Chemicals and Reagents
Wild-type C57BL/6 male mice were purchased from (Orient-Bio, Seongnam, Korea). Fetal bovine serum (FBS), horse serum (HS) and Dulbecco modified Eagle’s medium (DMEM) were purchased from Thermo Scientific (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 identified 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 reflux, and then filtered. The filtrate 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 filtered. The filtrate 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 Infinity 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 flow 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 flow 8 L/min, nebulizer pressure 35 psi, sheath gas temperature 350°C, sheath gas flow 11 L/min, 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 Profinder 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 filters was set at 1000 counts. Missing peaks were filtered 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 (Profinder Archive) file, which can be imported into Mass Profiler Professional (MPP) software (version 15.1, Agilent) for further data analysis. Normalization (percentile shift), defining the sample sets, baselining (median of all samples), filtering by frequency, and significance 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 specific metabolites (Table S1) for IO1 were identified 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 identification 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 identified 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% confidence 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 transmission, skeletal muscle contraction, muscle contraction mainly due to the presence of acetylcholine receptor subunits in the network. Important target nodes were identified 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 sacrificed. 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 sacrificed. Then, they were victimized on Day 3 and Day 21 after injection. All animals were sacrificed 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 Modified Eagle Medium high glucose; Thermo Scientific, 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 confluence 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 quantified using MTT colorimetric assay. In briefly, 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]. Briefly, 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]. Briefly, 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 ECLIPSE TE-2000U microscope and NIS-Elements F software (Nikon, Tokyo, Japan). To analyze the efficiency of myotube formation, the MHC-positive myotubes containing two to nine, or ten or more nuclei, were quantified 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 fields. Quantification of myotube diameter was performed with IMAGE J software. Average myotube diameter is presented as means determination of six fields ± 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 Table 1.
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-1α | Forward | 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’ |
Cox4 | Forward | 5’-CTATGTGTATGGCCCCATCC-3’ |
Reverse | 5’-AGCGGGCTCTCACTTCTTC-3’ |
Ucp2 | Forward | 5’-ACTGTCGAAGCCTACAAGAC-3’ |
Reverse | 5’-CACCAGCTCAGTACAGTTGA-3’ |
Tnfα | Forward | 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 fiber 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 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 fixation, permeation, and incubation with primary antibodies against MyhIIa and MyhⅡb (DSHB) and laminin (Abcam). Images were captured and proceed with a Nikon ECLIPSE TE-2000U using NIS-Elements F software. Myofiber 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 efficiency 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 figure legends. Differences were considered statistically significant at or under values of P < 0.05.