β-Sitosterol Attenuates Dexamethasone-Induced Muscle Atrophy via Regulating FoxO1-Dependent Signaling in C2C12 Cell and Mice Model

Sarcopenia refers to a decline in muscle mass and strength with age, causing significant impairment in the ability to carry out normal daily functions and increased risk of falls and fractures, eventually leading to loss of independence. Maintaining protein homeostasis is an important factor in preventing muscle loss, and the decrease in muscle mass is caused by an imbalance between anabolism and catabolism of muscle proteins. Although β-sitosterol has various effects such as anti-inflammatory, protective effect against nonalcoholic fatty liver disease (NAFLD), antioxidant, and antidiabetic activity, the mechanism of β-sitosterol effect on the catabolic pathway was not well known. β-sitosterol was assessed in vitro and in vivo using a dexamethasone-induced muscle atrophy mice model and C2C12 myoblasts. β-sitosterol protected mice from dexamethasone-induced muscle mass loss. The thickness of gastrocnemius muscle myofibers was increased in dexamethasone with the β-sitosterol treatment group (DS). Grip strength and creatine kinase (CK) activity were also recovered when β-sitosterol was treated. The muscle loss inhibitory efficacy of β-sitosterol in dexamethasone-induced muscle atrophy in C2C12 myotube was also verified in C2C12 myoblast. β-sitosterol also recovered the width of myotubes. The protein expression of muscle atrophy F-box (MAFbx) was increased in dexamethasone-treated animal models and C2C12 myoblast, but it was reduced when β-sitosterol was treated. MuRF1 also showed similar results to MAFbx in the mRNA level of C2C12 myotubes. In addition, in the gastrocnemius and tibialis anterior muscles of mouse models, Forkhead Box O1 (FoxO1) protein was increased in the dexamethasone-treated group (Dexa) compared with the control group and reduced in the DS group. Therefore, β-sitosterol would be a potential treatment agent for aging sarcopenia.


Introduction
Sarcopenia refers to a decline in muscle mass and strength with age, causing significant impairment in the ability to carry out normal daily functions and increased risk of falls and fractures, eventually leading to loss of independence [1,2]. Sarcopenia is not a simple loss of muscle mass and strength but represents a precursor of frailty and a predictor of increased

Animal Study Design
The Animal Experimental Ethics Committee of Gyeongsang National University (GNU-180823-M0044) gave its approval to perform animal tests, and the research was carried out following the ethical protocol for animal experimentation. We purchased 6-week-old male C57BL/6 mice with an average body weight of 22 g from Core Tech Co., Ltd, (Seoul, Korea) for the experimental animals. These animals were used in the experiment after a week of adaptation in a light and dark cycle environment with a temperature of 24 ± 2 • C, relative humidity of 40-60%, an illuminance of 150-300 lux, and a Nutrients 2022, 14, 2894 3 of 13 12-hour interval. Normal food was provided during the adaption period, and sterile water was freely provided with drinking water. After the adaptation period, C57BL/6 mice were randomly divided into four groups (n = 9 per group): control group (Control), β-sitosterol treatment group (S), dexamethasone treatment group (Dexa), and dexamethasone + βsitosterol treatment group (DS).
After the experimental animal adaptation phase, dexamethasone (20 mg/kg of body weight of mice) was injected intraperitoneally into the Dexa and DS groups at 10~11 a.m. every day for two weeks to generate a muscle atrophy model. The saline injection was administered to the control group. The control and Dexa groups received no medications during the same period. From one week before dexamethasone treatment to the end of the experiment, the DS group was given β-sitosterol (S0040; Toyko Chemical Industry, Tokyo, Japan) 200 mg/kg body weight of mice orally once a day. Three days before and after dexamethasone treatment, body weights were measured. The total experimental period for treatment was 3 weeks. On the day of the end of the experiment, all groups were euthanized, and the tibialis anterior and gastrocnemius muscles were quickly frozen in liquid nitrogen and stored at −80 • C for use in identifying the target protein through western blot.

Grip Strength Test
The grip strength of all test animals was assessed the day before the end of the experiment. The Bioseb Grip Strength Test was used to determine grip strength in grams (BIO-GS3; BIO-Science and Experimental Biology, Pinellas Park, FL, USA). The grip strength was assessed by tugging the tail at a steady speed (2 cm/s) until the grasp was released, and the stainless-steel T-bar of the experimental tool was gripped with both front paws of the test animal. The average value was calculated after each animal received five measurements.

Treadmill Analysis
Treadmill equipment (Panlab, Barcelona, Spain) was used to measure the speed, duration, and distance of the mouse running test, which was then changed using software (SeDaCom v2.0.02, Panlab, Barcelona, Spain). The adaptive gait speed was set to 10 cm/s for 3 min, then increased to 4 cm/s every 4 min until the maximum speed of 75 cm/s was attained, at which point the test was stopped. All groups were given the same adaptive walking and running speeds, as well as electric stimulation (1.1 mA) behind each treadmill rail to force them to run. The time to fatigue was calculated by placing the front legs on the rails for 3 s and the back legs on the electrical device for 3 s.

Histological Analysis of Muscle Tissue
The right tibialis anterior, gastrocnemius muscles, and extensor digitorum longus muscles were cryosectioned after being instantly frozen with Optimal Cutting Temperature (OCT) compounds (Lab-Tek; Miles Laboratories, Inc., Naperville, IL, USA). A cryostat (Leica CM1950; Heidelberg, Germany) was used to cut 5 mm thick muscle slices from the frozen samples, and 10 percent goat serum was used to block them for 1 h at room temperature. Wheat germ agglutinin, Alexa Fluor488 conjugate (W11261; Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) antibody was diluted to a concentration of 1:500 and stained at 4 • C overnight, and an upright microscope (Nikon Eclipse ni DSRi2; Nikon, Tokyo, Japan) was used to observe the extracellular matrix. The fiber cross-sectional area (CSA) and Min Feret diameter were measured with an ImageJ application after the samples were imaged with a microscope at 100 magnification.

In Vitro Study Design
Mouse C2C12 myoblast was purchased from the American Type Culture Collection (Manassa, VA, USA). C2C12 myoblasts were seeded at 3 × 10 5 /well on a 6-well culture plate containing 90% Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 100 units/mL penicillin and streptomycin (PS), and cultured at 37 • C CO 2 . Myoblasts were differentiated to myotube cells using a differentiation medium containing 2 percent horse serum (HS) and 100 unit/mL PS, which was cultured for 7 days with the differentiation medium being changed every 2 days. As a muscle reduction cell model, muscular atrophy was generated by treating cells with dexamethasone (1 µM) for 48 h. At this time, β-sitosterol was mixed for 48 h on the 5th day. All in vitro data were obtained from multiple experiments.

Cell Viability
C2C12 cells were seeded in 24-well plates at a concentration of 5 × 10 4 cells per well. After 24 h incubation, the specified dose of β-sitosterol or dimethyl sulfoxide (DMSO) was added to the medium for 24 h. Afterwards, the cells in each well were treated according to the instructions of the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Briefly, 10 µL per well of the CCK-8 solution was added and incubated for 1 h at 37 • C in a humidified, 5% CO 2 atmosphere. The amount of formazan dye generated by cellular dehydrogenase activity was measured by absorbance at 450 nm with a microplate reader (Molecular Devices, San Jose, CA, USA).

Total RNA Isolation and qPCR
Total RNA was extracted using Trizol solution after C2C12 myoblasts were washed twice with cold PBS. The iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) was used to convert the extracted 2 µg RNA into cDNA, and mRNA expression was measured using the ViiATM7 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) and TaqMan analysis. Amplification was done under the following conditions: 95 • C, 10 min; 40 cycles at 95 • C, 15 s and 60 • C, 60 s. MuRF1 (Mm01185221 m1), MAFbx (Mm00499523 m1), MyoD (Mm00440387 m1), MyoG (Mm00446194 m1), and Myostatin (Mm01254559 m1) were used as primers in this study. After amplification, the data were evaluated 40 times at intervals.

Statistical Analysis
For statistical analysis, GraphPad Prism (Version 5.01; GraphPad Software, San Diego, CA, USA) was used. All the tests were carried out three times, and the results were expressed as mean ± SD or mean ± SE. To determine the significance between two groups, a Student's t-test was used and among three or more groups, a one-way ANOVA analysis was used. A p-value of less than 0.05 was considered significant.

β-Sitosterol Protects Mice from Dexamethasone-Induced Muscle Atrophy
There was a significant difference in body weight between the Control group and the Dexa group after dexamethasone administration ( Figure 1A). Next, the backfoot muscles, the gastrocnemius, and tibialis anterior muscles were cut off and weighed. As a result, the weight of these muscles was dramatically reduced in the Dexa group. However, in the DS group, it weighed more compared to the Dexa group. The extensor digitorum longus also showed similar results, but there were no significant differences between the Dexa group and the DS group ( Figure 1B). Generally, sarcopenia leads to the loss of muscle mass, which is associated with reduced muscle fiber number and size. Therefore, to assess the histological properties of the morphological change of the muscle fibers, immunofluorescence staining was done on the tibialis anterior muscle, the gastrocnemius muscle, and extensor digitorum longus muscle of the muscle atrophy mouse model. In all three types of muscles, the size of muscle fibers decreased in the Dexa group and recovered in the DS group ( Figure 1C). In the quantification of myofiber size of gastrocnemius muscles and tibialis anterior muscles, total muscle fiber atrophy in the Dexa group resulted in a decrease in muscle fiber thickness when compared to the control group. On the other hand, the gastrocnemius muscle of the DS group showed an increase in muscle fiber thickness ( Figure 1D). Because all catabolic pathways of skeletal muscle induce up-regulation of MuRF-1 and MAFbx, we investigated the effect of muscle catabolism by evaluating changes in the expression of MuRF-1 and MAFbx [46]. Changes in protein expression of MAFbx and MuRF according to β-sitosterol treatment were confirmed in gastrocnemius muscle and tibialis anterior muscle. MAFbx increased in the Dexa group and decreased in the DS group, but there was no significant difference ( Figure 1E). The time to exhaustion of treadmill exercise was dramatically accelerated in the Dexa group but recovered in the DS group in an experiment comparing the time to exhaustion of treadmill exercise conducted with the dexamethasone-induced muscle atrophy mouse model ( Figure 2A). Grip strength was also decreased in the Dexa group compared to the control group and recovered in the DS group ( Figure 2B). Likewise, serum creatine kinase (CK) activity was increased in the Dexa group and recovered in the DS group ( Figure 2C).

In Vitro Verification of Muscle Loss Inhibitory Efficacy of β-Sitosterol in Dexamethasone-Induced Muscle Atrophy in C2C12 Myotube
β-sitosterol was evaluated in C2C12 myotubes differentiated from C2C12 myoblasts to find small molecules that protect muscles from dexamethasone-induced muscle atrophy ( Figure 3A). There was no significant difference in the viability of cells even when sitosterol was added to the C2C12 culture medium with various concentrations (0.25, 0.5, 1, and 2 mM) ( Figure 3B). The myotube width was significantly decreased in the dexamethasone treatment group, but it was increased in the dexamethasone + β-sitosterol treatment group ( Figure 3C,D). In the fusion index, the total nuclei of myotube were significantly decreased in the Dexa group but recovered in the DS group ( Figure 3E). The gene expression of sarcopenia-associated genes of myotube was evaluated. In the dexamethasone treatment group, the expression of MuRF1 and MAFbx was increased. However, in the dexamethasone + β-sitosterol 500 µM treatment group, it was reduced ( Figure 4A). The protein expression of MuRF1 and MAFbx was also evaluated. The protein expression of MAFbx showed a significant increase in the dexamethasone treatment group and a significant decrease in the dexamethasone + β-sitosterol group. MuRF1 also showed similar results to MAFbx but did not show a significant difference ( Figure 4B). Nutrients 2022, 14, x FOR PEER REVIEW 6 of 13

In Vitro Verification of Muscle Loss Inhibitory Efficacy of βSitosterol in Dexamethasone-Induced Muscle Atrophy in C2C12 Myotube
βsitosterol was evaluated in C2C12 myotubes differentiated from C2C12 myoblasts to find small molecules that protect muscles from dexamethasone-induced muscle atrophy ( Figure 3A). There was no significant difference in the viability of cells even when sitosterol was added to the C2C12 culture medium with various concentrations (0.25, 0.5, 1, and 2 mM) ( Figure 3B). The myotube width was significantly decreased in the dexamethasone treatment group, but it was increased in the dexamethasone + βsitosterol treat ment group ( Figure 3C,D). In the fusion index, the total nuclei of myotube were significantly decreased in the Dexa group but recovered in the DS group ( Figure 3E). The gene expression of sarcopenia-associated genes of myotube was evaluated. In the dexamethasone treatment group, the expression of MuRF1 and MAFbx was increased. However, in the dexamethasone + βsitosterol 500 μM treatment group, it was reduced ( Figure 4A). The protein expression of MuRF1 and MAFbx was also evaluated. The protein expression of MAFbx showed a significant increase in the dexamethasone treatment group and a significant decrease in the dexamethasone + βsitosterol group. MuRF1 also showed similar results to MAFbx but did not show a significant difference ( Figure 4B).

Induced Muscle Atrophy in C2C12 Myotube
βsitosterol was evaluated in C2C12 myotubes differentiated from C2C12 myoblasts to find small molecules that protect muscles from dexamethasone-induced muscle atrophy ( Figure 3A). There was no significant difference in the viability of cells even when sitosterol was added to the C2C12 culture medium with various concentrations (0.25, 0.5, 1, and 2 mM) ( Figure 3B). The myotube width was significantly decreased in the dexamethasone treatment group, but it was increased in the dexamethasone + βsitosterol treat ment group ( Figure 3C,D). In the fusion index, the total nuclei of myotube were significantly decreased in the Dexa group but recovered in the DS group ( Figure 3E). The gene expression of sarcopenia-associated genes of myotube was evaluated. In the dexamethasone treatment group, the expression of MuRF1 and MAFbx was increased. However, in the dexamethasone + βsitosterol 500 μM treatment group, it was reduced ( Figure 4A). The protein expression of MuRF1 and MAFbx was also evaluated. The protein expression of MAFbx showed a significant increase in the dexamethasone treatment group and a significant decrease in the dexamethasone + βsitosterol group. MuRF1 also showed similar results to MAFbx but did not show a significant difference ( Figure 4B).

βSitosterol Inhibited FoxO1-Mediated Protein Degradation, Reducing Dexamethasone Induced Atrophy
Next, FoxOs, which is an up-regulator of MuRF1 and MAFbx, expression levels measured in the C2C12 myotube [47]. First, FoxO1 decreased in the dexamethasone ment group. In contrast, FoxO3 protein was increased in the dexamethasone treat group, and the expression level increased when βsitosterol was administered ( Figure  However, in the gastrocnemius and tibialis anterior muscles, FoxO1 protein was incre in the Dexa group compared with the control group and reduced in the DS group. F protein was increased in the Dexa group, but there were no changes in the DS group

β-Sitosterol Inhibited FoxO1-Mediated Protein Degradation, Reducing Dexamethasone-Induced Atrophy
Next, FoxOs, which is an up-regulator of MuRF1 and MAFbx, expression levels were measured in the C2C12 myotube [47]. First, FoxO1 decreased in the dexamethasone treatment group. In contrast, FoxO3 protein was increased in the dexamethasone treatment group, and the expression level increased when β-sitosterol was administered ( Figure 5A). However, in the gastrocnemius and tibialis anterior muscles, FoxO1 protein was increased in the Dexa group compared with the control group and reduced in the DS group. FoxO3 protein was increased in the Dexa group, but there were no changes in the DS group ( Figure 5B,C). These results showed that the FoxO3 muscle atrophy-related signal transduction pathway is not thought to be a regulatory mechanism for β-sitosterol's protective action. Therefore, β-sitosterol prevents dexamethasone-induced muscle atrophy, and β-sitosterol is expected to play a key role in limiting the ubiquitin-proteasome pathway triggered by reducing FoxO1.
ure 5B,C). These results showed that the FoxO3 muscle atrophy-related signal transduction pathway is not thought to be a regulatory mechanism for βsitosterol's protective action. Therefore, βsitosterol prevents dexamethasone-induced muscle atrophy, and βsi tosterol is expected to play a key role in limiting the ubiquitin-proteasome pathway triggered by reducing FoxO1.
Several studies demonstrated that the concentration of plasma β-sitosterol was shown to be considerably lower in type 2 diabetes patients, suggesting that β-sitosterol
Several studies demonstrated that the concentration of plasma β-sitosterol was shown to be considerably lower in type 2 diabetes patients, suggesting that β-sitosterol may play a role in decreasing blood glucose levels [52]. The study of Pandey et al. reported that β-sitosterol-D-Glucopyranoside (BSD) extracted from Cupressus sempervirens stimulates estrogenic analog effects and glucose uptake, affecting skeletal muscle cells. They found that BSD-induced GLUT4 translocation stimulates skeletal muscle cells by increasing glucose uptake through the PI-3K/AKT mechanism [53].
In skeletal muscle, β-sitosterol has been studied to improve mitochondrial ATP production's responsiveness to increased energy demand. Hoi Shan Wong (2015) et al. reported that β-sitosterol extracted from Citanches Herba fraction (HCF1) induces redox-sensitivity induction of mitochondrial segregation in C2C12 myotubes and activation of AMPK/PGC-1. They did not report the results of animal experiments on the inhibition of muscle loss but demonstrated the anti-obesity effect of body energy expenditure in skeletal muscle in high fat-dieted, obese-induced mice [54]. After that, Hoi Shan Wong et al. and others reported through further study that β-sitosterol can increase the reaction of mitochondrial membrane to fluidization and energy demand in the mitochondrial electron transfer system, thereby suppressing mitochondrial-related muscle function degradation by not affecting C2C12 muscle separation induction [55]. The fact that there are many investigations of antioxidant-related effects in prior studies is also thought to disprove the results of the study. On the contrary, Hwang et al. investigated the regulation of fat and glucose metabolism by the AMP-activated protein kinase of β-sitosterol. Their experiments using L6 myotube cells demonstrated that β-sitosterol enhances TG inhibition and glucose uptake through the AMPK mechanism. Their findings are contrary to the results of our AMPK inhibitory effect, and it seems necessary to analyze the differences in muscle cell lines and research settings used in cell experiments [52].
The present study found that β-sitosterol inhibited muscle atrophy in dexamethasonetreated muscle atrophy cells and animal models, a model of catabolism-induced muscle atrophy, which is one of the major mechanisms of aging sarcopenia. These results suggested that blocking the ubiquitin-proteasome pathway induced by suppressing FoxO1, which is an up-regulator of MAFbx, plays an important role ( Figure 6). In addition, β-sitosterol was reported in relation to foxo1 regulating the catabolic pathway by promoting several genes, including MAFbx expression [33,56,57]. Therefore, β-sitosterol might inhibit the catabolic pathway by downregulating FoxO1/MAFbx, thereby preventing protein catabolism, which is key to aging muscle loss. However, in vitro experiment of C2C12 myotube showed controversial results that FoxO1 was decreased in the dexamethasone treatment group. Therefore, further studies related to the in vitro experiment on FoxO1 protein expression is needed.
ported that β-sitosterol extracted from Citanches Herba fraction (HCF1) induces redo sensitivity induction of mitochondrial segregation in C2C12 myotubes and activation AMPK/PGC-1. They did not report the results of animal experiments on the inhibition muscle loss but demonstrated the anti-obesity effect of body energy expenditure in ske tal muscle in high fat-dieted, obese-induced mice [54]. After that, Hoi Shan Wong et and others reported through further study that β-sitosterol can increase the reaction mitochondrial membrane to fluidization and energy demand in the mitochondrial ele tron transfer system, thereby suppressing mitochondrial-related muscle function deg dation by not affecting C2C12 muscle separation induction [55]. The fact that there a many investigations of antioxidant-related effects in prior studies is also thought to d prove the results of the study. On the contrary, Hwang et al. investigated the regulati of fat and glucose metabolism by the AMP-activated protein kinase of β-sitosterol. Th experiments using L6 myotube cells demonstrated that β-sitosterol enhances TG inhi tion and glucose uptake through the AMPK mechanism. Their findings are contrary to t results of our AMPK inhibitory effect, and it seems necessary to analyze the differences muscle cell lines and research settings used in cell experiments [52].
The present study found that β-sitosterol inhibited muscle atrophy in dexam thasone-treated muscle atrophy cells and animal models, a model of catabolism-induc muscle atrophy, which is one of the major mechanisms of aging sarcopenia. These resu suggested that blocking the ubiquitin-proteasome pathway induced by suppressi FoxO1, which is an up-regulator of MAFbx, plays an important role ( Figure 6). In additio β-sitosterol was reported in relation to foxo1 regulating the catabolic pathway by prom ing several genes, including MAFbx expression [33,56,57]. Therefore, β-sitosterol mig inhibit the catabolic pathway by downregulating FoxO1/MAFbx, thereby preventing pr tein catabolism, which is key to aging muscle loss. However, in vitro experiment of C2C myotube showed controversial results that FoxO1 was decreased in the dexamethaso treatment group. Therefore, further studies related to the in vitro experiment on FoxO protein expression is needed. Figure 6. The overall β-sitosterol mechanisms related to muscle atrophy. β-sitosterol down-reg lates the transcriptional factor, FoxO1. Down-regulated FoxO1 becomes unable to affect the expr sion of MAFbx. Consequently, inhibited MAFbx cannot induce muscle atrophy via the catabo pathway. Figure 6. The overall β-sitosterol mechanisms related to muscle atrophy. β-sitosterol down-regulates the transcriptional factor, FoxO1. Down-regulated FoxO1 becomes unable to affect the expression of MAFbx. Consequently, inhibited MAFbx cannot induce muscle atrophy via the catabolic pathway.
In aging sarcopenia, it may be difficult to achieve desired muscle strength improvement and muscle mass increase even with appropriate exercise due to inflammation and degeneration of neuromuscular junctions. Nevertheless, the mechanism of the study of the mitochondrial electron transport system conducted by Wong et al. and the mechanism of inhibition of muscle degradation by blocking the ubiquitin-proteasome pathway demonstrated in our study proves the important possibility of using β-sitosterol for the inhibition of aging sarcopenia [54,55].

Conclusions
In the present study, we found that β-sitosterol has anticatabolic effects in skeletal muscles by regulating the FoxO1/MAFbx pathway, which causes muscle loss. This result was confirmed in dexamethasone-treated muscle atrophy C2C12 myotube and mouse models. Therefore, β-sitosterol would be a potential treatment agent for aging sarcopenia. Funding: This study was conducted with support from the Rural Development Administration (PJ014155052019).

Institutional Review Board Statement:
The animal study protocol was approved by the Animal Experimental Ethics Committee of Gyeongsang National University (GNU-180823-M0044).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.