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Inonotus obliquus upregulates muscle regeneration
and augments function through muscle oxidative
metabolism
Chang-Lim Yu
Sungkyunkwan University School of Medicine
Sang-Jin Lee
AniMusCure Inc
Jinwoo Lee
AniMusCure Inc
Tuan Anh Vuoung
AniMusCure Inc
Hye-Young Lee
AniMusCure Inc
Se Yun Jeong
Sungkyunkwan University
Akida Alishir
Sungkyunkwan University
Allison S. Walker
Vanderbilt University
Gyu-Un Bae
Sookmyung Women’s University
Ki Hyun Kim ( khkim83@skku.edu )
Sungkyunkwan University
Jong-Sun Kang
Sungkyunkwan University School of Medicine
Research Article
Keywords: Inonotus obliquus, PGC-1α, muscle regeneration, muscle atrophy, muscle oxidative
metabolism
Posted Date: February 7th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2542802/v1
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Abstract
Background Skeletal muscle wasting related to aging or pathological conditions is critically associated
with the increased incidence and prevalence of secondary diseases including cardiovascular diseases,
metabolic syndromes, and chronic inammations. Much effort is made to develop agents to enhance
muscle metabolism and function.
Inonotus obliquus
(
I
.
obliquus
; IO) is a mushroom popularly called
chaga and has been widely employed as a folk medicine for inammation, cardiovascular diseases,
diabetes, and cancer in Eastern Europe and Asia. However, its effect in muscle health has not been
explored.
ObjectiveHere, we aimed to investigate the benecial effect of IO extract in muscle regeneration and
metabolism.
MethodsThe effect of
I. obliquus
extract was investigated on myogenesis and myotube atrophy models
of C2C12 myoblasts and muscle regeneration model of mice. The muscle stem cell proliferation and
regeneration post muscle injury were employed to further conrm the effect of
I. obliquus
. The underlying
mechanism of
I. obliquus
was also investigated by the mitochondrial content and oxidative muscle
metabolism as well as the activation of AKT and PGC-1α expression.
Results The treatment of IO in C2C12 myoblasts led to increased myogenic differentiation and alleviation
of dexamethasone-induced myotube atrophy. Network pharmacological analysis using the identied
specic chemical constituents of IO extracts predicted protein kinase B (AKT)-dependent mechanisms to
promote myogenesis and muscle regeneration. Consistently, IO treatment resulted in the activation of
AKT, which suppressed muscle-specic ubiquitin E3 ligases induced by dexamethasone. IO treatment in
mice improved the regeneration of cardiotoxin-injured muscles accompanied by elevated proliferation
and differentiation of muscle stem cells. Furthermore, it elevated the mitochondrial content and muscle
oxidative metabolism accompanied by the induction of peroxisome proliferator-activated receptor γ
coactivator α (PGC-1α).
Conclusions Our current data suggest that
I. obliquus
is a promising natural agent in enhancing muscle
regenerative capacity and oxidative metabolism thereby preventing muscle wasting.
Introduction
Inonotus obliquus
(
I. obliquus
) is a mushroom belonging to the family Hymenochaetaceae
(Basidiomycota) popularly called chaga in Russian folk medicine [1]. Chaga mushroom is a parasitic
fungus that grows on birch in the forests of Northern European countries [2]. The mushroom is widely
consumed as tea, syrup, bath agents, or concentrate [1, 3] and has been widely employed as a folk
medicine for inammation, cardiovascular diseases, diabetes, and cancer in Eastern Europe and Asia [1,
4, 5]. The main bioactive compounds of
I. obliquus
are polysaccharides, polyphenols, melanin, and
triterpenes [6]. By using whole
I. obliquus
extract or its components, studies have shown antitumor, anti-
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inammatory, immunomodulatory, antioxidant, hypoglycemic, and hypolipidemic activity of
I. oliquus
.
However, its potential effect on myogenesis and muscle regeneration has not been investigated so far.
Decline in skeletal muscle mass and function is a serious public health issue without effective cure. It not
only severely compromises body movement and daily activities, but also increases the incidence and
prevalence of secondary diseases leading to frailty and reduced healthy life span [7]. The age-associated
decline in muscle mass and strength, which is referred to as sarcopenia, is attributed to reduced
regenerative capacity of the muscle, declined mitochondrial function, and dysregulated immune
responses [8, 9]. Among diverse strategies, so far exercise seems to be most effective measure to improve
muscle function in sarcopenic patients so far [10, 11]. Thus, much efforts have been made to elucidate
the underlying molecular mechanisms of the exercise effect on muscle mass and function. Peroxisome
proliferator-activated receptor γ coactivator-1 alpha (PGC-1α) is a key regulator exerting exercise effect by
eliciting gene expression related to mitochondrial biogenesis, fatty acid oxidation, angiogenesis,
suppression of muscle atrophy and muscle regenerative capacity [12]. Mice lacking PGC-1α display
impaired oxidative muscle metabolism and exercise capacity [13, 14]. Thus, elevation of PGC-1α seems
to be an attractive strategy to intervene muscle atrophy and weakness related to aging. Indeed, several
studies have reported the benecial effect of PGC-1α increase in aging related metabolic diseases and
muscle wasting [15, 16]. Two PGC-1α inducers, AICAR (AMPK agonist) and metformin (anti-diabetes)
have shown to replicate the effect of exercise on muscle function [17–19]. Protein kinase B (AKT)
signaling is another promyogenic signaling pathway critical for cell survival and differentiation of
myoblasts triggered by the external signals like insulin-like growth factor [20–23]. AKT can activate the
action of myogenic regulatory factors like MyoD and MEF2 thereby inducing muscle specic gene
expression contributing to myogenesis and muscle regeneration [22, 24]. AKT signaling also plays a key
role in anabolic responses associated with increased protein synthesis and resulting in muscle growth
[23, 25].
The wide range of biological activity of
I. obliquus
in cells to modulate oxidative stress, immune
response, and metabolic properties make the natural resource an attractive candidate to promote muscle
health. Therefore, we investigated the effect of
I. obliquus
extract on myogenesis and myotube atrophy
models of C2C12 myoblasts and muscle regeneration model of mice. We show that
I. obliquus
treatment
improves myoblast differentiation and prevents myotube atrophy induced by dexamethasone (DEX). In
addition,
I. obliquus
enhances muscle stem cell proliferation and regeneration post muscle injury. Further
analysis revealed an increase in mitochondrial content and oxidative muscle metabolism in
I. obliquus
-
treated muscles. Finally, we present the activation of AKT and PGC-1α expression as the potential
underlying mechanism of
I. obliquus
’s biological activities in muscle.
Materials And Methods
Chemicals and Reagents
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Wild-type C57BL/6 male mice were purchased from (Orient-Bio, Seongnam, Korea). Fetal bovine serum
(FBS), horse serum (HS) and Dulbecco modied Eagle’s medium (DMEM) were purchased from Thermo
Scientic (Waltham, MA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DEX and all
other chemicals were from Sigma-Aldrich (St. Louis, MO). Antibodies were purchased as following:
Myosin heavy chain (MHC, Developmental Studies Hybridoma Bank (DSHB), Iowa, IA), MyoD (Novus
biologicals, Littleton, CO), Myogenin, Myoglobin, total-OxPHOS (Abcam, Cambridge, MA), total AKT,
phospho-AKT, total p38, phospho-p38, β-actin (Cell Signaling Technology, Beverly, MA), MuRF1, Atrogin-1,
HSP90 (Santa Cruz Biotechnology, Santa Cruz, CA), PGC-1α (Calbiochem, San Diego, CA) and β-tubulin
(Zymed, South San Francisco, CA).
Plant material
The fruiting bodies of chaga mushroom (
I. obliquus
) were purchased at Kyungdong herbal market, Seoul,
Korea, in July 2019, and were identied by one of the authors (K. H. Kim). A voucher specimen (SKKU CG-
2019-07) has been deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University,
Suwon, Korea
Extraction procedure of IO1 and IO2
I. obliquus
(500 g) were dried, chopped, and the chopped material was extracted using distilled water (3 ×
500 mL × 5 h) under reux, and then ltered. The ltrate was evaporated under a vacuum to obtain an
aqueous extract (20.2 g) of
I. obliquus
(IO1). Dried fruiting bodies of
I. obliquus
(500 g) were partially
chopped and extracted with 70% EtOH (3 × 500 mL) for 2 days at room temperature and ltered. The
ltrate was concentrated under vacuum pressure, generating a crude EtOH extract (22.5 g) of
I. obliquus
(IO2). The IO1 and IO2 extracts were prepared as stock solutions at 1 mg/mL in dimethyl sulfoxide
(DMSO, Sigma-Aldrich). All stock solutions were aliquoted and stored at − 80ºC until use.
UPLC Conditions
The samples were analyzed on an Agilent 1290 Innity II UPLC coupled to a G6545B Q-TOF MS system
with dual ESI source (Agilent Technologies, USA). All samples were separated on an Agilent ZORBAX
RRHD Eclipse Plus C18 column (50 × 2.1 mm, 1.8 µm) using 0.1% formic acid-deionized water (A) and
acetonitrile (B). The optimized gradient elution program was as follows: 0–10 min, 10–100% B; 10–12
min, 100% B; 12–15 min, 10% B. The temperature was set at 20°C, and the injection volume was 1 µL.
The spectral acquisition rate and time were set at 1 spectra/s and 1000 ms/spectrum each. The ow rate
was 0.3 mL/min. The wavelength was set at 210 nm. The concentration of samples (distilled water
extract and EtOH extract) was prepared as 1000 ppm and injected 3 times in cases of positive ion-mode
and negative ion-mode respectively.
ESI Q-TOF MS Analysis
The Agilent Q-TOF G6545B mass spectrometer (Agilent Technologies) was operated in positive-ion and
negative-ion mode. The parameters of the ESI source were optimized as follows: gas temperature 320°C,
gas ow 8 L/min, nebulizer pressure 35 psi, sheath gas temperature 350°C, sheath gas ow 11 L/min,
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capillary voltage 3500 V, nozzle voltage 1000 V, and fragmentor voltage 100 V. Internal references (Purine
and HP-0921) were adopted to modify the measured masses in real time. The reference masses in
positive ion mode were at
m/z
121.0508 and 922.0097. The reference masses in negative ion mode were
at
m/z
119.0363 and 1033.9881. The full scan range of the mass spectrometer was
m/z
100–1700 for
MS.
Data processing
Data processing process was conducted in cases of positive-ion mode and negative-ion mode
respectively. The obtained UPLC Q-TOF MS raw data were further processed by Agilent MassHunter
Pronder software (version 10.0, Agilent, America). The batch recursive feature extraction (small
molecules/peptide) algorithm was applied to extract compounds from the total ion chromatograms
(TICs) according to their molecular features including
m/z
, retention time and ion intensities, and the
main parameters of MFE were optimized. Also, this algorithm used to bind and align compounds within
the batch by retention time and mass tolerance; the tolerance windows of retention time and accurate
mass were 0.3 min and 20 ppm, respectively. The restrict retention time range and
m/z
range were set at
0.5–13.0 min and 100 to 1700
m/z
respectively. Low-abundance ions can be hard to identify if the
precursor ion intensity is low, generally below 1000 counts for an Agilent Q-TOF. To produce a matrix
containing fewer biased and redundant data, the thresholds of peak lters was set at 1000 counts.
Missing peaks were ltered according to their frequency, and metabolites that appeared in 100% of
samples in at least one group were retained. All the extracted compounds were output to create a .pfa
(Pronder Archive) le, which can be imported into Mass Proler Professional (MPP) software (version
15.1, Agilent) for further data analysis. Normalization (percentile shift), dening the sample sets,
baselining (median of all samples), ltering by frequency, and signicance analysis (T-test;
p
-value cut-
off: 0.05; fold change cut-off: 2.0) were applied to process the data. The generated data was then
processed for principal component analysis (PCA) by MPP software (version 15.1, Agilent). The
successfully obtained specic metabolites (Table S1) for IO1 were identied by their accurate mass-to-
charge ratio (
m/z
) values and the MS/MS fragmentation ion for each of the corresponding accurate
m/z
values aided by CFM-ID 4.0, a software tool for MS/MS spectral prediction and MS-based compound
identication at http://cfmid3.wishartlab.com as well as literature survey of
I. obliquus
compounds
reported and comparison to authentic standards.
Network pharmacology analysis
Predicted protein targets of metabolites were identied using the STITCH [26], SwissTargetPrediction [27],
and ChEMBL [28] databases. We included a target in the network if it was a human protein and had a
greater than 90% probability in SwissTarget or was predicted to be active at 90% condence in ChEMBL
or if a connection was present between the molecule and the target in the STITCH network. Under these
criteria, there were no hits from SwissTarget or STITCH, but there were hits from ChEMBL. A network of
these compounds and targets was built using STITCH and a network of the protein-protein interactions
was built using STRING [29]. In the STRING network, we observed that the following gene ontologies
relevant to muscular function were enriched: skeletal muscle tissue growth, neuromuscular synaptic
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transmission, skeletal muscle contraction, muscle contraction mainly due to the presence of
acetylcholine receptor subunits in the network. Important target nodes were identied by comparing the
degree, betweenness centrality, and closeness centrality to the median values. Betweenness and
closeness centrality were calculated using the Networkx python package [30].
Animal experimental design
The wild-type C57Bl/6 male mice were obtained from Orient-Bio (Seongnam, Korea) and maintained until
sacriced. All mice were maintained at 23℃ with a 12:12 h light-dark cycle and free access to food and
water. The mice were orally administered a daily dose of 4 mg/kg IO for 4 weeks (4-month-old mice,
young) and control mice were administered the same amount of vehicle drinking water. For the muscle
atrophy experiment, the 4-month-old mice were orally administered either vehicle or IO for 1 week prior to
injecting cardiotoxin (CTX) and were administered until be sacriced. Then, they were victimized on Day 3
and Day 21 after injection. All animals were sacriced after fasting for 16 h with ad libitum to water, and
their muscles were harvested 4 h after the last administration of IO. All animal experiments were
approved by the Institutional Animal Care and Research Advisory Committee at Sungkyunkwan University
school of Medicine (SUSM) and complied with the regulations of the institutional ethics committee.
Cell culture and cell viability assay
C2C12 myoblasts were cultured as previously described [31]. They were grown in DMEM (Dulbecco’s
Modied Eagle Medium high glucose; Thermo Scientic, Waltham, MA) containing 15% FBS (growth
medium, GM), 10 units/mL penicillin and 10 µg/mL streptomycin (Welgene, Daegu, Korea) at 37℃, 5%
CO2. To induce differentiation of C2C12 myoblasts, cells at near conuence were changed growth
medium into DMEM containing 2% HS (differentiation medium, DM) and myotube formation was
observed at 2 or 3 days after differentiation. For the DEX-induced atrophy study, C2C12 cells were
induced to differentiate in differentiation medium for 3 days and treated with 100 µM DEX and indicated
concentration of IO, followed by incubation in differentiation medium for additional 1 day [32].
Cell viability assay was quantied using MTT colorimetric assay. In briey, C2C12 cells were seeded in a
96-well plate (5 x 104 cells/well) overnight and treated with the indicated concentration of IO for 24 h.
MTT solution was added to the each well, and the cells were incubated for 4 h at 37℃. The optical
density was measured at 540 nm.
Western blotting and immunostaining
Western blot analysis was performed as previously described [33]. Briey, cells were lysed in cell
extraction buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing
complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), followed by SDS-PAGE and
incubation with primary and secondary antibodies.
Immunostaining for MHC expression was carried out as previously described [33]. Briey, the
differentiated cultures were then immunostained for MHC antibodies and Alexa 568- or 488-conjugated
secondary antibodies (Molecular Probes, Eugene, OR). Images were captured and processed with a Nikon
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ECLIPSE TE-2000U microscope and NIS-Elements F software (Nikon, Tokyo, Japan). To analyze the
eciency of myotube formation, the MHC-positive myotubes containing two to nine, or ten or more nuclei,
were quantied at least three times and measured using IMAGE J software (version 1.53e; National
Institutes of Health, Bethesda, MD). MHC-positive myotubes with 10 and more nuclei were measured
transverse diameter in six elds. Quantication of myotube diameter was performed with IMAGE J
software. Average myotube diameter is presented as means determination of six elds ± 1 standard
deviation (SD).
RNA isolation and quantitative RT-PCR
Total RNA extraction and quantitative RT-PCR analysis was performed as previously described [31].
Tissues were homogenized by FastPrepR-24 (MP Biomedicals, Santa Ana, CA) and extracted using the
easy-spin Total RNA Extract kit (iNtRON, Seongnam, Korea). Gene expression fold change was
normalized against the expression of 18S ribosomal RNA. The sequences of the primers used in this
study are provided in Table1.
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Table 1
The primers used in this study
Primer Sequence
eMHC Forward 5’-CTGGAGTTTGAGCTGGAAGG-3’
Reverse 5’-CAGCCTGCCTCTTGTAGGAC-3’
Myogenin Forward 5’-ATCTCCGCTACAGAGGCGGG-3’
Reverse 5’-TAGGGTCAGCCGCGAGCAAA-3’
Atrogin-1 Forward 5’-CAACATTAACATGTGGGTGTAT-3’
Reverse 5’-GTCACTCAGCCTCTGCATG-3’
MuRF1 Forward 5’-GAGAACCTGGAGAAGCAGCT-3’
Reverse 5’-CCGCGGTTGGTCCAGTAG-3’
MyhI Forward 5’-ACAAGCTGCAGCTGAAGGTG-3’
Reverse 5’-TCATTCAGGCCCTTGGCAC-3’
MyhIIa Forward 5’-CCAGCTGCACCTTCTCGTTTGCCAG-3’
Reverse 5’-CATGGGGAAGATCTGGTCTTCTT-3’
MyhIIb Forward 5’-CCTGGAACAGACAGAGAGGAGCAGGAGAG-3’
Reverse 5’-GTGAGTTCCTTCACTCTGCGCTCGTGC-3’
MyhIIX Forward 5’-TGCAACAGTTCTTCAACCAC-3’
Reverse 5’-GCCAGGTCCATCCCAAAGT-3’
Pgc-1αForward 5’-ATGTGTCGCCTTCTTGCTCT-3’
Reverse 5’-CGGTGTCTGTAGTGGCTTGA-3’
Mtco1 Forward 5’-CTACTATTCGGAGCCTGAGC-3’
Reverse 5’-GCATGGGCAGTTACGATAAC-3’
Mcad Forward 5’-GGTTTGGCTTTTGGACAATG-3’
Reverse 5’-TGACGTGTCCAATCTACCACA-3’
Sdhb Forward 5’-CAGAGTCGGCCTGCAGTTTC-3’
Reverse 5’-GGTCCCATCGGTAAATGGCA-3’
Cox7a1 Forward 5’-GTCTCCCAGGCTCTGGTCCG-3’
Reverse 5’-CTGTACAGGACGTTGTCCATTC-3’
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Primer Sequence
Cox4 Forward 5’-CTATGTGTATGGCCCCATCC-3’
Reverse 5’-AGCGGGCTCTCACTTCTTC-3’
Ucp2 Forward 5’-ACTGTCGAAGCCTACAAGAC-3’
Reverse 5’-CACCAGCTCAGTACAGTTGA-3’
TnfαForward 5’-AGCCCCCAGTCTGTATCCTT-3’
Reverse 5’-CTCCCTTTGCAGAACTCAGG-3’
Il6 Forward 5’-GGTGACAACCACGGCCTTCCC-3’
Reverse 5’-AAGCCTCCGACTTGTGAAGTGGT-3’
Il10 Forward 5’-GCCAAGCCTTATCGGAAATG-3’
Reverse 5’-CACCCAGGGAATTCAAATGC-3’
Il1RA Forward 5’-TTCTTGTTGCCTCTGCCACTCG-3’
Reverse 5’-GATTGGTCTGGACTGTGGAAGTG-3’
Ccl5 Forward 5’-TGCCCACGTCAAGGAGTATTT-3’
Reverse 5’-TTCTCTGGGTTGGCACACACT-3’
Ccl22 Forward 5’-AAGACAGTATCTGCTGCCAGG-3’
Reverse 5’-GATCGGCACAGATATCTCGG-3’
Cxcl1 Forward 5’-TGAGCTGCGCTGTCAGTGCC-3’
Reverse 5’-AGAAGCCAGCGTTCACCAGA-3’
Pax7 Forward 5’-GAGTTCGATTAGCCGAGTGC-3’
Reverse 5’-CGGGTTCTGATTCCACATCT-3’
MyoD Forward 5’-GATGGCATGATGGATTACAGCGGC-3’
Reverse 5’-GTGGAGATGCGCTCCACTATGCTG-3’
18S rRNA Forward 5’-AGGGGAGAGCGGGTAAGAGA-3’
Reverse 5’-GGACAGGACTAGGCGGAACA-3’
Cryosections, staining analysis, and ber size measurement
Muscle tissue was embedded in Tissue-Tek OCT Compound (Sakura Finetek, Nagano, Japan), and 7mm
thick serial sections for staining were cut using a cryomicrotome. To analyze the NADH dehydrogenase
activity, we dried the sectioned tissues for 10 min in room temperature and incubated in 0.9 mM NADH
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and 1.5 mM nitro blue tetrazolium (NBT; Sigma-Aldrich) in 3.5 mM phosphate buffer (pH 7.4) for 30 min.
To analyze the succinate dehydrogenase (SDH) activity, we incubated the sections for 1 h in 50 µM
sodium succinate and 0.3 mM nitro blue tetrazolium in 114 mM phosphate buffer containing K-EGTA
(Sigma-Aldrich). Myh immunostaining of muscle tissue sections was performed in the sequence of
xation, permeation, and incubation with primary antibodies against MyhIIa and Myhb (DSHB) and
laminin (Abcam). Images were captured and proceed with a Nikon ECLIPSE TE-2000U using NIS-
Elements F software. Myober area was measured with ImageJ software. For muscle histology, the
cryosections were stained with Mayer’s hematoxylin and eosin (BBC Biomedical, McKinney, TX). The
images were captured using a Nikon ECLIPS TE-2000U.
PGC-1α Luciferase Assay
PGC-1α luciferase assay was performed as previously described [34]. C2C12 cells were transfected with
an expression plasmid for luciferase responsive to the 2 kb promoter region of PGC-1α (Addgene plasmid
#8887, Addgene, Cambridge, MA, USA), PGC-1α promoter luciferase delta CRE site (Addgene plasmid
#8888, Addgene) and PGC-1α promoter luciferase delta MEF site (Addgene plasmid #8889, Addgene)
using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 24 h of
transfection, the cells were incubated in differentiation media with IO1. The cells were lysed with Reporter
Lysis Buffer (Promega), and luciferase assays were performed using a Luciferase Assay System kit and a
luminometer (Berthold Technology, Bad Wildbad, Germany). The Transfection eciency was normalized
based on the co-transfected b-galactosidase enzyme activity measured using an assay kit (Promega).
Experiments were performed in triplicates and repeated at least three times independently.
Statistical analysis
Values are expressed as mean ± SD or ± standard error of mean (SEM), as indicated in the gure legends.
Differences were considered statistically signicant at or under values of
P
< 0.05.
Results
Effects of Inonotus obliquus extracts on myogenic differentiation of C2C12 myoblasts
To investigate the effect of
Inonotus obliquus
extract (IO) on myogenic differentiation, we prepared two
types of IO: IO1 (aqueous extract) and IO2 (ethanol extract). C2C12 myoblasts were induced to
differentiate for 1.5 days in the presence of vehicle, IO1, or IO2 in DM and subjected to the assessment of
myoblast differentiation by quantitative real-time polymerase chain reaction (qRT-PCR). IO1 treatment
greatly elevated the expression of muscle-specic genes, embryonic myosin heavy chain (eMHC) and
Myogenin, compared to vehicle or IO2 treatment (Fig.1A). To further examine the promyogenic effects of
IOs, myotube formation was assessed by MHC immunostaining. Both IO1 and IO2 treatment in C2C12
myoblasts promoted the formation of larger MHC-positive multinucleated myotubes compared to vehicle-
treated cells (Fig.1B and C). In particular, IO1 treatment effectively elevated the proportion of larger MHC-
positive myotubes containing ten or more nuclei. Therefore, we decided to place more emphasis on the
investigation of IO1 (hereafter designated as IO). IO did not show cytotoxicity up to 1 µg/mL as assessed
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by MTT assay (Fig.1D). IO-treated C2C12 cells displayed incremental increase in the expression of MHC,
MyoD, and Myogenin in a dose-dependent manner (Fig.1E), which was also conrmed by
immunostaining of MHC (Fig.1F). For the quantication of myotube formation, the nuclei MHC-positive
myotubes were counted. Treatment with IO signicantly increased the proportion of larger myotubes with
20 or more nuclei, while mononucleated myocytes decreased in a concentration-dependent manner
(Fig.1G). These data suggest that IO enhances C2C12 myoblast differentiation without overt cytotoxicity.
Characterization of the chemical constituents in the active
IO and network pharmacological analysis
To investigate the potential underlying mechanism of the effects of
I. obliquus
in muscle, the chemical
constituents of
I. obliquus
extracts were analyzed by ultra-high performance liquid chromatography
(UPLC) coupled with quadrupole time-of-ight mass spectrometry (Q-TOF MS). Since IO1 showed greater
enhancement of C2C12 myoblast differentiation than IO2, we presumed that the specic metabolites in
the aqueous extract of
I. obliquus
(IO1) are the potential bioactive compounds. The obtained typical total
ion chromatograms (TICs) for the two types of IO were similar (Fig. S1) with most of the major peaks
appearing in TICs of both IO1 and IO2. However, the principal component analysis (PCA) of the UPLC-Q-
TOF MS data showed that the IO1 and IO2 samples were clearly separated (Fig.2A), indicating the
different metabolite proles for IO1 and IO2. In the positive-ion mode, there were 201 chemical
constituents in IO1 and 328 chemical constituents in IO2. In the negative-ion mode, there were 69
chemical constituents in IO1 and 138 chemical constituents in IO2. To verify the specic metabolites in
IO1, pairwise analysis of IO1 versus IO2 was performed using a Venn diagram (Fig.2B). The results
indicated that 27 and 3 specic metabolites of IO1 were found in the positive-ion mode and negative-ion
mode, respectively. The specic metabolites obtained were checked manually, since they contained
redundant signals caused by different isotopes and in-source fragmentation, and the redundant signals
were manually removed. Finally, the highly reproducible and non-redundant metabolite signals were taken
as 6 specic chemical constituents in IO1 (Fig.2C).
Next, network pharmacology analysis was performed to explore the potential mechanisms of
I. obliquus
on myoblast differentiation and muscle regeneration using the identied specic chemical constituents
of IO1. We used STITCH [26], SwissTargetPrediction [27], and ChEMBL [28] to identify possible protein
targets of IO1. The ChEMBL database was the only tool that identied candidate target proteins with high
condence. We then constructed a network of the target proteins identied by ChEMBL and proteins of
interest associated with skeletal muscle tissue growth, contraction, and neuromuscular synaptic
transmission using STRING [29] and STITCH [26]. In this network, the neuronal acetylcholine receptor was
a predicted target of di-
n
-butyl sebacate, 1-monopalmitin, and 12-hydroxyoctadecanoic acid and the
acetylcholine receptor was a predicted target of methyl 3-methoxypropionate (Fig.2D). The acetylcholine
receptors were in turn linked to many of the proteins of interest including protein kinase B (AKT1),
myoblast differentiation protein (MYOD), myogenin (MYOG), myosin heavy chain 2, 3, 8 (MYH2, MYH3,
MYH8), E3 ubiquitin-protein ligase (TRIM63), and peroxisome proliferator-activated receptor g coactivator
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(Pgc-1α). In addition, 12-hydroxyoctadecanoic acid was predicted to interact with focal adhesion kinase 1
(PTK2), which was identied to be a major node that links between the acetylcholine receptors and
downstream kinases such as AKT and MAP in the network. Accordingly, the network pharmacological
analysis suggests that the most likely mechanism of IO1 activity is through activation of acetylcholine
receptors and PTK2 leading to downstream activation of the AKT and MAPK pathways to promote
myoblast differentiation.
Preventive effects of IO (IO1) on DEX-induced myotube
atrophy through AKT activation
AKT functions as a promyogenic kinase in myoblast differentiation and as an essential regulator of
muscle protein synthesis and hypertrophy [33, 35]. To examine whether the promyogenic effect of IO
(IO1) is through AKT activation as predicted from the network pharmacological analysis, C2C12
myoblasts were induced to differentiate in DM for 1 day, treated with the indicated concentrations of IO,
and the activation status of AKT and p38 was assessed by immunoblotting. The treatment of IO
dramatically increased p-AKT levels in a dose-dependent manner without changes in the levels of p-p38
and total form of AKT and p38 (Fig.3A). AKT activation attenuates the induction of muscle-specic
ubiquitin ligases triggered by synthetic glucocorticoid DEX thereby preventing muscle atrophy [36]. To
examine whether IO can protect DEX-induced C2C12 myotube atrophy, C2C12 cells were treated with DEX
alongside with vehicle DMSO or IO and subjected to MHC immunostaining to assess the thickness of the
myotubes. The DEX-elicited myotube atrophy was suppressed by IO treatment, as evident by the larger
multinucleated myotubes in IO-treated cultures (Fig.3B and C). In addition, the IO treatment abrogated the
DEX-induced elevation of protein levels of muscle-specic E3 ligases, Atrogin-1 and MuRF-1 (Fig.3D).
The rescue effect of IO treatment on DEX-induced atrophic myotubes was also reected by the restored
MHC expression close to the level of vehicle control. Furthermore, DEX-treated myotubes exhibited
decreased level of p-AKT while IO treatment in DEX-treated myotubes abrogated this decrease. Consistent
with the immunoblotting analysis, qRT-PCR analysis showed that DEX treatment greatly elevates the
expression of muscle-specic E3 ubiquitin ligases, Atrogin-1 and MuRF-1, while the treatment with IO in
DEX-treated myotubes signicantly diminishes the mRNA level of Atrogin-1 and MuRF-1 (Fig.3E). Taken
together, these results suggest that IO rescues DEX-induced myotube atrophy through inhibition of
muscle-specic ubiquitin ligases mediated by Akt activation.
Effect of IO on skeletal muscle regeneration in CTX-injury
mouse model
To further examine the role of IO in muscle regeneration, acute injury was induced by cardiotoxin (CTX)
injection in the tibialis anterior (TA) muscle of four-month-old wild type male mice. Starting from a week
before CTX injection, mice were orally administrated with vehicle or 4 mg/kg IO daily and dissected on
day 21 after CTX injection (Fig.4A). IO treatment did not incur changes in body weight, food intake, and
blood glucose level compared with the vehicle treatment (Fig.4B-D). The hindlimb muscles of the IO-
treated mice appeared darker than those of control mice, which could be readily detected in
Page 14/30
gastrocnemius (GAS) muscle (Fig.4E). Among four hindlimb muscles, TA muscle displayed the most
signicant increase in mass per body weight with approximately 12% increase in the IO-fed mice
compared to the vehicle-fed mice (Fig.4F). Sections of the TA muscle also revealed an increase in the
cross-sectional areas (CSA) of myobers by IO treatment (Fig.4G-I). In addition, treatment with IO
increased the number of myonuclei per CSA in TA muscle bers compared to the treatment with vehicle
(Fig.4J). Taken together, these data suggest that IO treatment improves muscle regeneration in CTX-
injured muscles.
To investigate the effect of IO on myober types, CTX-injected TA muscle sections were analyzed by
immunostaining with antibodies against MyhIIa and MyhIIb. The IO treatment signicantly increased the
number and CSA of MyhIIb-positive myobers and increased the number and CSA of MyhIIa-positive
myobers without statistical signicance compared with the vehicle treatment (Fig.4K-M). Furthermore,
qRT-PCR analysis of TA muscles from vehicle- and IO-fed mice revealed that IO treatment signicantly
increases the expression of both oxidative myober markers, MyhI and MyhIIa, and glycolytic myober
markers, MyhIIb and MyhIIx (Fig.4N). These data indicate that IO treatment enhances the regeneration of
both oxidative and glycolytic myober types.
Effect of IO on muscle stem cell proliferation in
regenerating muscles at day 3 post-CTX-injury
To investigate the effect of IO at the early stage of muscle regeneration, four-month-old wild type male
mice were orally administered with either vehicle or 4 mg/kg IO daily starting from 7 days prior to CTX
injection in TA muscles and the muscles were harvested post 3 days of injury (Fig.5A). The IO treatment
did not induce changes in body weight and relative TA muscle weight compared with the vehicle
treatment (Fig.5B and C). To monitor the proliferation in the regenerating muscles, cryosections of TA
muscles were subjected to immunostaining for BrdU incorporation and Ki67. IO treatment elevated the
number of BrdU and Ki67-positive cells compared to the vehicle treatment (Fig.5D and E). Furthermore, IO
treatment signicantly increased the expression levels of genes expressed during cell proliferation
(Ccnb1, Ccnb2, Ccne1, Ccnf, Aurkb, Mcm6, and p21) and markers for activated proliferating muscle stem
cells (Pax7 and MyoD), suggesting that the improved muscle regeneration by IO treatment is attributable
to the increased proliferation of muscle stem cells (Fig.5F and G). Aged human myoblasts treated with IO
also exhibited increased proliferation compared with the vehicle-treated cells, further verifying the effect
of IO on the muscle stem cell proliferation (Fig.5H and I). In parallel with Fig.5H & I, IO treatment
signicantly increased the Ki67 expression (Fig.5J). These results suggest that IO enhances the
activation and proliferation of muscle stem cells in response to muscle injury.
Effect of IO on the oxidative muscle metabolism
Since IO-treated mice exhibited darker hindlimb muscles and substantially increased expression of MyhI
and MyhIIa compared with the vehicle-treated mice (Fig.4E and N), we presumed that IO may also have
an effect in promoting oxidative muscle metabolism. TA muscles of 4-month-old mice treated with either
vehicle or 4 mg/kg of IO for 4 weeks were subjected to histochemical staining for the activity of
Page 15/30
mitochondrial enzyme succinate dehydrogenase (SDH). IO treatment elevated the proportion of myobers
with strong (dark) and intermediate staining for SDH compared to the vehicle treatment (Fig.6A and B).
In addition, IO signicantly enhanced the expression of total OxPHOS (Atp5a, Uqcr2, Mtco1, NdufB8, and
Sdhd) and mitochondrial genes (Mtco1, Mcad, Sdhd, Cox7a1, Cox4, and Ucp2) (Fig.6C-E). The relative
mitochondrial DNA content was also signicantly increased by IO treatment, indicating that IO enhances
oxidative muscle metabolism (Fig.6F). To conrm these effects of IO, C2C12 myoblasts were induced to
differentiate with the indicated concentrations of IO in DM and subjected to immunoblotting analysis. In
agreement with the results from the mice model, IO treatment signicantly increased the expression of
total OxPHOS and mitochondrial genes in a dose dependent manner (Fig.6G and H). Finally, IO-treated
C2C12 cells exhibited increased mitochondrial membrane potential as evidenced by the increased JC-1
polymer/monomer ratio compared with the vehicle-treated cells (Fig.6I and J). These results suggest that
IO enhances the oxidative muscle metabolism through upregulation of mitochondrial gene expression.
Effect of IO on the expression of PGC-1α in young mice skeletal muscle and C2C12 cells
PGC-1α is a transcriptional coactivator that activates the expression of genes involved in mitochondrial
biogenesis, stimulation of fatty acid oxidation, and resistance to muscle atrophy [12]. Thus, we
investigated whether IO regulates the oxidative muscle metabolism through regulation of PGC-1α
expression. The mRNA and protein levels of PGC-1α and myoglobin were elevated in the IO-treated TA
muscles compared with the vehicle-treated muscles (Fig.7A and B). In agreement with the data of TA
muscles, IO treatment increased the level of PGC-1α mRNA as well as protein up to approximately 3.0-fold
in C2C12 myoblasts (Fig.7C and D). To further dene the mechanism of IO in the activation of PGC-1α
expression, the reporter activity of full-length or mutant PGC-1α promoters (with a deletion of either MEF2
or CRE motif) was measured in C2C12 cells. The full-length promoter-driven luciferase activity was
signicantly upregulated by IO1 in a dose-dependent manner, with 1 µg/mL IO treatment resulting in
higher luciferase activity than the treatment with 0.5 mM (1.313 mg/mL) AICAR, which is a known
activator of PGC-1α through AMPK activation (Fig.7E). The deletion of MEF2 or CRE motif abrogated the
reporter activity upregulation by IO to a greater degree than and it did to the reporter activity upregulation
by AICAR (Fig.7F), suggesting for the requirement of both elements to mediate the effect of IO on PGC-1α
expression. Taken together, these data suggest that IO upregulates the expression of PGC-1α to promote
oxidative muscle metabolism.
Discussion
I. obliquus
has been reported to have various health promoting properties which include antitumor,
antioxidant, antiviral, anti-inammatory, and immunomodulatory activity without prominent side effects
(reviewed in [37]; [38]). However, most studies were performed in the context of cancer cells with a few
exceptions of normal cell lines from digestive system such as pancreatic and hepatic cell lines. There
have been limited investigation to our knowledge on its effect on myogenesis and regeneration of
skeletal muscle. In this study, we investigated the effect of IO treatment on muscle health and the
potential mechanism underlying the effect. IO treatment promoted C2C12 myoblast differentiation and
Page 16/30
alleviated DEX-induced myotube atrophy. IO treatment also improved muscle regeneration of CTX-injury
mouse accompanied by an increase in muscle stem cell proliferation and oxidative muscle metabolism.
Network pharmacological analysis of IO predicted that the promyogenic function of IO is mediated by the
activation of AKT pathway, which was conrmed in C2C12 myoblasts. Taken together, the data here
present a positive effect of IO on myogenesis and muscle regeneration in both in vivo and in vitro model
of muscle atrophy through AKT-dependent mechanisms.
Investigation on the bioactive ingredients of IO and the molecular mediators of its biological effect is yet
minimal and still ongoing. By comparing the metabolite proles of IO1 and IO2, we identied 6 chemical
constituents of IO as the potential bioactive compounds responsible for the myogenic effect of IO
(Fig.2C; Table S1). Based on the network pharmacological analysis using the identied 6 chemical
constituents of IO, we propose two major mediators of IO in the skeletal muscle: AKT and PGC-1α.
Previous studies have shown the activation of PI3K/AKT signaling pathway by treatment of IO or its
component in other contexts [39, 40]. In C2C12 myoblasts, we also observed an increase in
phosphorylated AKT in response to IO. AKT activity regulates skeletal muscle growth by downregulating
muscle-specic ubiquitin E3 ligases, Atrogin-1 and MuRF-1. It is also involved in the proliferation of
muscle stem cells through regulation of mTOR [41]. In the current study, IO treatment decreased the
expression of Atrogin-1 and MuRF-1 in DEX-treated C2C12 myoblasts and promoted muscle stem cell
activity and proliferation in TA muscles of CTX-injury mice and aged human myoblasts. We present here
the AKT activation as one of the potential mechanisms underlying the rescue effect of IO on muscle
atrophy.
PGC-1α is a key regulator of muscle metabolism via modulating mitochondria biosynthesis [42]. Of the 6
potential bioactive compounds of IO, 10-oxo-
cis
-12-octadecenoic acid and 12-hydroxyoctadecanoic acid
were shown to strongly activate peroxisome proliferator-activated receptors (PPARs), which in turn
regulate the activity and expression of PGC-1α [43, 44]. The expression of PGC-1α was indeed
signicantly increased by IO treatment in both mice and C2C12 myoblast (Fig.7A-D). Moreover, increased
oxidative muscle metabolism by IO treatment was indicated by the increase in SDH enzymatic activity,
expression of total OxPHOS and mitochondrial genes, and relative mitochondrial DNA content in TA
muscles of IO-treated mice (Fig.6A-F). Since mitochondrial function is important during regeneration for
energy production, homeostasis of reactive oxygen species, cross talk with immune cells, and modulation
of stem cell fate, the enhanced mitochondrial function by IO through PGC-1α may also contribute to the
improved muscle regeneration.
Taken together, our study demonstrates a promyogenic effect of IO in the context of muscle regeneration
following injury through modulation of AKT signaling pathway and oxidative muscle metabolism. As
imbalance in protein homeostasis and mitochondrial dysfunction are the key characteristics of aged and
diseased muscle, IO is a promising drug candidate to promote muscle health.
Conclusion
Page 17/30
IO treatment resulted in the activation of AKT, which inhibits the muscle-specic ubiquitin E3 ligase
induced by dexamethasone. IO treatment showed improved regeneration of muscle with increased
proliferation and differentiation of muscle stem cells and increased mitochondrial content and muscle
oxidative metabolism with induction of PGC-1α. These results suggest that IO is considered a promising
drug candidate for promoting muscle health with promyogenic effects that overcome the imbalance of
protein homeostasis and mitochondrial dysfunction seen in aging through modulation of the AKT
signaling pathway and oxidative muscle metabolism.
Abbreviations
AICAR, aminoimidazole carboxamide ribonucleotide; AKT, protein kinase B; Atp5a, ATP synthase subunit
alpha; Atrogin-1, F-box only protein 32; Ccl22, c-c motif chemokine ligand 22; Ccl5, c-c motif chemokine
ligand 5; Cox4, cytochrome c oxidase subunit 4; Cox7a1, cytochrome c oxidase subunit 7a1; CSA, cross
section area; Cxcl1, c-x-c motif chemokine ligand 1; DEX, dexamethasone; DMSO, dimethyl sulfoxide; EDL,
extensor digitorum longus; GAS, gastrocnemius; H&E, hematoxylin and eosin; Hsp90, heat shock protein
90; Il10, interleukin-10; Il1Ra, interleukin-1 receptor antagonist; Il6, interleukin-6; Mcad, medium-chain acyl-
coenzyme a dehydrogenase; MHC, myosin heavy chain; Mtco1, mitochondrially encoded cytochrome c
Oxidase 1; MuRF-1, E3 ubiquitin-protein ligase TRIM63; Myh, myosin heavy chain; MyoD, Myoblast
determination protein 1; MyoG, myogenin; NdufB8, NADH dehydrogenase ubiquinone 1 beta subcomplex
subunit 8; p38MAPK, p38 mitogen-activated protein kinases; Pax7, paired box 7; Pgc-1α, peroxisome
proliferator-activated receptor γ coactivator α; SDH, succinate dehydrogenase; Sdhb, succinate
dehydrogenase complex iron sulfur subunit B; SOL, soleus; TA, tibialis anterior; Tnfα, tumor necrosis
factor-alpha; Upc2, mitochondrial uncoupling protein 2.
Declarations
Supplementary Information
The online version contains supplementary material available at https:
Acknowledgements
Not applicable.
Authors’ contributions
The authors’ responsibilities were as follows—GUB, KHK, and JSK designed the project; CLY, SJL
performed most of the experiments and analyzed data; CLY, SJL, JL, TAV, and HYL performed the
molecular biology and cell biology experiments; SYJ and AA performed extraction and LC/MS analysis;
Page 18/30
ASW performed network pharmacology analysis; CLY, SJL, GUB, KHK, and JSK wrote the manuscript; All
authors contributed and approved the nal version of the manuscript.
Funding
This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean
government (MSIT) (grant numbers 2019R1A5A2027340, 2020R1A2C1007555 and
2021R1A2C2007937). This work was supported by Industry cooperation grant funded by Sungkyunkwan
University (grant number S-2021-1372-000).
Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author
on reasonable request.
Ethics approval and consent to participate
The present study was reviewed and approved by the Sungkyunkwan University Animal Ethics
Committee. All experimental procedures were performed in accordance with the Korea Food and Drug
Administration (KFDA) Guidelines for the Care and Use of Laboratory Animals, and animal handling
followed the dictates of the national animal Welfare Law of Korea. All experimental procedures were
performed in accordance with guidelines of the Committee for the Purpose of Control and Supervision of
Experiments on Animals of Sungkyunkwan University.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Figures
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Figure 1
Effect of IO on myogenic differentiation of C2C12 myoblast cells. (A) Comparison of IO1 and IO2 in the
degree of myoblast differentiation. Expression of eMHC and Myogenin in C2C12 cells was analyzed by
qRT-PCR. The data were normalized using 18s RNA. eMHC and Myogenin expression levels were further
normalized to the expression level of vehicle (DMSO). (B) Immunostaining for MHC expression in C2C12
cells treated with DM for 1 day and treated with vehicle, IO1 (1.0 µg/mL), or IO2 (1.0 µg/mL) for 1 day in
DM. Scale bar, 50 µM. (C) Quantication of myotube formation from data shown in panel B. (D) Cell
viability of C2C12 cells in the presence of 0, 0.1, 0.5, and 1.0 µg/mL IO for 1 day. (E) Immunoblot analysis
of C2C12 cells treated with indicated concentration of IO and differentiated in DM for 2 days. Cell lysates
were subjected to antibodies against MHC, MyoD, Myogenin, and β-tubulin. (F) Immunostaining of C2C12
cells treated with indicated concentration of IO for MHC expression (green). Scale bar, 50 µM. (G)
Quantication of myotube formation from data shown in panel F. Data from three (A, C and G) or four (D)
Page 23/30
independent experiments were presented as the means ± SD. Asterisks indicate signicant difference
from the control. *
P
< 0.01, **
P
< 0.05, and ***
P
< 0.001.
Figure 2
Analysis of the chemical constituents in IO1 and network pharmacological analysis. (A) Principal
component analysis (PCA) of IO1 and IO2 samples based on the metabolomics analysis in negative-ion
mode. (B) Venn diagrams of pairwise analyses (IO1 versus IO2). (C) The identied specic chemical
constituents in IO1. (D) Pharmacological network of IO1. Pharmacological network was built using
SwissTargetPrediction, ChEMBL, STRING, and STITCH. The specic chemical constituents identied from
IO1 are shown in red, predicted targets in blue, proteins of interest in green, and proteins added to the
network by STRING or STITCH in yellow. Edges hypothesized to be important in the mechanism of action
based on node importance and literature search are highlighted in red.
Page 24/30
Figure 3
Effect of IO on destruction of DEX-induced muscle atrophy model through AKT activation. (A) C2C12 cells
were differentiated in DM for 1 day and treated with indicated concentration of IO. Cell lysates were
subjected to immunoblotting with antibodies against p-AKT, AKT, p-p38, p38, and β-tubulin. (B)
Immunostaining for MHC expression in C2C12 cells treated with normal DM for 3 days and treated with
vehicle, IO (1 µg/mL), DEX (100 µM), or both IO and DEX for 1 day in DM. Scale bar, 50 µm. (C)
Quantication of MHC-positive myotube diameter per eld. (D) C2C12 cells were differentiated in DM for
3 days and treated with vehicle, IO (1 µg/mL), DEX (100 µM), or both IO and DEX for 1 day in DM. Cell
lysates were subjected to immunoblotting with antibodies against Atrogin-1, MuRF-1, MHC, p-AKT, AKT,
and β-tubulin. (E) Expression of Atrogin-1 and MuRF-1 in C2C12 cells treated with normal DM for 3 days
Page 25/30
and treated with vehicle, IO (1 µg/mL), DEX (100 µM), or both IO and DEX for 1 day in DM. The data were
normalized using 18s RNA. Atrogin-1 and MuRF-1 expression levels were further normalized to the
expression level of vehicle (DMSO). Values are means of triplicate ± SD. To determine statistical
signicance, an unpaired two-tailed Student’s
t
-test was used. *
P
< 0.05, **
P
< 0.01 and ***
P
< 0.001.
Figure 4
Page 26/30
Effect of IO on skeletal muscle regeneration in
in vivo
mice model at day 21 post-CTX-injection.(A)
Experimental set-up. (B-D) Body weights (B), changes in food intake (C), and blood glucose level (D) of
Vehicle- and IO-treated mice. (E) Photographs of isolated hindlimb muscles from 4-month-old mice
ingested with control or 4 mg/kg IO for 4 weeks. (F) Weights of four muscle types from control or 4
mg/kg IO ingested mice for 4 weeks. (G) Representative H&E and Laminin staining of TA muscle sections
from vehicle- and IO-administrated mice. (H) Distribution of the myobers based on their cross-sectional
area (CSA) from panel G. (I) Quantication of the average myober CSA from panel G. (J) Quantication
of myonuclei per CSA from panel G. (K) Immunostaining of MyhIIa (green), MyhIIb (green), and laminin
(red) in the TA muscles of control or 4 mg/kg IO-ingested 4-month-old mice for 4 weeks. (L) Distribution
of MyhIIa- and MyhIIb- positive myobers based on their CSA and average myober CSA from panel K.
Quantication of. (M) Quantication of ber-type content in TA muscle. (N) Expression of slow and fast
muscle-associated genes in TA muscles. MyhI is a slow muscle type, whereas MyhIIa, MyhIIb, and MyhIIX
are fast muscle types. Data from ve (Vehicle) and four (IO) independent experiments were presented as
the means ± SD. Asterisks indicate signicant difference from the control. *
P
< 0.05 and **
P
< 0.01.
Figure 5
Effect of IO on muscle stem cell proliferation in
in vivo
mice model at 3 days post-CTX-injection.(A) The
experimental scheme. (B) The change of body weight for total 10 days of the experiment. (C) The relative
TA muscle weight normalized to body weight. (D) Immunostaining of BrdU incorporation and
quantication of BrdU-positive cells in Veh- or IO-treated TA muscles post 3 days of CTX injury (n = 3). (E)
Immunstaining for Ki67 and quantication of Ki67-positive cells in TA muscles (n = 3). (F & G)
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Quantitative RT-PCR analysis of cell cycle markers (F) and early myogenic regulatory factors (G) in TA
muscles. The data were normalized to 18s RNA levels and were further normalized to the expression level
of vehicle. (H) Immunostaining of BrdU incorporation in Veh- or IO-treated aged human myoblasts (66-old
years). (I) Quantication of BrdU-positive cells from data shown in panel H. (J) Quantitative RT-PCR
analysis of Ki67 in IO-treated aged human myoblasts (66-old-years). Data from three independent
samples were presented as the means ± SD. Asterisks indicate signicant difference from the control. *
P
< 0.05, **
P
< 0.01 and ***
P
< 0.001.
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Figure 6
Effect of IO on the oxidative muscle metabolism. (A) Histochemical staining for SDH enzymatic activities
in TA muscle. Scale bar, 100 µm. (B) The staining intensities of SDH are quantied as three different
grades (dark, intermediate, and pale) and plotted as a percentile (
n
= 3). (C) Western blot analysis for the
expression of total-OxPHOS in TA muscles from 4-month-old mice ingested with control or 4 mg/kg IO for
4 weeks. (D) Quantication of the relative levels of total-OxPHOS proteins from panel C (
n
= 5). (E) The
expression of genes involved in the regulation of mitochondrial function examined by qRT-PCR analysis.
The data were normalized using 18s RNA and were further normalized to the expression level of vehicle.
(F) Relative mitochondrial DNA content in TA muscles from 4-month-old mice ingested with control or 4
mg/kg IO for 4 weeks. (G) Western blot analysis for the expression of total-OxPHOS in C2C12 cells
treated with indicated concentration of IO. (H) Quantication of the relative levels of total-OxPHOS
proteins from panel G (
n
= 3). (I) JC-1 uorescence in C2C12 cells treated with either Vehicle or IO1. (J)
Quantication of the relative levels of JC-1 from panel J. To determine statistical signicance, an
unpaired two-tailed Student’s
t
-test was used (B, D, E, and G). *
P
< 0.05, **
P
< 0.01 and ***
P
< 0.001.
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Figure 7
Effect of IO on the expression of PGC-1α in young mice skeletal muscle and C2C12 cells. (A) Relative
expression of PGC-1α in TA muscles from 4-month-old mice ingested with vehicle or 4 mg/kg IO for 4
weeks. (B) Western blot analysis and quantication of the relative levels of PGC-1α and myoglobin
proteins in TA muscles from 4-month-old mice ingested with vehicle or 4 mg/kg IO for 4 weeks (
n
= 5). (C)
Western blot analysis for the expression of PGC-1α in C2C12 cells treated with indicated concentration of
IO. (D) Quantication of the relative levels of PGC-1α protein from panel C. (E) The relative PGC-1α
luciferase activity in C2C12 cells treated with vehicle (DMSO) or indicated concentration of IO or AICAR
(0.5 mM, as a positive control). (F) The relative PGC-1α luciferase activity in C2C12 cells treated with
Page 30/30
vehicle (DMSO), IO (1.0 µg/mL), or AICAR (0.5 mM). The Values are means of quintuplicate (B), triplicate
(D and E) and quadruplicate (F) ± SD. To determine statistical signicance, an unpaired two-tailed
Student’s
t
-test was used (B, D, E and F). Asterisk indicates statistical signicance. *
P
< 0.05, **
P
< 0.01
and ***
P
< 0.001 (IO vs. Vehicle).
Supplementary Files
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Supplementarydata20230202.docx
