Protective effect of Luffa cylindrica Roemer against dexamethasone-induced muscle atrophy in primary rat skeletal muscle cells

Glucocorticoids (GCs) are commonly used in the treatment of chronic inflammatory conditions. However, the administration of high doses and long-term use of GCs can induce muscle atrophy (MA) in patients, leading to a decline in quality of life and increased mortality. MA leads to protein degradation in skeletal muscle, resulting in a reduction of muscle mass. This process is triggered by GCs like dexamethasone (DEX), which induce the expression of E3 ubiquitin ligases, namely Atrogin-1 and muscle RING-finger protein-1 (MuRF1). In this study, we examined the anti-MA potential of Luffa cylindrica Roemer (LCR) on DEX-treated primary skeletal myotubes. Primary skeletal myotubes stimulated with LCR alone resulted in a significant upregulation of myotube development, characterized by an increase in both the number and diameter of myotubes. Contrastingly, combined treatment with LCR and DEX reduced the expression of Atrogin-1, while treatment with DEX alone induced the expression of MuRF1. Furthermore, LCR treatment successfully restored the number and diameter of myotubes that had been diminished by DEX treatment. These findings suggest that LCR holds potential for treating MA, as an accelerating effect on muscle development and anti-MA effects on primary skeletal muscle cells were observed. Supplementary Information The online version contains supplementary material available at 10.1007/s10974-023-09661-5.


Introduction
Muscle atrophy (MA) refers to changes that occur in muscles as a result of various pathophysiological stimuli, including aging, starvation, muscle inactivity, and diseases.It is characterized by a reduction in protein content, ber diameter, and force production (Jackman and Kandarian 2004).MA not only leads to decreased functional capacity and weakness in patients but also contributes to obesity, thereby negatively impacting the quality of life and increasing mortality rates among affected individuals (Foletta et al. 2011;Marcell 2003).
Glucocorticoids (GCs) are a class of steroid hormones known for their potent anti-in ammatory and immunosuppressive effects.They are commonly prescribed for the treatment of chronic in ammatory diseases like systemic lupus erythematosus, rheumatoid arthritis, and bronchial asthma (Troncoso et al. 2014; Ma et al. 2003).However, the prolonged and high dose use of GCs can result in MA and frailty due to their catabolic effects on skeletal muscle tissue (Hasselgren 1999; Hermans and Van den Berghe 2015).In muscle cells, excessive GCs bind to the GC receptor, triggering MA.This process involves the upregulation of speci c E3 ubiquitin ligases, such as Atrogin-1/muscle atrophy F-box and muscle RINGnger protein-1 (MuRF1), which play a role in GC-induced MA (Mishra et al. 2022).
Dexamethasone (DEX), a synthetic GC, has been employed to investigate the cellular and molecular mechanisms of MA and assess the catabolic effects of GCs (Liu et al. 2016).The extent of MA improvement has been explored using a skeletal muscle-wasting model with a high dose of DEX on skeletal myotubes (Qin et al. 2013) Studies on MA commonly employ immortalized cell lines such as C2C12 and L6, due to their convenient culturing process and rapid proliferation rates.These well-established cell lines are extensively used in research.In contrast, primary cell lines, isolated from tissues, have a limited number of passages but exhibit properties similar to the cells in vivo and retain important tissue-speci c characteristics and functions (Smith and Merrick 2010).
In this study, we used an in vitro model of primary skeletal muscle treated with DEX to simulate physiological characteristics, including intracellular calcium homeostasis and muscle regenerative capacity.Our investigation focused on assessing the anti-MA effect of LCR by examining changes in cell viability, MA markers, and the diameter and number of myotubes.This study offers insights into the fundamental pathophysiological mechanisms of MA and explores the potential therapeutic effects of a natural compound with minimal side effects, even after prolonged usage.Our ndings aim to identify a new drug candidate and promote the development of healthier dietary options.

Preparation of LCR extracts
The LCR used in this study was obtained by Green M. P. Pharmaceutical Co. Ltd. (Gyeonggido, Korea).To extract LCR, it was subjected to heat treatment with distilled water for 3 h at 105°C.The resulting mixture was then ltered through lter papers.After ltration, the ltrate was rapidly frozen to -70°C and subsequently lyophilized using a freeze dryer (Ilshin BioBase Co., Ltd, Gyeonggido, Korea).The lyophilized extract was kept at -20°C until further use.

Isolation and culture of primary skeletal myoblasts
Ethical approval for this study was obtained from the Jaseng Animal Care and Use Committee (Approval number: JSR-2022-07-001-A).Primary skeletal myoblasts were obtained from 1-d-old Sprague-Dawley rats (Samtako Bio, Korea), and isolated as previously described (Musaro and Carosio 2017; Boscolo Sesillo et al. 2020).Brie y, after postnatal rats were sacri ced, the tibialis anterior muscle from the hindlimb was immediately placed in a petri dish containing cold Dulbecco's Modi ed Eagle Medium (DMEM; Gibco BRL, Grand Island, NY, USA).The muscle tissue was digested using a skeletal muscle dissociation kit (Miltenyi, Bergisch Gladbach, Germany), and 10 ml of DMEM was added.The resulting suspension was then passed through a 70-µm strainer and centrifuged at 200 ×g for 20 min at room temperature (RT).The cell pellets were resuspended to a mixture of DMEM and Ham's F-10 nutrient mix (Gibco BRL) at a 1:1 ratio, supplemented with 20% fetal bovine serum (Gibco BRL), 1% penicillinstreptomycin (PS, Gibco BRL), and 10 ng/ml broblast growth factor-basic (bFGF; Peprotech, NJ, USA).The obtained primary myoblast were seeded in culture dishes or plates coated with Matrigel (Corning, New York City, NY, USA) (Scheme 1).

Extract treatment
When cell density reached 90% con uency after myoblast seeding, the growth medium was replaced with a differentiation medium consisting of DMEM supplemented with 5% horse serum (Gibco BRL) and 1% PS.After 4 d of incubation in the differentiation medium, primary skeletal myotubes were treated with different concentrations of LCR alone or LCR and 200 µM DEX (Sigma-Aldrich, St. Louis, MO, USA), or DEX alone for 48 h (Scheme 1).

Cell viability assay
Following the DEX stimulation, the myotubes were evaluated for cell viability using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) utilized as per the manufacturer's instructions.The absorbance of the samples was measured at 450 nm using a microplate reader (Epoch; BioTek, Winooski, VT, USA).

Western blotting
Following the treatment, the myotubes were homogenized in RIPA buffer supplemented with phosphatase and protease inhibitors (Millipore, Burlington, MA, USA) for 30 min.Protein lysates were then separated using an 8% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) and then transferred onto a polyvinylidene di uoride membrane (Millipore) at 100 V for 90 min.The membrane was blocked in 5% nonfat skim milk (BD Biosciences, Franklin Lakes, NJ, USA) for 1 h at RT. Subsequently, the membrane was incubated with primary antibodies (Table 1) and then with secondary antibodies for 2 h at RT.Protein bands were visualized on an Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden) imaging system using an enhanced chemiluminescence (ECL) system (Bio-Rad, Hercules, CA, USA).Quanti cation of protein levels was performed using ImageJ (NIH, Bethesda, Maryland, USA).Immunocytochemistry Following stimulation, the myotubes were xed using 4% paraformaldehyde for 30 min at RT and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 10 min.The myotubes were blocked with 2% normal goat serum for 1 h before incubation with primary antibodies (Table 1) and then treated with uorescein isothiocyanate (FITC)-conjugated secondary antibodies (Jackson; west grove, Pennsylvania, USA) for 2 h at RT. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at RT.The stained cells were visualized using a confocal microscope at 100× or 400× magni cation (Nikon, Tokyo, Japan).

Real-time polymerase chain reaction (PCR)
After DEX stimulation, TRIzol (15596018, Ambion) was added to each well for RNA extraction.The extracted RNA was reverse transcribed to cDNA using an Accupower RT PreMix (Bioneer, Daejeon, Korea).
Real-time PCR was performed using SYBR Green Master Mix (170-8882AP; Bio-Rad).The primer sequences used for PCR are listed in Table 2.

LCR enhances cell viability and protects against DEXinduced muscle atrophy in primary myotubes
Initially, primary myoblasts were prepared from the tibialis anterior (TA) muscle of 1-d-old rats (Fig. 1A) and their differentiation into myotubes prompted.When initiating fusion to form the myotubes, spontaneous beating occurred, forming myotube-like structure (Video S1).To determine whether LCR possesses cytotoxicity towards differentiated myotubes, we performed a CCK-8 assay.Following treatment with LCR at varying concentrations (1 to 400 µg/ml), cell viability increased signi cantly for all concentrations > 25 µg/ml (Fig. 1B).Furthermore, we used DEX-treated primary myotubes as an in vitro model of MA.When myotubes were subjected to DEX treatment alone, cell viability was signi cantly decreased, while LCR co-treatment restored cell viability in a dose-dependent manner in the model (Fig. 1C).Our ndings suggest that LCR is not cytotoxic to primary myotubes and has potential protective activity against DEX-induced MA in myotubes.

LCR promotes myotube formation in the primary skeletal muscle of rats
To investigate the effect of LCR in promoting myotube formation, staining for MHC was performed after exposure to LCR for 48 h without DEX treatment.LCR treatment resulted in the formation of more MHC + myotubes and thicker myotubes in a dose-dependent manner (Fig. 2a).Further quantitative analysis substantiated these ndings, as LCR treatment demonstrated a signi cant and progressive improvement in myotube formation, with an increased number observed at higher concentrations of LCR (Fig. 2b).Moreover, the diameter of the myotubes increased in a dose-dependent manner, with a signi cant enhancement evident upon treatment with 200 and 400 µg/ml LCR (Fig. 2c).When quantitatively analyzed by calculating the percentage of the myotubes with the average myotube diameter according to LCR concentration, a signi cant concentration-dependent increase was observed in the LCR-treated groups (Fig. 2d).Collectively, these observations suggest that LCR facilitates myotube formation by enhancing both the number and diameter of myotubes.

LCR suppresses DEX-induced upregulation of Atrogin-1 and MuRF1 in primary myotubes
To evaluate the inhibitory effect of LCR on MA-related ubiquitin ligases, namely MuRF1 and Atrogin-1 (Foletta et al. 2011), we examined their mRNA and protein levels in primary myotubes.Initially, primary myotubes were treated with 50, 100, and 200 µM DEX to establish optimal concentrations of DEX, a known MA inducer (Fig. 3a).Subsequently, we evaluated myotube diameter as an indicator of MA induction.The results revealed a signi cant reduction in myotube diameters following treatment with 200 µM DEX (Fig. 3b).As shown in Fig. 3c and Fig. 3d, the expression levels of MuRF1 and Atrogin-1 mRNA were signi cantly increased following DEX treatment compared with those in the blank group, whereas LCR decreased these expression levels dose dependently, especially at 200 and 400 µg/mL concentrations.To con rm these ndings at the protein level, further analysis through western blotting was performed (Fig. 3e) and the protein expression levels of MuRF1 and Atrogin-1 were signi cantly increased by DEX treatment compared with those in the blank group, whereas LCR decreased these levels concentration dependently (Fig. 3f, g).Notably, Atrogin-1 protein expression signi cantly decreased only at a concentration of 400 µg/ml (Fig. 3g).Our ndings suggest that LCR administration restored the expression of Atrogin-1 and MuRF1 to normal levels, effectively counteracting the DEX-induced upregulation.
LCR restores the number and diameter of primary myotubes reduced by DEX-induced MA In light of these observations, we evaluated the potential protective effect of LCR against DEX-induced MA by confocal analysis.Primary myotubes were stained for MHC, which indicated the nal stage of myogenesis (Fig. 4a).DEX treatment signi cantly decreased both the myotube number and diameter compared with those in the control group.However, LCR effectively restored these parameters in a dose-dependent manner (Fig. 4b, c).Furthermore, the ratio of the number of myotubes to their diameter, indicative of myotube quality, was signi cantly improved at LCR concentrations of 200 and 400 µg/ml (Fig. 4d).These results demonstrate that LCR treatment led to the recovery of myotube number, diameter, and overall myotube quality, thereby mitigating the detrimental effects of DEX on primary myotubes.

Discussion
In the present study, we evaluated the effects of LCR on skeletal muscle differentiation and its protective role against GC-induced MA.Treatment with LCR resulted in an increased number of myotubes containing MHC, a speci c marker of mature myotubes, with a concomitant increase in their diameter.These ndings demonstrate that LCR mitigates the detrimental effects of DEX on primary myotubes.
DEX can induce either MA or hypertrophy, depending on the stage of differentiation of C2C12 cells in vitro.Treatment with DEX during the myoblast stage promotes myoblast proliferation, myotube enlargement, and expression of MHC, a protein associated with terminal differentiation while reducing the expression of atrophy markers, such as myostatin and Atrogin-1, thereby inducing hypertrophy.However, when myotubes are exposed to DEX, the expression of atrophy markers, myostatin, and Atrogin-1, increases in a concentration-dependent manner, whereas the expression of myoblast markers, pax7, and MHC, decreases.This suggests that the process of muscle atrophy is in uenced by the ubiquitinproteasomal pathway (Guerriero and Florini 1980 In conclusion, our study provides evidence that LCR promotes skeletal muscle differentiation and exerts an anti-muscle atrophy effect.Furthermore, this study highlights the antioxidant, anti-in ammatory, and immunomodulatory properties of LCR and evaluates its potential for protecting against DEX-induced MA.These results highlight the potential therapeutic value of LCR in counteracting muscle atrophy and preserving skeletal muscle integrity.
Potential    Supplementary Files DEX treatment resulted in the induction of MA by upregulating the expression of Atrogin-1 and MuRF1, consequently leading to a reduction in muscle mass with decreased myotube diameter and thickness (McRae et al. 2017; Castillero et al. 2013).
protective effect of Luffa cylindrica Roemer (LCR) by increasing cell viability in primary myotubes (a) Isolation image of primary myoblasts obtained from postnatal day 1 rats.(b, c) Cell viability of primary myotubes treated with LCR or DEX treatment only or DEX and different concentrations of LCR for 48 h.Data are expressed as means ± SD.The results were evaluated using a one-way analysis of variance (*p<0.05,**p<0.01,***p<0.001,and ****p<0.0001 vs. DEX group; ####p<0.0001 vs. blank group).

Figure 4 Restorative
Figure 4 (Pan et al. 2009s Luffa cylindrica Roemer (LCR), commonly known as sponge gourd, which belongs to the Cucurbitaceae family.LCR has been traditionally used in Korean medicine for various therapeutic effects, including fever reduction and promoting hemostasis.It contains functional components such as phenolics, avonoids, anthocyanins and ascorbic acid, saponin, and vitamin A. LCR extract has also demonstrated anti-in ammatory, antioxidant, and immunomodulatory activities(Pan et al. 2009; Alge et al. 2006; Zhang et al. 2020; Khajuria et al. 2007; Kao, Huang, and Chen 2012; Dubey et al. 2015).While the effects of LCR have been investigated in various disease models, limited research has been conducted on its impact on MA.
(Bagherniya et al. 2022 have begun exploring the potential effects of medicinal plant extracts in MA(Bagherniya et al. 2022).

Table 1
Primary antibodies used for western blot and immunocytochemistry

Table 2
List of primer sequences used for Real-time PCR (Hakim et al. 2005o 2017).Local injection of DEX alone does not induce muscle atrophy, as con rmed by histological evaluations, reduction in local in ammatory cytokines, and increased contractile tension, indicating bene cial effects on muscle strain(Hakim et al. 2005).These ndings indicate that the use of DEX in muscle-related studies should be carefully considered based on the speci c objectives.The results of our study revealed that LCR, in primary myotubes, reduces the expression of Atrogin-1 and MuRF1 by inhibiting the ubiquitinproteasomal pathway activated by DEX.Additionally, LCR increases the number and diameter of myotubes, suggesting it as a natural compound with promising potential for protecting skeletal muscle.This study has some limitations.Although we assessed MHC levels, which are expressed in myotubes and myo bers and are most abundant in myocytes, we did not analyze other muscle differentiation markers, such as MyoD and myogenin.However, considering the number and diameter of MHC-stained myotubes at the nal stage of muscle differentiation, we hypothesize that LCR promotes skeletal muscle differentiation in a concentration-dependent manner.However, further studies are needed for the investigation of the molecular mechanism of skeletal muscle development by LCR.