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Sarcopenia, characterized by reduced muscle mass and fiber number leading to muscular atrophy, has been asso ‑ ciated with serious socioeconomic challenges among the elderly in developed countries. Therefore, preventing sarcopenia could be a promising strategy for achieving a healthy aging society. Nutmeg (Myristica fragrans) has been used as a spice to increase flavor and prevent putrefaction of food. Nutmeg contains various bioactive components that improve muscle activity. To determine the potential effect of lignan‑ enriched nutmeg extract (LNX) on sarco‑ penia, LNX (100 mg/kg body weight)‑fed aged mice were subjected to forced exercise. Herein, aged (22‑month‑ old) mice fed LNX for three weeks exhibited a shortened and thickened soleus muscle. The ratio of the soleus muscle mass (%) to body weight was significantly increased in LNX ‑fed aged mice. The relative increase in muscle mass in LNX ‑fed aged mice improved exercise activities, including rotarod, swimming, and grip strength test results. Proteome profiles of the soleus muscle of LNX‑fed mice were used to analyze protein–protein interaction network. Several myosin heavy chain isoforms were found to interact with actin, ACTA1, which functions as a hub protein. Furthermore, the expres‑ sion of myogenic proteins, such as MYH1, MYH4, and ACTA1, was dose‑ dependently increased in vivo. In result, our functional proteomic analysis revealed that feeding LNX restored muscle proteins in aged mice. Keywords Nutmeg, Sarcopenia, Exercise activity, Protein–protein interaction network Je‑Ho Lee, Hyuno Kang, Gyung‑ Tae Ban, Beom Kyu Kim and JaeHyeon Lee equally contributed in this study *Correspondence: Kun Cho email@example.com Jong‑Soon Choi firstname.lastname@example.org Geron Biotech Ltd., Daejeon 34133, Republic of Korea Division of Analytical Science, Korea Basic Science Institute, Daejeon 34133, Republic of Korea Department of Cancer Preventive Material Development, College of Korean Medicine, Kyung Hee University, Seoul 02447, Republic of Korea Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju 28119, Republic of Korea EastW ‑ est Cancer Center, Seoul Korean Medicine Hospital of Daejeon University, Seoul 05836, Republic of Korea Center for Research Equipment, Korea Basic Science Institute, Cheongju 28119, Republic of Korea Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Lee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 2 of 10 and safrole, are responsible for such toxicity (Du et al. Introduction 2014). Among these, myristicin is the most abundant Skeletal muscle is the largest organ by mass in the human toxic substance in nutmeg (Maeda et al. 2008). Report- body, in which muscle status indicates health and lon- edly, the methanol extract of nutmeg contains an aver- gevity of an individual. It reveals various physiological age of 2.1% myristicin (Hallström and Thuvander 1997). symptoms as it ages, including deterioration of func- One of the key aspects in preparation of nutmeg to con- tions, such as movement, respiration, vision, body tem- sume is minimizing myristicin concentration below non- perature control, and metabolic homeostasis. Human toxic level and maximizing beneficial ingredients such as muscle mass peaks during 30s, subsequently decreasing nectandrin B. by approximately 1% annually, and falling sharply in 70s In the present study, we aimed to investigate whether (Kalyani et al. 2014). This loss of muscle mass, called sar - exercise performance was enhanced in aged mice fed a copenia, can result in early death and reduce the qual- lignan-enriched nutmeg extract (LNX). We have estab- ity of life, ultimately leading to fall-related fractures and lished the optimal conditions for preparing LNX, which increased mortality rates (Xie et al. 2021). Currently, would comprise minimal levels of toxic myristicin (less there is no therapeutic agent available to prevent sarco- than 0.5%) and maximum nectandrin B, an active ingre- penia. Consumption of adequate proteins, vitamin D and dient (Jang et al. 2019). After oral administration of LNX strength training have been recommended to minimize for three weeks in C57BL/6 mice, the hindlimb muscles muscle loss (Uchitomi et al. 2020). Long-term use of cre- were dissected to estimate the anatomical indices. Exer- atine supplements, known as muscle boosters, can cause cise performance was evaluated as previously described liver and kidney damage among the elderly. Addition- (Xie et al. 2021). Rotarod, wire-hanging, swimming and ally, whey protein concentrate, often used as a protein grip strength tests were performed to evaluate muscle supplement, contains lactose, which can cause digestive activity in young and aged control, and LNX-fed aged problems in this population (Souza et al. 2009). Pharma- mice. Muscle proteomes of LNX-fed aged mice were cological strategies for muscle development include the cataloged, and a protein network was constructed from use of growth and sex hormones, insulin-like factors and the soleus muscle using a well-established shotgun pro- myostatin inhibitors. Bimagrumab (Novartis), RG6206 teomic method (Jang et al. 2020). We found that myo- (Roche) and ACE-031 (Acceleron) have been developed sin heavy chain isoforms in muscles were increased in and examined to prevent muscular dystrophy; however, LNX-fed aged mice, and their expression levels were long-term use of these drugs could induce renal toxicity verified by immunoblotting analysis. The findings of the (Rooks et al. 2017; Dao et al. 2020; Campbell et al. 2017). present study provide further insights into the potential Several studies have explored natural products hav- of lignan compounds as therapeutic agents to overcome ing potentials in improving muscle performance. For sarcopenia. example, natural herbal plants from tropical regions like Indonesia reportedly exhibit potential health benefits. In Materials and methods addition, some herbal plants are rich in bioactive ingredi- Animal treatment ents, such as catechins, resveratrol and curcumin, known Herein, we used C57BL/6 male mice supplied by the to contribute to skeletal muscle development (Pratiwi Animal Facility of Aging Science at Gwangju Center in et al. 2018). In particular, nutmeg is known to exhibit the Korea Basic Science Institute (KBSI). All animals (3- potential effects on various age-related diseases (Lestari and 22-month-old mice) were maintained at 22 ± 2 °C, et al. 2012). Nutmeg (Myristica fragrans), mostly cul- 55 ± 15% humidity, and a 12 h light/12 h dark cycle. The tivated in India, South Africa and Indonesia, has been experimental group was divided into four groups: young used as a flavoring agent in foods and as an oriental control (3-month-old), aged control (22-month-old), and medicine for diarrhea, abdominal distension, vomiting 10 and 100 mg/kg body weight (mpk) LNX-fed aged mice and appetite loss. Nutmeg extract contains bioactive lig- groups. LNX was orally administered once daily for three nan compounds, such as nectandrin B, fragransin A and weeks. Animal experiments were approved and followed tetrahydrofuroguaiacin B (Jang et al. 2019), which are the regulations of the KBSI Institutional Animal Care and beneficial for treating obesity, type 2 diabetes and other Use Committee (KBSI-20–30 and KBSI-IACUC-21–28). metabolic disorders (Hien et al. 2011). In animal mod- els, nectandrin B was shown to prevent adult diseases Measurement of muscle mass by activating the AMPK pathway (Nguyen et al. 2010). The body weights of C57BL/6 mice were measured daily Despite known medicinal benefits, excessive ingesting of before LNX administration. Upon completion of exercise nutmeg can induce toxic side effects, leading to delirium. paradigms, all mice were anesthetized using isoflurane Alkylbenzene derivatives, such as myristicin, elemicin L ee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 3 of 10 and then sacrificed by exsanguination, cutting the pos - the force at which the wire was released on pulling the terior vena cava and abdominal aorta. After skinned tail was recorded using the Chatillon Digital Force Gauge and photographed hindlimbs, the soleus and the gas- PC program. trocnemius muscles were independently dissected, and the length and the width were measured using a caliper. Protein sample preparation Each muscle was weighed using an electronic scale (HR- The mouse soleus muscle was selected as the protein 250AZ; Coretech, Hwasung, Korea). source for proteomic analysis. Using a surgical scalpel, the muscle tissue was cut into 5 mm × 5 mm × 5 mm pieces, placed in an Eppendorf tube, and washed three Histological observation of skeletal muscle times for 10 min with 50 ml of saline containing a pro- The gastrocnemius and the tibialis anterior muscles of tease inhibitor (Protease Inhibition Cocktail Tablet, hindlimbs were independently separated and fixed in 4% Roche). Muscle tissue (100 mg) was homogenized in 1 ml paraformaldehyde for 48 h. After dehydration and paraf- of mammalian protein extraction buffer (GE Healthcare, fin embedding, 5-μm-thick cross-sections were prepared Chicago, IL). The protein concentration was quantified using a microtome (Microm STP 120, Thermo Fischer using a BCA assay kit (Thermo Fischer, Seoul, Korea). Scientific, Waltham, MA). Serial sections were stained For proteomic analysis, in-solution trypsin digestion with hematoxylin and eosin using Vitroview Mas- was performed as described previously (Lee et al. 2015). son’s trichrome staining. Images of muscle fibers were Trypsin proteolysis was performed by reduction with obtained using a microscope (Nikon Eclipse Ti, Tokyo, dithiothreitol and alkylation with indoleacetic acid. Japan) with a camera (Nikon DS-Ri2, Tokyo, Japan) oper- Trypsin digestion was performed at 37 °C for 16 h, fol- ated by NIS-Elements BR software. lowed by lyophilization prior to mass spectrometry (MS). Physical exercise performance Mass spectrometry analysis The rotarod test was conducted using the Rotarod instru - MS analysis was performed as previously described ment (SB Technology, Miami, FL), as described previ- (Michalski et al. 2011). Dried peptides were reconstituted ously (Vaught et al. 1985). The rotarod measurements in 0.02% formic acid and 0.5% acetic acid to a final con - were performed in fixed and accelerating modes, rotat - centration of 100 ng/μl. Peptide sample (10 μL) was eluted ing at 4 rpm and 4–40 rpm, respectively. Mice were using a nanoACQUITY UPLC 2G-V/V Trap 5 μm Sym- acclimatized for 1 h before testing, and re-training was metry C (180 μm × 20 mm) trapping column (Waters, performed for 1 min in fixed mode. The main experiment Ireland) with 10 μL/min for 10 min, with the flow rate was performed in the accelerating mode, and the time maintained at 120 nL/min. The specification of analyti - taken to fall off the rotarod was recorded. cal column is 3 µm C AQ (100 µm × 15 cm) (NanoLC, In the wire-hanging test, mice were hung on a hori- Thermo Scientific, Waltham, MA). The peptides were zontal bar, approximately 50 cm above the ground. The eluted with a gradient of 0–65% acetonitrile for 80 min. strength of the forelimb was determined by measuring All MS and tandem MS spectra were detected using a the duration and latency before falling from the bar. The Q-Exactive Mass Spectrometer (Thermo Fischer Scien - test was repeated after intervals of at least 1 min. tific, Waltham, MA) in a data-dependent manner. Each The swimming test was performed as previously full MS was scanned in the range of 300–2000 m/z, and described (Nunez 2008). Briefly, mice were placed on a the most abundant precursor ion peaks were obtained platform (touchable by their feet) at one end of a 120 cm from MS spectra. All MS/MS spectra were acquired in round water tank, which was sufficiently wide to prevent a data-dependent mode for fragmentation of the 5 most limbs from touching the walls. Non-fat dry milk was abundant peaks from the full MS scan with 35% normal- added to the tank water to ensure the tank bottom was ized collision energy. The separated peptide ions eluted not visible. The test mice were allowed to swim with their from the analytical column were entered into the mass backs to the platform using different starting directions. spectrometer at an electrospray voltage of 2.2 kV. The An animal behavior tracking system recorded the move- dynamic exclusion duration was set at 180 s and exclu- ment route to calculate the swimming speed. sion mass width 0.5 Da. MS spectra were acquired with a The grip strength test was performed using a digi - mass range of 150–2000 m/z. tal force gauge (Ametek, Berwyn, PA), as previously described (Seto et al. 2011). The equipment comprised a Protein identification and quantification wire (1 mm in diameter) installed 35 cm high. The mouse Raw MS data were analyzed using the MaxQuant soft- was allowed to grip the wire using only the forelimbs, and ware (ver. 22.214.171.124, www. maxqu ant. org), as described Lee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 4 of 10 previously (Cox and Mann 2008). The MS/MS spectra overnight at 4 °C with primary antibodies (Santa Cruz were identified as proteins using forward and reverse Biotechnology, Dallas, TX) against Titin, Myh1, Myh4, sequences from the UniProt mouse database (http:// and Acta1. The blot was washed five times for 5 min with www. unipr ot. org; released in 2018_10, 59,345 entries) TTBS and incubated with horseradish peroxidase (HRP)- in the Andromeda search engine (integrated with Max- conjugated goat anti-mouse IgG (1:5000) secondary Quant). The precursor and fragment masses were antibody. Signals were detected using enhanced chemi- matched at initial mass tolerance levels of 6 and 20 ppm, luminescence (MilliporeSigma, Burlington, MA) and respectively. Two miscleavages were allowed, and Cys- visualized using a Chemi-Doc Imaging System (Bio- carbamidomethylation (+ 57) and Met-oxidation (+ 16) Rad, Hercules, CA). The relative expression of the target were considered fixed and variable protein modifications, protein was normalized to that of β-actin, an endogenous respectively. Protein quantification of the liquid chro - housekeeping protein. matography with tandem MS (LC–MS/MS) spectra was analyzed using a semi-quantitative method with Max- Statistical analysis Quant ion scoring. Qualified scoring peptides were con - Experimental animal data were analyzed using the sidered to satisfy the threshold set above p < 0.05, peptide Mann–Whitney U test. Comparisons were statistically level ≤ 0.05, and false discovery rate < 0.05 in the decoy significant at a 95% confidence level. One-way analy - database. Peptides were identified with monoisotopic sis of variance (ANOVA) was used to compare multiple mass selected, a precursor mass tolerance of 25 ppm, and groups. Data analyses were performed using GraphPad a fragment mass tolerance of 25 ppm, two missed trypsin Prism 5.0 (GraphPad Software). Statistical evaluation cleavage and fixed modification of carbamidomethyl of the proteome network analysis was performed using cysteine. Threshold score/expectation value for accepting Student’s t-test, and p ≤ 0.05 were deemed statistically individual spectra was based on Mascot ion score thresh- significant. old (0.05) as the standard ion score threshold specifically calculated by Mascot for each database search. Results and discussion In the present study, we evaluated the exercise perfor- Bioinformatics analysis mance of LNX-fed aged mice and further performed a Gene Ontology (GO) was analyzed with qualified protein proteomic network analysis of skeletal muscle to iden- data using InterPro2Go mapping (www. ebi. ac. kr/ GOA/ tify core proteins involved in muscular enhancement. Inter Pro2GO). GO annotation used search parameters in LNX, the test substance, was obtained by two-step eth- InterProScan. The top-ten BLAST hits were arranged by anol extraction, as previously reported (Nguyen et al. –3 listing those with an E-value of ≤ 1 × 10 for each query 2010). We concentrated nectandrin B, an active ingredi- sequence. Statistical significance was determined using ent, by up to 6.1% after the second extraction with 80% an individual t-test module from the scipy.stats module ethanol, whereas the content of myristicin, the toxic (scipy.org), with an in-house program coded by Python compound, was only 0.04% (data not shown). Isolation 3.8, where significant proteins were selected by p < 0.05 and enrichment of LNX by ethanol-extraction resulted and fold-change > 1.5. Protein–protein interaction (PPI) in the following yield of principle lignans: nectandrin B network was constructed using the Search Tool for (41.66 mg/g), fragransin A2 (0.18 mg/g), and verrucosin the Retrieval of INteracting Genes/proteins (STRING) (2.32 mg/g) (Additional file 1: Fig. S1). The final dried database (string-db.org), maximizing the networks as a extracts of LNX were subjected to subsequent animal default setting. experiments and proteomic analysis. After feeding naturally aged C57BL/6 mice with LNX Immunoblot analysis for three weeks, the debilitated fore- and hindlimb mus- Soleus muscle proteins were extracted with extraction cles were improved (Additional file 1: Fig. S2A). Micro- buffer [50 mM Tris–HCl (pH 7.4), 2 mM EDTA, 100 μg/ scopic observation of hindlimb muscles (gastrocnemius ml leupeptin, 20 μg/ml aprotinin, and 100 mM NaCl]. and tibialis anterior) revealed that the diameters of Protein samples (40 μg) were subjected to 10% sodium hindlimb muscle fibers in aged mice were smaller than dodecyl sulfate–polyacrylamide gel electrophoresis those in young mice. In addition, myofibers of tibialis (SDS-PAGE), and the gel was transferred to a polyvi- anterior muscle in aged mice were more loosely arranged nylidene difluoride (PVDF) membrane. The membrane than young mice. However, skeletal muscles of LNX (100 was blocked for 1 h with 5% skimmed milk in TTBS con- mpk)-fed aged mice showed marked recovery to the taining 50 mM Tris–HCl (pH 8.2), 0.1% Tween 20, and level of young mice in terms of myofiber diameter and 150 mM NaCl. Subsequently, the blots were incubated muscle fiber compactness (Additional file 1: Fig. S2B). L ee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 5 of 10 Testosterone, myostatin inhibition, and exercise are comparable with that of a previous report, demonstrat- known to increase contractile proteins (actin and myo- ing that Schisandrae fructus can enhance both gastroc- sin) in muscle fibers via anabolic intervention, resulting nemius and splenic muscles in aged mice (Kim et al. in hypertrophy (Verdijk et al. 2009). Conversely, an age- 2018), whereas LNX specifically increased the ratio related decrease in muscle mass and strength are typical of the soleus muscle mass to body weight. Likewise, it features of sarcopenia. has been reported that continuous feeding of C57BL/6 Skeletal muscles of LNX-fed aged mice were charac- mice with a natural product, 2,6-dimethoxy-1,4-ben- terized by gain of body weight and muscle indices. The zoquinone, for 7 weeks can increase muscle mass and anatomical indices included width, length and mass strength (Yoo et al. 2021). per body weight of the calf muscles. The gastrocnemius Several studies have reported the potential of phy- and the soleus muscles in hindlimbs were dissected, tochemicals to suppress or recover from progressive and anatomical indices were measured. Although low- sarcopenia, affording a sarcopenia recovery program. dose LNX-fed mice showed slight weight loss when Schizandra chinensis exhibits an inhibitory effect on compared with untreated controls, this tendency was muscle fiber in mice and suppresses muscular dystro - not observed in high-dose LNX-fed old mice. Thus, phy by increasing protein synthesis in human myocytes the effect of LNX on body weight gain was not cor - (Choi 2017; Kim et al. 2016). Recently, nutmeg extract related between LNX-treated and untreated groups, was found to increase skeletal muscle weight in aged rats, regardless of age (Fig. 1A). However, the morphology of mainly via IGF1-Akt-mTOR signaling and autophagy skeletal muscles revealed a clear relationship with the inhibition (Pratiwi et al. 2018). Therefore, we evaluated LNX feeding dose. In LNX-fed aged mice, the lengths the effect of LNX on exercise performance in C57BL/6 of gastrocnemius and soleus muscles were decreased, mice. Examination of several indices confirmed in vivo whereas the widths of both muscles were increased efficacy of LNX in terms of exercise performance using in a dose-dependent manner (Fig. 1B, C). LNX feed- rotarod, wire-hanging, swimming and grip strength tests. ing specifically affected both anatomical indices of In the rotarod test, endurance time in the accelerating the soleus muscle. In high-dose LNX-fed aged mice, mode (4 to 40 rpm) was increased in both LNX-treated the soleus muscle mass to body weight ratio was sig- aged groups; in particular, LNX feeding showed a signifi - nificantly increased (p < 0.05; Fig. 1D). This finding is cant increase specifically in aged mice (p < 0.05; Fig. 2A). Fig. 1 Phenotypical and anatomical changes of LNX‑fed mice. A Body weight gain. B Images of dissected gastrocnemius (Gas) and soleus (Sol) muscles. Each gradation on a rule is 1 mm scale. C Dimensions of dissected Gas and Sol muscles. D Weight of each Gas and Sol muscle divided by body weight. Data represent the mean ± standard deviation of triple or quadruple experiments. Statistical significance * p < 0.05, ** p < 0.01 Lee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 6 of 10 (A) (B) mpk - 10 100 - 10 100 mpk - 10 100 120 LNXCre Young Old Old (C) (D) 0.25 20 * 0.20 ** 0.15 0.10 0.05 0.00 - 10 100 - 10 100 mpk - 10 100 mpk YoungOld Old Fig. 2 Improved effects of LNX on exercise activities in LNX ‑fed mice. A Rota‑rod, B wire ‑hanging, C swimming, and D grip strength tests were performed in LNX‑fed mice. Data represent mean ± standard deviation of triple or quadruple experiments. Statistical significance * p < 0.05, ** p < 0.01 The wire-hanging test revealed an increasing pattern of B could reduce intracellular reactive oxygen species endurance time in LNX-fed aged mice with no statisti- levels in human fibroblasts and prevent senescence by cal significance (Fig. 2B). Creatine, known as a muscle activating AMPK signaling (Jang et al. 2019). In a reju- booster, can increase wire-hanging endurance time, simi- venation program, concurrent application of anticancer lar to LNX. However, administration of 120 mpk creatine drugs, dasatinib and quercetin, in mice as a senolytic for three weeks caused severe cirrhosis, while the livers cocktail therapy was shown to improve muscle strength, of LNX-fed old mice appeared normal (Additional file 1: treadmill exercise ability, and enhance grip strength (Xu Fig. 3). Creatine is used as an energy source by phospho- et al. 2018). Administering the senolytic drug ABT263 creatine kinase in muscles; however, excessive creatine for 4 weeks could improve skeletal muscle (Chang et al. use in the elderly may burden the liver and kidney (Poort- 2016), and aged mice fed ruxolitinib (INCB18424) for mans and Francaux 2000). 10 weeks increases hanging duration and grip strength Considering the swimming test, no difference was (Xu et al. 2015). Therefore, the senolytic potential of LNX detected in young mice fed in the presence and absence needs to be explored. of LNX (Fig. 2C). However, swimming speed of aged To identify core proteins induced by LNX feeding, mice was approximately 50% less than that of young shotgun proteomic analysis was performed with the mice. Interestingly, swimming speed was recovered by soleus muscle of mouse hindlimb. Shotgun proteomics up to 89% of the young control in 100 mpk LNX-fed was used with modifications, except for the MaxQuant aged mice. In particular, low- and high-dose LNX-fed database from a previous method (Lee et al. 2015). Con- aged mice showed statistical significance at p < 0.05 and sidering the experimental design, five groups were clas - p < 0.01, respectively. The forelimb grip strength was sified as young (3-month-old) and aged (22-month-old) measured in aged mice fed 10 or 100 mpk LNX. Grip controls, and LNX (10, 30, 100 mpk)-fed aged mice. The strength was significantly increased in 100 mpk LNX-fed distribution of protein abundance from 15 samples is aged mice (P < 0.05; Fig. 2D). Collectively, these results shown using a box plot (Additional file 1: Fig. S4). The suggest that nectandrin B, as the main lignan compound average values for all groups were constant at 18, and the in LNX, enhanced exercise activity by increasing mus- degree of variance was similarly normalized, suggest- cle myofibers. Previously, we reported that nectandrin ing that the proteomic conditions were well established. Swim Speed (cm/sec) Drop Time (sec) Grip Strength (kgf) Drop Time (sec) L ee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 7 of 10 Table 1 Summary of quantitative proteome analysis. Data are presented in three groups: muscles of young, old, and LNX (100 mpk)‑ fed old mice Group Fold change Number of proteins Remarks Old vs Young p* < 0.05 |FC|≥ 1.5 35 Additional file 2: Table S1 − 1.5 < FC < 1.5 55 p ≥ 0.05 96 LNX‑ Old vs Old p* < 0.05 |FC|≥ 1.5 16 Additional file 2: Table S2 − 1.5 < FC < 1.5 6 p ≥ 0.05 13 *p-value was calculated from three independent proteins altered in old mice compared to that in young mice or in LNX-fed old mice compared to that in old mice The number of proteins indicated in this table was determined using general proteome search criterions A total of 221 proteins were summarized according to enhanced expression of zinc finger in muscle cells under the comparison group, statistical significance, and fold- muscular dystrophy conditions (Lynch et al. 2019). If change (Table 1). Among them, 35 proteins between these zinc finger proteins are marker proteins of aging, aged and young mice groups showed a fold-change of ˃ LNX could restore their expression to normal levels and 1.5, with statistical significance at p < 0.05 (Additional afford clues regarding aging. Another interesting protein file 2: Table S1). Furthermore, 16 proteins were signifi - is LIM-binding protein, in which LIM and cysteine-rich cantly altered between LNX (100 mpk)-fed and -unfed domain 1 (LMCD1) regulate skeletal muscle hypertro- aged mice, with a fold-change of ˃1.5 (Additional file 2: phy (Ferreira et al. 2019). Likewise, in our proteomic Table S2). Heat map analysis revealed that 13 proteins analysis, the expression of LIM-binding protein 3 (Ldb3) were significantly recovered by LNX feeding in aged was increased in aged mice; however, LNX administra- mice (Additional file 1: Fig. S5), including LNX-induced tion decreased this expression, resulting in a young-like up- and down-regulated proteins in aged mice (Table 2). phenotype. Interestingly, increased expression of heat shock 70 pro- To address the biological function, proteins identi- tein (Hspa1b) was consistent with that reported in mus- fied in the soleus muscles of aged mice and LNK (100 cular dystrophic mdx mice (Carberry et al. 2014). In mpk)-fed aged mice were subjected to GO assessment addition, zinc finger proteins, Zfp882 and Zc3b13 were via InterProScan. Based on the top priority of GO increased in senescent mouse muscle tissue, similar to groups, components related to skeletal muscle were Table 2 List of recovered muscle proteins in LNX‑fed old mice from up ‑ or down‑regulated proteins of old versus young mice Acc. # Protein name Description Mr (Da) Fold change (O Fold vs Y ) change (LX-O vs O) Proteins altered by LNX from down-regulated proteins in old mice Q99LX0 Park7 Protein/nucleic acid deglycase DJ‑1 20,021 − 1.76 + 1.58 P63038 Hspd1 60 kDa heat shock protein, mitochondria 61,055 − 1.58 + 2.31 Proteins altered by LNX from up-regulated proteins in old mice E9PYJ9 Ldb3 LIM domain‑binding protein 3 77,135 + 1.77 − 5.61 D3Z7G7 Zfp882 Zinc finger protein 882 (fragment) 63,994 + 2.80 − 3.33 Q8CGP6 Hist1h2ah Histone H2A type 1‑H 13,950 + 2.04 − 2.93 E9Q784 Zc3h13 Zinc finger CCCH domain‑ containing protein13 203,755 + 2.06 − 2.53 P17879 Hspa1b Heat shock protein 70 kDa protein 1B 70,176 + 1.69 − 2.23 Q6P6L5 Mybpc1 Myosin‑binding protein C, slow‑type 126,197 + 1.51 − 2.12 Q6IFZ6 Krt77 Keratin, type II cytoskeletal 1b 61,359 + 2.34 − 1.86 P29387 Gnb4 Guanine nucleotide‑binding protein subunit β‑4 37,379 + 1.76 − 1.83 A2AT66 Ttn Titin (fragment) 76,372 + 1.56 − 1.76 F6RSJ3 Ttn Titin (fragment) 130,483 + 1.54 − 1.70 Q62000 Ogn Mimecan 34,012 + 1.85 − 1.64 Lee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 8 of 10 assigned as the main GO groups (Additional file 2: verified by immunoblot analysis (Fig. 4). As shown in Table S3). LNX-induced myofiber restoration was well the heat map analysis (Additional file 1: Fig. 5), LNX matched with the GO analysis, in which myofibers slightly decreased Ttn expression; however, the expres- consist of actin and myosin. This finding is consistent sion levels of Myh1, Myh4, and Acta1 significantly with that of a previous study, demonstrating that the increased in a dose-dependent manner. The expression expression of myosin, tropomyosin, and the small heat of contractile proteins was in good agreement with the shock protein Hsp27 was reduced in aged mice (Car- results of previous proteomic studies (Dowling et al. berry et al. 2012). Using the STRING database, a PPI 2019). A muscle restoration study with a single com- network was constructed using whole soleus muscle pound, such as nectandrin B, verrucosin or fragransin proteomes from young, aged and LNK-fed aged mice A2, known components of LNX, could afford promis- (https:// string- db. org). Several myosin heavy chain ing results for developing potent therapeutic drug can- protein isoforms (Myh) were detected in the core didates against sarcopenia. cluster, with actin Acta1 as a hub protein (Fig. 3A). In addition, Ldb3 interacts with the surrounding actin Conclusions proteins in the local network Group 3 (Fig. 3B). It can Herein, we aimed to determine the effect of LNX, com - be speculated that Ldb3 regulates myosin and actin via prising the major lignan, nectandrin B, on exercise per- PPI while acting antagonistically against LMCD1 as a formance and examine the working mechanism using positive regulator of muscle mass (Ferreira et al. 2019). protein network analysis via shotgun proteomics. After Keratin 77 levels increased with age but were sig- a three-week treatment, LNX-fed aged mice showed nificantly decreased by LNX feeding. Krt77 interacts increased soleus muscle mass to body weight ratio and with Myh4, a component of the main protein network enhanced exercise performance in rotarod, swimming cluster. and grip strength tests. Comparing rejuvenation-related When proteins were unpacked, it was confirmed proteins in LNX-fed aged mice, zinc finger protein, heat that the actin isoforms, including muscle fiber pro- shock protein 70, and keratin were expressed, as reported teins such as Group 1, form a network. Accordingly, in previous studies. Muscle protein network analysis immunoblotting confirmed the expression of Myh1 revealed the muscle contraction protein Acta1 cluster and Myh4 interacting with Acta1 in LNX-fed aged and upregulation of Myh1, Myh4, and Acta1 confirmed mouse muscles. The expressional changes in the by immunoblotting. In conclusion, LNX could be effec - soleus muscles of aged mice fed different LNX con- tive for aging-related sarcopenia, and bioactive lignans of centrations, centering on myogenic proteins, were LNX could be developed as potential therapeutic agents. Fig. 3 Protein network analysis of whole mouse soleus muscle proteomes. Protein network was constructed using STRING database (https:// string‑ db. org). A Full view of protein–protein interaction network. B Main protein–protein network (group 1) and satellite networks (group 2–4). Red and blue circle indicates actin and myosin isoforms, respectively L ee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 9 of 10 Fig. 4 Immunoblot analysis of soleus muscle proteins identified from LNX ‑fed old mice. A Gel images of immunoblotting. B Quantitative analysis of protein markers such as TTN, MYH1, MYH4, and ACTA1. Relative expression was presented as %control of old mice using the normalized levels of β‑actin as internal control. ns, not significant, statistical significance at * p < 0.05, ** p < 0.01, ***p < 0.001 Acknowledgements Supplementary Information We remember the initiation of the present work by the late Dr. Ik‑Soon Jang. The online version contains supplementary material available at https:// doi. We thank the Dae‑Han‑ Cell‑Pharm and their researchers for giving us the nut ‑ org/ 10. 1186/ s40543‑ 023‑ 00377‑2. meg extract samples. Dr. G‑ T. Ban would like to acknowledge Geron Biotech Ltd., KBSI Research Company, for the first publication. Additional file 1. Figure 1 Isolation and enrichment of ethanol‑ extracted LNX. (A) UV chromatogram of ethanol‑ extracted LNX and yields of main Author contributions lignan compounds from LNX. Mean±St Dev. Standard deviation was HK, H–SY, KC and J‑SC designed the study. J‑HL, HK, G‑ TB, BKK and JHL calculated by triplicate experiments. (B) Chemical structures of main performed material preparations, animal studies, histology and biochemical lignan compounds from LNX. Analysis condition: equipment, Agilent experiments. HH and KC performed and analyzed proteomics. HK, G‑ TB, H– 1200 series HPLC system; mobile phase, acetonitrile: 0.1% formic acid (4:6); SY, K.C. and J‑SC analyzed the data and wrote the paper with input from all stationary phase, Waters C18 column (5 μm pore size, 25 cm in length); authors. All authors read and approved the final manuscript. J‑HL, HK, G‑ TB, detection wavelength, 234 nm; flow rate, 1.5 ml/min. Figure 2 Dissection BKK and JHL equally contributed. and microscopic images of skeletal muscles of LNX‑fed mice. (A) Images of skeletal muscles. Left and right panel on each picture indicates fore Funding and hind limb, respectively. Scale bar is 1 cm. (B) H&E staining of cross This work was supported by a grant from the Korea Basic Science Institute to sections from gastrocnemius muscle and tibialis anterior. Abbreviations: J‑S. Choi (C380300). mfb, microfibrils; mf, microfiber; pm, perimysium. Arrows indicate nuclei staining. Scale bar = 20 μm. Figure 3 Anatomical changes of livers in (A) Availability of data and materials young, (B) LNX (100 mpk) and (C) creatine (120 mpk)‑fed old mice. Scale The datasets used and/or analyzed during the current study are available from bar = 1 cm. Figure 4 Box plots of all proteome quantitates from four the corresponding author on reasonable request. groups: young, old, LNX‑fed old mice. Doses of LNX applied to old mice were 10, 30, and 100 mpk. Proteomic sample source was soleus muscle Declarations from each group. Figure 5 Heat map analysis of mouse soleus muscle proteomes. Significantly altered 16 proteins in LNX ‑fed old vs. old mice Competing interests soleus muscles. Red and blue color intensity means the degree of up‑ and The authors declare that they have no competing interests. down‑regulation, respectively. Additional file 2. Table 1 List of significantly altered proteins in old mice muscle compared to young mice control. Table 2 List of significantly Received: 22 September 2022 Accepted: 29 January 2023 altered proteins in LNX‑fed old mice muscle compared to old mice control. Table 3 List of GO‑analyzed proteins identified commonly in old versus LNX (100 mpk)‑fed old mice muscle. Top ‑ten ranked GO groups in number were listed according to (A) biological process, (B) molecular function, and (C) cellular function. Lee et al. Journal of Analytical Science and Technology (2023) 14:11 Page 10 of 10 References Michalski A, Cox J, Mamm M. 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Journal of Analytical Science & Technology – Springer Journals
Published: Feb 16, 2023
Keywords: Nutmeg; Sarcopenia; Exercise activity; Protein–protein interaction network
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