Exploring the Potential of Crassostrea nippona Hydrolysates as Dietary Supplements for Mitigating Dexamethasone-Induced Muscle Atrophy in C2C12 Cells

Muscle atrophy is a detrimental and injurious condition that leads to reduced skeletal muscle mass and disruption of protein metabolism. Oyster (Crassostrea nippona) is a famous and commonly consumed shellfish in East Asia and has become a popular dietary choice worldwide. The current investigation evaluated the efficacy of C. nippona against muscle atrophy, which has become a severe health issue. Mammalian skeletal muscles are primarily responsible for efficient metabolism, energy consumption, and body movements. The proteins that regulate muscle hypertrophy and atrophy are involved in muscle growth. C. nippona extracts were enzymatically hydrolyzed using alcalase (AOH), flavourzyme (FOH), and protamex (POH) to evaluate their efficacy in mitigating dexamethasone-induced muscle damage in C2C12 cells in vitro. AOH exhibited notable cell proliferative abilities, promoting dose-dependent myotube formation. These results were further solidified by protein expression analysis. Western blot and gene expression analysis via RT-qPCR demonstrated that AOH downregulated MuRF-1, Atrogin, Smad 2/3, and Foxo-3a, while upregulating myogenin, MyoD, myosin heavy chain expression, and mTOR, key components of the ubiquitin–proteasome and mTOR signaling pathways. Finally, this study suggests that AOH holds promise for alleviating dexamethasone-induced muscle atrophy in C2C12 cells in vitro, offering insights for developing functional foods targeting conditions akin to sarcopenia.


Introduction
Sarcopenia is a health condition that leads to diminished skeletal muscle mass, muscle fiber number, and muscle area with increasing age.It destructively impacts a healthy life and is a high-risk factor for the physical disability known as muscle atrophy.Muscle atrophy leads to an interruption in the balance between protein synthesis and degradation, lessening the protein content, reducing fiber diameter, and forming fatigue resistance in the body [1].At ages above 60, the potential occurrence of muscle atrophy-related sarcopenia increases to approximately 5-13% [2].Hence, these unfavorable conditions influence the regular lifestyle of humans and impair their overall well-being.Therefore, ubiquitin-proteasome systems [34].DEXA also delivers the necessary environmental conditions to downregulate the mammalian target of the rapamycin mTOR pathway to promote the breakdown of protein synthesis inside the cell [2].Therefore, it is important to investigate therapeutic approaches to treat this condition to prevent DEXA-induced muscle atrophy, introduce anti-muscle atrophy compounds to prevent important clinical implications, and investigate anti-muscle atrophy drugs or foods to treat these conditions effectively.Numerous studies have shown that enzyme-assisted extraction methods provide outstanding outcomes, including efficient extraction of bioactive compounds with greater extraction yields and protein content, which are highly recognized for superlative muscle growth [6,[35][36][37][38][39].
Therefore, this study aimed to prepare oyster hydrolysates from C. nippona hydrolyzed with three different enzymes, alcalase, flavourzyme, and protamex, and evaluate their ability to prevent or delay muscle atrophy in DEXA-induced C2C12 cells in vitro.Moreover, a comparative study was also performed to determine which C. nippona hydrolysates obtained from alcalase, flavourzyme, and protamex treatments can be applied convincingly for product development in downregulating DEXA-induced muscle atrophy in skeletal muscles of C2C12 cells in vitro.In addition, given that the development of functional foods and the demonstration of the suitability of enzyme hydrolysates of C. nippona is also necessary in the modern world, this study determined the effectiveness of these samples in the development of functional foods to treat atrophic disorders, such as sarcopenia, in a promising way.

General Composition of the Enzyme Hydrolysates of Crassostrea Nippona
The proximate analyses of AOH, FOH, POH, and DWO are summarized in Table 1.Polysaccharides, polyphenols, proteins, lipids, ash, and yield were determined on a dryweight basis.The yields of AOH, FOH, POH, and DWO were obtained as 75.33 ± 1.08%, 68.33 ± 0.58%, 61.33 ± 0.58%, and 14.00 ± 1.00%, respectively.The yields of all hydrolysates were significantly higher than those of the DWO sample, with that of AOH being the highest.The highest protein content was acquired in AOH as 45.90 ± 2.71%, while FOH, POH, and DWO had protein recovery percentages of 36.02 ± 2.42%, 43.68 ± 1.57%, and 40.05 ± 0.43%, respectively.AOH delivered a higher polysaccharide yield than FOH, POH, and DWO.As expected, the polyphenol content of all four samples was very low, and no significant differences were observed in the lipid profiles of all samples analyzed in this study.

Cell Cytotoxicity and Proliferation Activity of the Enzyme Hydrolysates of Crassostrea Nippona
The muscle growth activity of the three different enzyme hydrolysates obtained from C. nippona was evaluated by assessing cell cytotoxicity, proliferation, and differentiation.The toxic effects and proliferative ability of C. nippona in C2C12 cells were determined using CCK8 and BrdU assays, respectively, in a dose-dependent manner.According to the consideration of cell toxicity degrees, results confirmed that all three hydrolysate samples (AOH, FOH, and POH) and DWO were nontoxic to C2C12 cells at 25, 50, and 100 µg/mL concentrations (Figure 1a-d).
The muscle growth activity of the three different enzyme hydrolysates obtained from C. nippona was evaluated by assessing cell cytotoxicity, proliferation, and differentiation.The toxic effects and proliferative ability of C. nippona in C2C12 cells were determined using CCK8 and BrdU assays, respectively, in a dose-dependent manner.According to the consideration of cell toxicity degrees, results confirmed that all three hydrolysate samples (AOH, FOH, and POH) and DWO were nontoxic to C2C12 cells at 25, 50, and 100 µg/mL concentrations (Figure 1a-d).
Cell proliferation results showed that both AOH and POH had a significant cell proliferation influence over the DEXA-treated group in C2C12 cells in a dose-dependent manner.FOH did not perform significantly on cell proliferation at the low-concentration ranges of 25 and 50 µg/mL.At the same time, the DWO group was unable to perform any cell-productive effect at any range over the DEXA-treated group.DEXA concentration was maintained constantly throughout the differentiation process at 10 µM [40].The significance of each hydrolysate sample was assessed over the DEXA treatment group (Figure 1e-h).

Immunostaining and Myotube Diameters on MyHC Expression
Muscle growth was assessed by immunostaining with myosin heavy chain (MyHC) primary antibodies, followed by analysis of the myotube diameters of each stained caption relevant to specific hydrolysate groups and respective concentrations.MyHC observations, nuclear staining of 4′,6-diamidino-2-phenylindole (DAPI), and merged images of MyHC and DAPI for 25, 50, and 100 µg/mL concentrations for AOH and POH are shown in Figure 2a,b, respectively.Myotube quantification data for AOH and POH at 25, 50, and 100 µg/mL concentrations is shown in Figures 2c,d.The results indicate that MyHC expression and myotube diameters in differentiated C2C12 cells treated with AOH and POH were significantly upregulated by DEXA in a dose-dependent manner (Figure 2).Cell proliferation results showed that both AOH and POH had a significant cell proliferation influence over the DEXA-treated group in C2C12 cells in a dose-dependent manner.FOH did not perform significantly on cell proliferation at the low-concentration ranges of 25 and 50 µg/mL.At the same time, the DWO group was unable to perform any cell-productive effect at any range over the DEXA-treated group.DEXA concentration was maintained constantly throughout the differentiation process at 10 µM [40].The significance of each hydrolysate sample was assessed over the DEXA treatment group (Figure 1e-h).

Immunostaining and Myotube Diameters on MyHC Expression
Muscle growth was assessed by immunostaining with myosin heavy chain (MyHC) primary antibodies, followed by analysis of the myotube diameters of each stained caption relevant to specific hydrolysate groups and respective concentrations.MyHC observations, nuclear staining of 4 ′ ,6-diamidino-2-phenylindole (DAPI), and merged images of MyHC and DAPI for 25, 50, and 100 µg/mL concentrations for AOH and POH are shown in Figures 2a and 2b, respectively.Myotube quantification data for AOH and POH at 25, 50, and 100 µg/mL concentrations is shown in Figure 2c,d.The results indicate that MyHC expression and myotube diameters in differentiated C2C12 cells treated with AOH and POH were significantly upregulated by DEXA in a dose-dependent manner (Figure 2).

Molecular Weight Determination and Amino Acid Composition
The average molecular weight of the AOH-treated differentiated C. nippona was determined and is shown in Figure 3.The protein band was visualized as a smear distributed over the 15-75 kDa range and highly concentrated in the 63-75 kDa range.The amino acid compositions of the same sample (AOH) are presented in Table 2.

Molecular Weight Determination and Amino Acid Composition
The average molecular weight of the AOH-treated differentiated C. nippona wa termined and is shown in Figure 3.The protein band was visualized as a smear distrib over the 15-75 kDa range and highly concentrated in the 63-75 kDa range.The amino compositions of the same sample (AOH) are presented in Table 2.

Protein Expression after Treatment with AOH
Western blot analysis was conducted on the AOH samples to determine the expression of MyHC, myogenin, and MyoD, as shown in Figure 4.The protein expression of MuRF1, Atrogin, Smad 2/3, and Foxo-3a are illustrated in Figure 5. Relative expressions for each were analyzed through ImageJ.MyHC, myogenin, and MyoD were expressively upregulated over the DEXA-treated group, and MuRF-1, Atrogin, p-Smad 2/3, and p-foxo-3a were strongly downregulated in the AOH samples treated with DEXA (10 µM) in a dose-dependent manner.GAPDH was used as an internal reference.

Discussion
Oyster (C.nippona) is a famous and commonly consumed shellfish in East Asian countries and has become a popular dietary choice worldwide [23].Investigating the potential of marine shellfish in muscle growth has also become vital on the industrial scale to develop functional foods and pharmaceutical products [41].Hence, the current study aimed to investigate the potential of C. nippona for human consumption to develop as an impactful functional food for sarcopenia-like muscle atrophic conditions effectively.Therefore, the present study examined the value of AOH on muscle atrophy.Specifically, the authors focused on its potential anti-atrophic effects and explored underlying mechanisms.The overall findings assuredly demonstrate that AOH declined muscle atrophy.Through comprehensive analysis of gene and protein expression levels, the potential of AOH for the regulation of key myokines involved in this process was revealed.Notably, both negative and positive myokines were affected, shedding light on the intricate regulatory network governing muscle preservation.
Proteins, lipids, and carbohydrates are critical determinants of skeletal muscle growth, with their composition playing a pivotal role in the development of functional foods and dietary supplements.Among these components, proteins are particularly essential for muscle growth and development.Conversely, polysaccharides serve as vital energy sources for cellular metabolism and play a crucial role in hormone modulation and regulation.Notably, the general composition of enzyme hydrolysates revealed significantly higher levels of proteins, lipids, and carbohydrates in AOH, encouraging further investigations into its anti-atrophic activity [41].
Considering both cytotoxicity and cell proliferation, this study shows that the AOH and POH are not toxic to differentiating cells and have significant effects on

Discussion
Oyster (C.nippona) is a famous and commonly consumed shellfish in East Asian countries and has become a popular dietary choice worldwide [23].Investigating the potential of marine shellfish in muscle growth has also become vital on the industrial scale to develop functional foods and pharmaceutical products [41].Hence, the current study aimed to investigate the potential of C. nippona for human consumption to develop as an impactful functional food for sarcopenia-like muscle atrophic conditions effectively.Therefore, the present study examined the value of AOH on muscle atrophy.Specifically, the authors focused on its potential anti-atrophic effects and explored underlying mechanisms.The overall findings assuredly demonstrate that AOH declined muscle atrophy.Through comprehensive analysis of gene and protein expression levels, the potential of AOH for the regulation of key myokines involved in this process was revealed.Notably, both negative and positive myokines were affected, shedding light on the intricate regulatory network governing muscle preservation.
Proteins, lipids, and carbohydrates are critical determinants of skeletal muscle growth, with their composition playing a pivotal role in the development of functional foods and dietary supplements.Among these components, proteins are particularly essential for muscle growth and development.Conversely, polysaccharides serve as vital energy sources for cellular metabolism and play a crucial role in hormone modulation and regulation.Notably, the general composition of enzyme hydrolysates revealed significantly higher levels of proteins, lipids, and carbohydrates in AOH, encouraging further investigations into its anti-atrophic activity [41].
Considering both cytotoxicity and cell proliferation, this study shows that the AOH and POH are not toxic to differentiating cells and have significant effects on downregulating muscle atrophy.Based on these criteria, AOH and POH were selected for the immunostaining analysis.The present study reveals that myotube formation was repressed by DEXA (Figure 2).When AOH and POH were subjected to analysis of the formation of myotubes through immunofluorescence techniques, the results confirmed that both AOH and POH were sufficient for their ability to downregulate muscle atrophy in a dose-dependent manner.MyHC was used as a marker to evaluate differentiation.The decline in MyHC expression suggests the incapacitation of myotube formation, which indicates declining muscle regeneration and strength.A similar trend was reported in recent studies of Ishige okamurae (IO) and diphloroethohydroxycarmalol (DPHC) against palmitic acid-impaired skeletal myogenesis in C2C12 cells [42,43].A significant increase in myotube diameters in AOH and POH leads to enhanced myotube fusion of myoblasts to form active mature muscle fibers for treating muscle atrophy [43].
Comparative analysis revealed that protein expression was in an optimum state to abate muscle atrophy, detected only in AOH.Therefore, AOH was selected from the entire screening process to conduct this study.The enzyme hydrolysates of C. nippona contain a mixture of proteins with a wide range of molecular weights, and SDS-PAGE was used to analyze the molecular weight distribution of the AOH.This aids in our further studies regarding the peptide isolation from AOH depending on the significance of the biological activities.Amino acids also play a vital role in muscle development, and composition analysis of AOH revealed that arginine represented the highest MOL% (15.45%), which is significant for muscle growth, strength, and regeneration.Leucine, isoleucine, and valine are represented as branching amino acids in a considerable range compared to others.These amino acids are highly responsible for the downregulation of muscle breakdown and the assembly of proteins in muscles.Leucine activates mTOR, which is a necessary component of protein synthesis [28].Isoleucine promotes myogenesis and intramyocellular lipid accumulation, which also facilitates significant protein turnover in the body.Moreover, valine affects the advancement of skeletal tissues, the synthesis of neurotransmitters, energy production, muscle synthesis, and maintenance of immunological parameters effectively [44][45][46].Additionally, substantial amounts of alanine and glycine are also present in this sample, significantly contributing to muscle growth [47].Hence, this amino acid profile obtained from AOH was highly beneficial for the development of muscles as well as showing their potential to suppress the atrophic conditions generated in the body due to various stress factors.Alcalase is a serine endopeptidase synthesized from Bacillus lichenifomis that has a superior potential for the effective breakdown of proteins from numerous sites, in the formation of a diverse range of peptide fragments, which leads to vital environmental conditions for treating muscle growth and abating muscle atrophy [48].Therefore, in consideration of the broad view of our future studies regarding peptide isolation and purification, AOH shows high potential for the development of functional foods.
A Western blotting experiment was performed to evaluate the myokine expression and its regulation by AOH in the DEXA-induced C2C12 cells.Results obtained from Western blotting showed that AOH downregulated muscle atrophic conditions by synchronizing the major constituents contributing to the ubiquitin-proteasome and mTOR signaling pathways.MuRF-1 and Atrogin are E3 ubiquitin ligases involved in the ubiquitinproteasome system, which leads to the degradation of proteins in the cells [49].FoxO3a forms a complex with Smad2/3 and activates MuRF-1 transcription.An alternative study revealed that increasing the expression of phosphorylated forms of Smad2/3 and Foxo 3a leads to the upregulation of positive regulators of atrogein [50,51].Therefore, overexpression of MuRF-1 and Atrogin activated the ubiquitin-proteasome pathway for protein degradation to promote muscle atrophy.The results obtained from Western blot analysis strongly expressed that, under DEXA-induced conditions, AOH has a significant effect on downregulating the ubiquitin ligases of MuRF-1 and Atrogin in a dose-dependent manner (Figure 4).One study reported similar effects by using a marine fish (Lutjanus guttatus), and another study detected quercetin-like flavonoids isolated from various plants for muscle atrophy potential [52,53].MyoD synchronizes the myofibrillar gene expression to facilitate muscle contraction and regeneration.Furthermore, myogenic differentiation is increased by myogenin [54,55].Due to the atrophy situation, Smad2 and Smad3 targeted MyoD and myogenin and inhibited myoblast and myofiber proliferation and differentiation [2].
The protein expression levels of MyoD and myogenin in the AOH-treated samples were significantly higher than in the DEXA group.Downregulation of the phosphorylated forms of Smad2/3 and Foxo-3a during this study signified an upsurge in the regulation of MyoD and myogenin to mitigate muscle atrophy in AOH-treated C2C12 cells.Hence, this study showed that AOH has a significant impact on abating muscle atrophic conditions and boosts muscle growth by facilitation through myoblast and myofiber proliferation, differentiation, muscle contraction, and regeneration effectively.
The major constituents required for protein synthesis in cells are mTOR [54] and the myosin-heavy chain which contributes to the contractile apparatus in muscle fibers [42].Hence, protein expressions and mRNA gene expressions showed a significant upregulation of MyHC and mTOR even under DEXA-induced conditions, signifying that AOH can promote sufficient protein and fibers for muscles to avoid atrophic conditions effectively.
Therefore, the results indicated that AOH has an exceptional effect on attenuating DEXA-induced muscle atrophy in C2C12 cells in an in vitro model by impending negative regulators such as MuRF-1, Atrogin, Smad2/3, and FoxO 3a according to the ubiquitinproteasome pathway and promoting the expression of positive regulators such as Myogenin, MyoD, mTOR, and MyHC, which correspond to the mTOR signaling pathway (Figures 4 and 5).
Analogous to the trend of protein expression, the AOH-treated differentiated C. nippona significantly downregulated the mRNA expression trend of MuRF-1, Atrogin, and FoxO 3a, and upregulated the expression of Myogenin, MyoD, and mTOR over in DEXA (Figure 5).Downregulation of Foxo-3a also signified that AOH effectively inhibited MuRF-1 and Atrogin in mRNA levels and amplified the gene levels of myogenin and MyoD.
Functional foods are the components that enclose higher nutritional value and maintain superior quality in terms of human health.Tasteful factors, along with the high capacity of safety levels and well-being impacts, are the profound beneficiaries of these products over others [56].C. nippona is a potent oyster shellfish used as an edible portion for human consumption.The outcomes obtained from this study revealed that the AOH of C. nippona comprises high nutritional values and works as an outstanding component to fulfill nutrient requirements during the new product development stage.
Protein expressions at the Act-RIIB and PI3K pathways and their respective receptor level expression analyses were not extensively addressed in this study.Furthermore, previous studies by Gilson et al., 2007, revealed that myostatin contributes a vital role to the formation of stress conditions in the cells followed by the activation of phosphorylated Smad2/3 during muscle atrophic conditions.Hence, further feasibility studies are recommended and encouraged to distinguish the receptor-level mechanism analysis and execute experiments in vivo to confirm the suitability of these hydrolysates for abating muscle atrophy.The isolation of purified peptides will help categorize the molecular mechanism of the active agent of AOH and deliver effective applications in the functional food industry in a meaningful way.

Preparation of Enzyme Hydrolysate of Crassostrea Nippona
Oysters (C.nippona) were purchased from the domestic seafood market in Tongyeongsi, Republic of Korea, from June to August 2022.The oysters' shells were carefully opened, and the shell was removed.The muscle tissue was then dissected from the remaining body, ensuring minimal inclusion of other tissues.To reduce salinity, the extracted muscle tissue underwent thorough rinsing with running water, followed by an additional rinse using distilled water.Subsequently, the oyster muscle tissue was ground into a fine paste, ensuring uniformity.Finally, the ground tissue was freeze-dried to remove moisture, resulting in powdered oyster samples utilized for further experiments.In this study, powdered oyster samples (20 g) were mixed with 200 mL of distilled water to achieve a 10% substrate concentration.The pH of the mixture was adjusted to 8. Next, three different enzymes-alcalase (7592870), flavourzyme (7612249), and protamex (PW2A1042)-were separately added to the sample, resulting in a final enzyme-substrate concentration of 5.00%.The prepared samples were placed in a shaking incubator (SFDSM06, Jeio Tech, Republic of Korea) and incubated at 55 • C for 24 h with a shaking speed of 130 rpm.Incubated enzymehydrolyzed samples were inactivated at 100 • C for 10 min followed by centrifugation at 10,000 rpm for 10 min at 4 • C. Supernatants from each enzymatic hydrolysis sample were collected through vacuum filtration by sourcing a Buchner funnel with Whatman number 4 double filter papers at a time (Cat:1004110, GE Healthcare, Amersham, UK) and triplicates of 1 mL samples were subjected to measure the yield and rest were freeze-dried for 72 h.Freeze-dried samples were collected and allocated to measure the general composition and muscle growth analysis and samples were kept at 4 • C until use for analysis.

Cell Culture and Cell Differentiation
DMEM supplemented with heat-inactivated 10% FBS, 1% penicillin, and streptomycin (Cat.LS 202-02) was used for cell culture experiments and DMEM enriched with 2% HS, (Ref.26170-043) 1% streptomycin and penicillin was used to differentiate the cultured cells at 37 • C and in a 5% CO 2 controlled environment (60915672, Sanyo Electric Co., Ltd., Osaka, Japan) [1][2][3][4].DEXA (CAS-No: 50-02-2) concentration was maintained at 10 µM throughout these experiments to determine whether selected enzyme hydrolysates of C. nippona had a significant effect against that in a dose-dependent way [25,60,61].Initially, the C2C12 cells were seeded into the concentration of 1 × 10 5 cells/mL in the DMEM medium.Differentiation was initiated once the cells reached 80% confluence, by shifting the medium to DMEM containing 2% HS and 1% PS.The differentiation procedure was followed three times and differentiated cells were subjected to immunostaining, Western blot analysis, and RT-qPCR [62].For immunostaining initially, cells were seeded into 48-well plates via Western blot and for RT-qPCR we used 5 mL cell culture dishes, respectively.Initially, the total volumes we used for the 48-well plates and 5 mL cell culture dishes were 250 µL and 4 mL, respectively.4.5.Cytotoxicity Evaluation using Cell Counting Kit-8 (CCK-8 assay) The cytotoxicity influence of oyster enzyme hydrolysates on the C2C12 cells was achieved using CCK8 assay [63].C2C12 myoblasts were seeded in a 96-well plate and incubated for 24 h at 37 • C with 5% CO 2 .Viable cell concentration was maintained at 1 × 10 5 cells/mL throughout the experiment period.Cells were counted by using a LUNAII automated cell counter machine.Here, we used Luna cell counting sides (Cat no: L12001).The concentrations of the enzyme extracts were maintained by considering the cytotoxicity results.The seeded cells were treated with three hydrolysates (AOH, FOH, POH) and DWO with three different concentrations of each extract, from 25 µg/mL, 50 µg/mL, and 100 µg/mL, and incubated for another 24 h at similar environmental conditions.Each well was treated with CCK8 solution and after 2 h the wells were evaluated by taking the absorbance at 450 nm by using a microplate reader (SynergyTM HT, Vermont, USA) [43].The absence of sample-treated wells was used as a control and calculated as 100% cell viability, and all absorbance readings of sample-treated wells were stated as mean percentage values compared to the untreated wells [64].

Cell Proliferation Evaluation using 5-Bromo-2 ′ -deoxyuridine (BrdU) Assay
To estimate the proliferation ability of AHO, FOH, POH, and DWO in C2C12 cells, the BrdU assay (Millipore, Billerica, MA, USA) was directed by handling Bromodeoxyuridine (BrdU) labeling and a detection kit (11647229001, RocheR Life Science Products, Penzberg, Upper Bavaria, Germany).Seeded C2C12 cells were treated with digested AHO, FOH, POH, and DWO during the cell differentiation period under a differentiation medium (DMEM + 2% HS + 1% PS).The proliferation capability of the cells treated with digested samples was compared with that of the DEXA-treated sample and DEXA was evaluated over control sample wells.The background effect of the media was eliminated by acquiring the absorbance by using the wells that contained only the media without any serum and cells.The absorbance of the samples and background were measured in an ELISA reader at 370 nm [62].

Immunostaining of Myosin Heavy Chain (MyHC)
Differentiated myotubes were washed three times with cold 1× PBS (20× PBS consisted of 2.7 M sodium chloride (NaCl), 54 mM potassium chloride (KCl), 86 mM sodium phosphate dibasic (Na 2 HPO 4 ), and 28 mM potassium phosphate monobasic (KH 2 PO 4 ), all dissolved in distilled water.The final pH was maintained at 7.2-7.5 and fixed with chilled methanol for 5 s.Fixed myotubes were washed three times with PBST (PBS + 1% Tween 20).Fixed and permeabilized cells were blocked with 1% BSA (Cat No. BSAS 0.1) containing 22.52 mg/mL glycine (CAS No. 56-40-6) and incubated with MHC-specific primary antibodies (Santa Cruz) (1:250 in 1% BSA) for 2 h at room temperature.After being washed three times with PBST, incubated cells were labeled with goat anti-mouse IgG H&L (Alexa Fluor TM Plus 555; A32727)-conjugated secondary antibodies (Santa Cruz) (1:1000 in 1% BSA) for 2 h at room temperature, followed by a similar washing step before nuclear staining.Cell nuclei were stained with 300 nm DAPI (40,6-diamidino-2-phenylindole; Sigma-Aldrich), and the final washing step was performed using PBST.Finally, fluorescent images of the stained myotubes were visualized using a confocal microscope (Carl Zeiss, Oberkochen, Germany) [44,65].Myotube diameters were calculated for control, DEXA, and DEXA-treated samples of AOH and POH using ImageJ software 1.49v (National Institutes of Health, Bethesda, MD, USA) according to the confocal microscope captions referring to each well, and significance was calculated for each sample over the DEXA-treated control value [62].

Molecular Weight Determination and Amino Acid Composition
After confirmation of cytotoxicity, cell proliferation ability, and myotube diameter, AOH was selected for Western blotting and RT-qPCR analysis.Molecular weight determination was conducted using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 4% stacking and 12% separating polyacrylamide gels, and amino acid composition of the chosen sample was performed using Agilent 1260 series HPLC conditions [66,67].An AOH sample (100 mg/mL) was subjected to SDS PAGE [45].Waters Nova-Pak C18 4 µm (3.9 × 300 mm) was the column used during the HPLC analysis.The two mobile phases applied in this analysis were 140 mM Sodium Acetate trihydrate, 0.15% TEA, 0.03% EDTA, 6% CH3CN, pH 6.1, and 60% CH3CN, 0.015% EDTA.The flow rate, run time, and equilibration time were 1.0 mL/min, 30 min, and 10 min, respectively.The HP 1100 Series at 254 nm was used to detect the peaks.AOH (100 mg/mL) was used for the SDS-PAGE analysis [68].

RT-qPCR
The selected AOH samples at 25, 50, and 100 µg/mL were differentiated as previously mentioned in Section 2.4.The cells were then harvested, and total Ribonucleic Acid (RNA) was extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer's instructions.RNA content was measured using a µDrop plate (Thermo Scientific, Waltham, MA, USA), and 2 µg of RNA was reverse-transcribed into cDNA using a first-strand cDNA synthesis kit (TaKaRa, Shiga, Japan).The primers used in the experiment are listed in Table 3.The PCR cycling conditions were as described by [65].Here, initial denaturation was performed at 95 • C/30 s.Annealing and extension were conducted in 30 cycles (95 • C/5 s, 55 • C/10 s, and 72 • C/20 s.Final extension 95 • C/15 s, and 60 • C/30 s).Livak and Schmittgen (2001) implemented a scheme to quantify the relative gene expression [68].GAPDH was used as an internal control to normalize the relative gene expression levels.

Gene
Primer Sequence

Statistical Analysis
To realize the final results, all the analyses were performed in triplicate.Data are expressed as means ± standard deviation.Statistical significance was evaluated using one-way analysis of variance (ANOVA) and Tukey's test.Two-tailed Student's t-tests were performed when two conditions were required for the analysis.The significance is denoted compared to the control; p < 0.0001 "####" and over DEXA level was defined; p < 0.05 "*", p < 0.01 "**", p < 0.001 "***", and p < 0.0001 "****" .

Conclusions
In conclusion, this study robustly supports the anti-muscle atrophic effect of AOH derived from C. nippona.Through a comprehensive analysis of gene and protein expression levels, AOH downregulates negative regulators (MuRF-1, Atrogin-1, Smad2/3, and FoxO 3a) associated with the ubiquitin-proteasome pathway.Simultaneously, AOH promotes the expression of positive regulators (Myogenin, MyoD, mTOR, and MHC) related to the mTOR signaling pathway.The study outcomes confirmed the muscle growth and anti-atrophy effects of AOH in a DEXA-induced muscle atrophy model using C2C12 cells.Amino acid composition and cell-protective aptitude analyses further highlight AOH's potential for treating muscle atrophy effectively compared to other hydrolysates.Receptorlevel analysis and in vivo experiments are recommended to strengthen these findings and facilitate impactful industrial product development for muscle atrophy treatment.

Table 1 .
Hydrolysate yield and proximate analysis of the protein hydrolysate of Crassostrea nippona on muscle growth.

Table 2 .
Amino acid composition of AOH of Crassostrea nippona.

Table 3 .
Sequence of primers used in this study.