1 in soleus (i), Muscle
atrophy F-box, MAFbX in soleus (j); P70 ribosomal protein
S6 kinase, P70S6K in soleus (k); FoxO1 in quadriceps femoris
(l); MAFbX in quadriceps femoris (m); P70S6K in quadriceps
femoris (n). All data were analyzed by one-way ANOVA fol-
lowed by Fisher’s LSD post-hoc test. All data are presented as
means ± SEM. *Represents p < 0.05; **Represents p < 0.01
higher in the gastrocnemius of the LAT group than
in that of the CON group (p < 0.01). In contrast, the
IGF-1 content of the gastrocnemius of the DET group
was significantly lower (p < 0.05) than those of the
CON and LAT groups (Fig. 5a).
Changes in the levels of serum and skeletal muscle
oxidative stress markers
Compared with that in the CON group, the serum
levels of 4-HNE (p < 0.05) and 8-OHdG (p < 0.05) in
the LAT group were significantly decreased; the lev-
els of 8-OHdG in the DET (p < 0.01) group were also
significantly decreased (Fig. 5b, c). Compared with
that in the gastrocnemius of the CON group, SOD2
(p < 0.01) content in the gastrocnemius of the LAT
group increased significantly, whereas the content of
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Fig. 4  Skeletal muscle inflammation-related proteins in each
group of rats. Western blotting bands of inflammation-related
proteins in soleus (a); Western blotting bands of inflamma-
tion-related proteins in quadriceps femoris (b). Adiponectin
in soleus (c); Tumor Necrosis Factor-α, TNF-α in soleus (d);
Fig. 5  Serum and skeletal muscle oxidative stress markers
in each group of rats. Insulin-like growth factors 1, IGF-1 in
gastrocnemius (a); 4-Hydroxynonena, 4-HNE in serum (b);
8-hydroxy-2′-deoxyguanosine, 8-OHdG in serum (c); Superox-
ide Dismutase 2, SOD2 in serum (d); Malondialdehyde, MDA
in gastrocnemius (e); 4-HNE in gastrocnemius (f); 8-OHdG in
gastrocnemius (g); SOD2 in gastrocnemius (h); Western blot-
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Adiponectin in quadriceps femoris (e); TNF-α in quadriceps
femoris (f). All data were analyzed by one-way ANOVA fol-
lowed by Fisher’s LSD post-hoc test. All data are presented as
means ± SEM. *Represents p < 0.05; **Represents p < 0.01
ting bands of SOD2 in soleus (i); Western blotting bands of
SOD2 in quadriceps femoris (j); SOD2 in soleus (k); Super-
oxide Dismutase 1(SOD1) and Catalase (CAT) in soleus (l);
SOD2 in quadriceps femoris (m); SOD1 and CAT in quadri-
ceps femoris (n). All data were analyzed by one-way ANOVA
followed by Fisher’s LSD post-hoc test. All data are presented
as means ± SEM. *Represents p < 0.05; **Represents p < 0.01
Biogerontology (2023) 24:753–769
4-HNE (p < 0.05) and 8-OHdG (p < 0.05) decreased
significantly (Fig. 5f–h). However, compared with
that in the gastrocnemius of the LAT group, the con-
tent of SOD2 in the gastrocnemius of the DET group
decreased significantly (p < 0.01), whereas that of
MDA (p < 0.01) increased significantly (Fig. 5e, h).
LAT significantly increased the protein expression
of SOD2 in the quadriceps femoris (p < 0.05) and
soleus (p < 0.01) compared with that in the CON
group (Fig. 5k, m). However, compared with that in
the muscles of the LAT group, SOD2 protein expres-
sion in the quadriceps femoris (p < 0.05) and soleus
(p < 0.01) muscles of the DET group decreased sig-
nificantly (Fig. 5k, m). Compared with the CON
group, LAT significantly increased CAT​ (p < 0.05)
and SOD1 (p < 0.05) mRNA expression in the quadri-
ceps femoris (Fig. 5n). However, compared with that
in the LAT group, CAT​ and SOD1 mRNA expres-
sion in the quadriceps femoris of the DET group
decreased, although the difference was not significant
(Fig. 5n). There were no significant differences in the
mRNA expression levels of CAT​ and SOD1 in the
soleus muscle of any group (Fig. 5l).
Changes in the Nrf2 pathway and ferroptosis‑related
mRNA and protein expression in the quadriceps
femoris and soleus
Compared with the CON group, LAT significantly
upregulated SES1 (p < 0.05), SES2 (p < 0.05), and
SES3 (p < 0.01) mRNA expression in the quadriceps
femoris but downregulated Keap1 (p < 0.01) mRNA
expression (Fig. 6a). However, compared with that
in the quadriceps femoris of the LAT group, SES3
(p < 0.05) mRNA expression was significantly down-
regulated in the quadriceps femoris of the DET
group, whereas Keap1 (p < 0.05) mRNA expression
was upregulated (Fig. 6a). The mRNA levels of SES1,
SES2, and NRF2 also decreased in the DET group,
but this difference was not significant (Fig. 6a). There
were no significant differences in the mRNA expres-
sion of SES1, SES2, SES3, Keap1, and Nrf2 in the
soleus between the groups (Fig. 6j). Compared with
that in the CON group, the expression levels of ses-
trin1 (SES1) (p < 0.05) and Nrf2 (p < 0.05) proteins
were significantly increased in the quadriceps femo-
ris in the LAT group, but there was no significant
difference in Keap1 protein expression (Fig. 6d–f).
However, the expression of SES1 (p < 0.05) and
761
Nrf2 (p < 0.05) proteins decreased in the quadri-
ceps femoris of the DET group compared with that
in the quadriceps femoris of the LAT group (Fig. 6d,
f), although there were no significant differences
in SES1, Keap1, and Nrf2 protein expression in the
soleus muscle of each group (Fig. 6m–o).
Compared with that in the quadriceps femo-
ris of the CON group, the expression of FTL
(p < 0.05) mRNA and ferritin heavy polypeptide 1
(FTH) (p < 0.01), glutathione peroxidase 4 (GPX4)
(p < 0.05), and solute carrier family 7member 11
(SLC7A11) (p < 0.05) proteins in the quadriceps
femoris of the LAT group increased significantly,
and NOX4 (p < 0.05) and ACSL4 (p < 0.05) mRNA
expression decreased significantly (Fig. 6b, g–i).
However, compared with that in the quadriceps femo-
ris of the LAT group, FTH (p < 0.05) and SLC7A11
(p < 0.05) protein expression was significantly down-
regulated in the quadriceps femoris of the DET
group, whereas HO-1 (p < 0.05) mRNA expression
was upregulated (Fig. 6b, g, i). The expression of
FTL and FPN (p < 0.05) mRNA and GPX4 protein
also decreased in the DET group compared with
that in the quadriceps femoris of the LAT group, but
the difference was not significant (Fig. 6b, h). Com-
pared with that in the CON group, the expression
of GPX4 (p < 0.01) and FTL (p < 0.01) mRNA and
FTH (p < 0.01) and SLC7A11 (p < 0.05) proteins was
upregulated significantly in the soleus of the LAT
group, and NOX4 (p < 0.01) and ACSL4 (p < 0.01)
mRNA expression was downregulated (Fig. 6k, p,
r). However, the expression of FTL (p < 0.05) mRNA
and FTH (p < 0.01), GPX4 (p < 0.05), and SLC7A11
(p < 0.01) proteins was significantly downregulated in
the soleus of the DET group, compared with that in
the soleus of the LAT group (Fig. 6k, p–r).
Discussion
Evidence has shown that the proportion of lean mass
decreases and the proportion of fat increases dur-
ing aging (Gordon et al. 2016), accompanied by the
redistribution of fat, which manifests as a decrease in
subcutaneous fat and an increase in visceral fat (Kuk
et al. 2009). Moreover, age-related changes in body
composition are closely related to abnormal skeletal
muscle function (Sternfeld et al. 2002), and the loss
of skeletal muscle mass and function can lead to a
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Fig. 6  Keap1/Nrf2 pathway and ferroptosis-related mRNA
and protein expression in quadriceps femoris and soleus
in each group of rats. The expression of Sestrin1(SES1),
Sestrin2(SES2), Sestrin3(SES3), Kelch-1ike ECH-associated
protein l(Keap1), and Nuclear factor-erythroid 2-related factor
2(Nrf2) mRNA in quadriceps femoris (a); The expression of
NADPH Oxidase 4(NOX4), Ferritin Light polypeptide(FTL),
Ferroportin(FPN), Acyl-CoA Synthetase Long-Chain Family
Member 4(ACSL4), and Heme Oxygenase-1(HO-1) mRNA
in quadriceps femoris (b); Western blotting bands of ferrop-
tosis and Nrf2 pathway related proteins in quadriceps femo-
decline in basic living ability and increase the risk
of falls and mortality among older people (Gschwind
et al. 2013). In the present study, we found that the
fat-to-lean mass ratio of aging rats was lower in the
CON and LAT groups than in the DET group. Moreo-
ver, in our previous study, we found that LAT signifi-
cantly reduced the BW and fat mass of aging rats, but
DET reversed this change (Sun et al. 2021). In addi-
tion, the ratio of lean mass to BW was lower in the
DET group than in the CON group, which is similar
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Biogerontology (2023) 24:753–769
ris (c); The expression of SES1 (d), Keap1 (e), Nrf2 (f), FTH
(g), GPX4 (h), and SLC7a11 (i) protein in quadriceps femo-
ris; The expression of SES1, SES2, SES3, Keap1, and Nrf2
mRNA in soleus (j); The expression of Glutathione Peroxidase
4 (GPX4), FTL, HO-1, NOX4, and ACSL4 mRNA in soleus
(k); Western blotting bands of ferroptosis and Nrf2 pathway
related proteins in soleus (l); The expression of SES1 (m),
Keap1 (n), Nrf2 (o), FTH (p), GPX4 (q), and SLC7a11 (r)
protein in soleus. All data were analyzed by one-way ANOVA
followed by Fisher’s LSD post-hoc test. All data are presented
as means ± SEM. *Represents p < 0.05; **Represents p < 0.01
to a previous study in which 4 weeks of detraining
increased obesity in rats (Sertie et al. 2019). These
results suggest that long-term detraining aggravates
fat accumulation and increases obesity in aging rats.
The accumulation of adipose tissue is also
related to increased inflammation levels in the aging
body (Yang et al. 2018), particularly in visceral adi-
pose tissue (Tchkonia et al. 2010). Lifelong aerobic
exercise effectively reduces the level of systemic
inflammation in naturally aging rats (Nilsson et al.
Biogerontology (2023) 24:753–769
2019). In addition, as an important cytoprotective
factor, Nrf2 may mediate the effect of exercise on
inflammatory gene expression in the skeletal muscle
(Done and Traustadóttir 2016; Tonolo et al. 2022).
Some studies have found that aerobic exercise with
50% of the maximal running speed can activate
Nrf2 and effectively alleviate inflammation in skele-
tal muscles (Toledo-Arruda et al. 2020). Consistent
with this, we also observed that LAT upregulated
Nrf2 protein expression, downregulated TNF-α pro-
tein expression, and increased adiponectin levels in
the quadriceps femoris, whereas DET reversed these
changes. Interestingly, LAT and DET did not induce
changes in Nrf2 protein or mRNA expression in the
soleus muscle. Consistent with these findings, there
were no significant differences in adiponectin and
TNF-α levels in the soleus muscle in each group.
These results indicate that compared with lifelong
exercise, long-term detraining may increase the
level of inflammation in skeletal muscle by inhibit-
ing the Nrf2 pathway, but there may be differences
in different muscle fiber types.
The occurrence of age-related skeletal muscle
atrophy is also related to an increase in inflamma-
tion levels in the skeletal muscle (Liang et al. 2022),
which can affect skeletal muscle hypertrophy and
atrophy by regulating the relevant pathways of skel-
etal muscle protein synthesis and degradation (Wang
2022). TNF-α can accelerate protein degradation
mediated by the PI3K/AKT pathway in the skeletal
muscle (Wang et al. 2014; Webster et al. 2020), and
adiponectin can reverse this process (Singh et al.
2017; Morinaga et al. 2021). As mentioned above,
compared with LAT, DET downregulated adiponec-
tin expression and upregulated TNF-α expression in
the quadriceps femoris. In addition, as a key growth
factor regulating both the anabolic and catabolic
pathways in the skeletal muscle (Yoshida and Dela-
fontaine 2020), IGF-1 can increase skeletal muscle
protein synthesis via the PI3K/Akt/mTOR path-
way and inhibit protein degradation mediated by
the PI3K/AKT/FoxO pathway (Timmer et al. 2018;
Yoshida and Delafontaine 2020). In present study,
we observed that LAT significantly increased IGF-1
content in the skeletal muscle but decreased it in the
DET group. These results indicate that DET reverses
the protective effect of LAT on aging skeletal mus-
cle atrophy. Therefore, we further explored changes
763
related to protein synthesis and skeletal muscle atro-
phy in rats of each group. LAT significantly upreg-
ulated the expression of PI3K, AKT, mTOR, and
P70S6K in the quadriceps femoris and soleus, sug-
gesting that lifelong aerobic exercise promotes pro-
tein synthesis. This is consistent with the increase in
the relative mass of skeletal muscle in the LAT group
compared with that in the CON group. However, DET
significantly downregulated the protein expression of
PI3K, AKT, and P70S6K and significantly upregu-
lated that of FoxO1 and MAFbX in the quadriceps
femoris. This is consistent with the observation that
the relative mass of skeletal muscle in the DET group
was significantly lower than that in the LAT group,
which further corroborates with our previous study’s
finding that DET significantly reverses the improve-
ment of grip power and tension force by LAT in aging
rats (Sun et al. 2021). These results suggest that the
lifelong exercise benefit of resisting aging skeletal
muscle atrophy is lost during 8 months of detrain-
ing. Taken together, these results show that lifelong
aerobic exercise can improve the anti-inflammatory
ability of aging rats and maintain the mass and func-
tion of aging skeletal muscles. However, 8 months of
detraining in the aging phase increased the level of
inflammation in aging rats, which led to an increase
in protein degradation and atrophy of aging skeletal
muscle.
In addition to its anti-inflammatory effects, adi-
ponectin has been shown to resist skeletal muscle
oxidative stress (Abou-Samra 2020). Specifically, adi-
ponectin can induce SOD1 and CAT​ mRNA expres-
sion in the skeletal muscle by targeting Nrf2 (Ren
et al. 2017), ultimately improving the antioxidant
capacity of the skeletal muscle. Moreover, exercise-
induced improvements in inflammation and skeletal
muscle function are related to reduced oxidative stress
(Bar-Shai et al. 2008; El Assar et al. 2022). There-
fore, we further explored whether LAT and DET
affected the corresponding changes in oxidative stress
in rats. We found that LAT reduced the serum levels
of 4-HNE and 8-OHdG. Simultaneously, LAT upreg-
ulated SOD2 protein expression in the quadriceps
femoris and soleus, particularly in the quadriceps
femoris. However, it is worth noting that LAT signifi-
cantly increased CAT​and SOD1 mRNA expression in
the quadriceps femoris but not in the soleus muscle.
LAT also upregulated adiponectin and Nrf2 protein
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expression in the quadriceps femoris instead of in
the soleus, which is consistent with previous research
(Jiménez-Maldonado et al. 2019). These findings
indicate that improvements in the antioxidant capac-
ity of aging skeletal muscle by lifelong aerobic exer-
cise may involve differences in muscle fiber types,
and adiponectin may mediate such differences. Con-
sistent with this conjecture, the expression of CAT​
and SOD1 mRNA and adiponectin protein was down-
regulated in the quadriceps femoris of the DET group
but did not change in the soleus muscle. Therefore,
DET may reverse the effect of LAT on the antioxi-
dant capacity of the aging quadriceps femoris. Taken
together, these results indicate that lifelong aerobic
exercise and detraining are more likely to increase
and decrease, respectively, the antioxidant capacity
of fast-twitch muscle fibers during aging, which may
be related to metabolic changes in adiponectin levels
in the skeletal muscle. In addition, we also observed
that the SOD2 content increased, whereas 4-HNE
and 8-OHdG levels decreased significantly in the gas-
trocnemius muscle of the LAT group. However, DET
reduced SOD2 content, while the amount of peroxide
products increased in the gastrocnemius, particularly
that of MDA, suggesting that detraining in the aging
stage reversed the improvement in the antioxidant
capacity of lifelong exercise and increased lipid per-
oxidation levels in aging skeletal muscle.
We further explored the effects of LAT and DET
on antioxidant capacity and lipid peroxidation-related
changes in aging skeletal muscle of different muscle
fiber types. We next analyzed the Keap1/Nrf2 path-
way and ferroptosis in the quadriceps femoris and
soleus muscles of rats in each group. Ferroptosis is a
form of cell death caused by increased lipid peroxida-
tion due to iron overload, which can increase skeletal
muscle inflammation and atrophy (Alves et al. 2022;
Lv et al. 2022). The Keap1/Nrf2 pathway as an anti-
oxidant defense mechanism in various tissues, and
activation of the Nrf2 pathway inhibits the occurrence
of ferroptosis (Bocalini et al. 2010; Lo et al. 2011).
Moreover, the Nrf2 pathway mediates the inhibi-
tory effect of adiponectin on ferroptosis (Feng et al.
2022). Based on previous findings, we found that
LAT significantly upregulated the mRNA and pro-
tein expression of SES1, SES2, SES3, and Nrf2 and
downregulated the mRNA expression of Keap1 in the
quadriceps femoris. Consistent with this, the mRNA
and protein expression of GPX4, FTL, and SLC7A11
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was upregulated, while the mRNA expression of
NOX4 and ACSL4 was downregulated in the quadri-
ceps femoris of the LAT group. This suggests that
lifelong aerobic exercise improves antioxidant capac-
ity and resists the occurrence of ferroptosis by acti-
vating the Keap1/Nrf2 pathway in aging quadriceps
femoris. However, the activation of the Keap1/Nrf2
pathway and inhibition of ferroptosis were reversed
in the quadriceps femoris of the DET group, which
indicated that the 8-month detraining in the aging
stage led to the complete loss of antioxidant capacity
of lifelong exercise. However, LAT and DET did not
alter SES1, SES2, KEAP1, and Nrf2 mRNA and pro-
tein expression in the soleus muscle, which is consist-
ent with the observed unchanged expression of anti-
oxidant-related genes and adiponectin in the soleus
muscle. Interestingly, LAT significantly upregulated
FTH, GPX4, and SLC7A11 protein and FTL mRNA
expression in the soleus muscle, whereas DET
reversed this change, indicating that LAT improves
ferroptosis in aging soleus muscles independently
of the Keap1/Nrf2 pathway (Fig. 7); however, this
requires further exploration.
It is worth noting that, compared with that in the
quadriceps femoris, LAT did not change the Nrf2
pathway or the expression of antioxidant-related
genes in the soleus muscle. This indicates differences
in muscle fiber types in the improvement of antioxi-
dant capacity of different skeletal muscles by aerobic
exercise, which may be related to the difference in the
metabolic adaptation of different types of muscle fib-
ers to aerobic exercise (Qaisar et al. 2016). Previous
studies also found that compared with fast muscle
fibers, slow muscle fibers exhibit higher SOD, GPX,
and CAT activities (Criswell et al. 1993; Lawler et al.
1994; Powers et al. 1994). These differences may
partly explain the lower plasticity potential of aero-
bic oxidation and antioxidant capacity of slow-twitch
muscle fibers under the stimulation of moderate-
intensity aerobic exercise. Meanwhile, it has been
reported that long-term aerobic exercise significantly
increases the proportion of type I muscle fibers and
decreases that of type II muscle fibers in the planta-
ris and soleus muscles in rats, with the plantaris mus-
cle being more evident than the soleus muscle (Hyatt
et al. 2019); this phenomenon has also been observed
in the gastrocnemius muscle in humans (Hoff et al.
2013). However, detraining reverses this change
(Hoff et al. 2013; Hyatt et al. 2019), possibly because
Biogerontology (2023) 24:753–769
Fig. 7  Effects of lifelong exercise and detraining on oxidative stress in different skeletal muscles
detraining decreases the proportion of type I muscle
fibers and increasing that of type IIx muscle fibers in
the skeletal muscle (Serrano et al. 2000). This indi-
cates that aerobic exercise may more easily induce the
conversion of fast muscle fibers to slow muscle fib-
ers in skeletal muscle dominated by fast muscle fib-
ers, which increased the oxidative metabolism and
antioxidant capacity of skeletal muscle to generate
adaptation to aerobic exercise. This is similar to our
results; the antioxidant capacity and oxidative stress
of fast-twitch muscle fibers changed more evident
after lifelong aerobic exercise and detraining. Taken
together, these findings suggest that aerobic exercise
and detraining may have greater effects on oxida-
tive metabolism and the antioxidant capacity in fast-
twitch muscle fibers than in slow-twitch muscle fibers
(Joseph et al. 2016), but further research is needed to
confirm this.
765
Conclusions
Lifelong exercise can maintain skeletal muscle mass
and reduce the levels of circulating oxidative stress
markers in aging rats; however, the benefits of life-
long exercise are gradually lost due to detraining dur-
ing the aging phase. Long-term detraining during the
aging phase reverses the lifelong exercise-induced
inhibition of aging skeletal muscle inflammation,
oxidative stress, and ferroptosis, which accelerates
aging skeletal muscle atrophy. These changes in the
quadriceps femoris are more evident than those in the
soleus, which may be related to the different changes
in the Keap1/Nrf2 pathway in different skeletal
muscles.
Acknowledgements We would like to thank those who sup-
ported this study. We would like to thank Editage (www.​edita​
ge.​cn) for English language editing.
Author contributions Z-ZW performed the experiments;
H-CX, H-XZ, and C-KZ analyzed the data; B-ML and J-HH
interpreted the results of the experiments; Y-QL and P-SN
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prepared the figures; Z-ZW drafted, edited, and revised the
manuscript; F-HL and X-MY conceived and designed the
research. All authors read and approved the final manuscript.
Funding This work was supported by the National Natu-
ral Science Foundation of China [Grant Nos. 31500961 and
31971099] and Interdisciplinary Studies at Nanjing Normal
University.
Data availability The datasets generated during and/or ana-
lysed during the current study are available from the corre-
sponding author on reasonable request.
Declarations
Competing interests The authors declare that they have no
known competing financial interests or personal relationships
that could have influenced the work reported in this study.
Ethical approval All experiments were approved by the ani-
mal experiment ethical checklist of Nanjing Normal University
(IACUU-1903006).
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