Bacterioruberin extract from Haloarchaea Haloferax marinum: Component identification, antioxidant activity and anti‐atrophy effect in LPS‐treated C2C12 myotubes

Abstract

Carotenoids are natural pigments utilized as colourants and antioxidants across food, pharmaceutical and cosmetic industries. They exist in carbon chain lengths of C₃₀, C₄₀, C₄₅ and C₅₀, with C₄₀ variants being the most common. Bacterioruberin (BR) and its derivatives are part of the less common C₅₀ carotenoid group, synthesized primarily by halophilic archaea. This study analysed the compositional characteristics of BR extract (BRE) isolated from ‘Haloferax marinum’ MBLA0078, a halophilic archaeon isolated from seawater near Yeoungheungdo Island in the Republic of Korea, and investigated its antioxidant activity and protective effect on lipopolysaccharide (LPS)‐induced C2C12 myotube atrophy. The main components of BRE included all‐trans‐BR, monoanhydrobacterioruberin, 2‐isopentenyl‐3,4‐dehydrorhodopin and all‐trans‐bisanhydrobacterioruberin. BRE exhibited higher antioxidant activity and DNA nicking protection activity than other well‐known C₄₀ carotenoids, such as β‐carotene, lycopene and astaxanthin. In C2C12 myotubes, LPS treatment led to a reduction in myotube diameter and number, as well as the hypertranscription of the muscle‐specific ubiquitin ligase MAFbx and MuRF1. BRE mitigated these changes by activating the Akt/mTOR pathway. Furthermore, BRE abolished the elevated cellular reactive oxygen species levels and the inflammation response induced by LPS. This study demonstrated that ‘Hfx. marinum’ is an excellent source of natural microbial C₅₀ carotenoids with strong antioxidant capacity and may offer potential protective effects against muscle atrophy.

INTRODUCTION

Carotenoids, naturally occurring red-orange pigments, can be classified as C₃₀, C₄₀, C₄₅ and C₅₀ carotenoids based on the number of carbons in their carotene backbones (Yabuzaki, 2017). Compared to ubiquitous C₄₀ carotenoids (i.e. β-carotene and lycopene), only a small number of C₅₀ carotenoids have been discovered. Bacterioruberin (BR) and its derivatives are a group of C₅₀ carotenoids abundant in halophilic archaea, which thrive in environments with salt concentrations approaching saturation (Oren, 2009). BR has 13 pairs of conjugated double bonds with no subsidiary conjugation arising from the terminal isoprenoid groups, and it contains four hydroxyl group functionalities solely at both ends (Jehlicka et al., 2013). The structure of BR has been proposed to function as a reinforcement for membranes, stabilizing phospholipid bilayers under conditions of high osmotic and oxidative stress and providing protection against UV light (Morilla et al., 2023). Additionally, these conjugated double bonds and functional groups contribute to their biological activities, such as anti-inflammatory activity by regulating the NF-κB pathway, as well as their antioxidative potential by scavenging free radicals through various chemical mechanisms (Albrecht et al., 2000; Fiedor & Burda, 2014; Morilla et al., 2023).

Haloferax species exhibit pink-red colour due to the production of mainly C₅₀ carotenoids, such as BR and its derivatives (Oren, 2009). ‘Haloferax marinum’ strain MBLA0078 is a halophilic archaeon isolated from seawater near Yeoungheungdo Island in the Republic of Korea, and it also produces C₅₀ carotenoids, which give rise to its distinctive red-coloured colonies (Cho et al., 2021). To advance our knowledge of C₅₀ carotenoids, the present study aimed to examine the chemical composition, antioxidant capacity and biological properties of BR and its derivatives extracted from ‘Hfx. marinum’.

Muscle atrophy, characterized by a decrease in the mass and function of skeletal muscle, occurs under various systemic conditions such as ageing, sepsis, AIDS and cancer (Cohen et al., 2015). During muscle atrophy, proteolytic systems are activated, leading to the removal of contractile proteins and organelles, which reduces muscle fibre size. Excessive muscle atrophy can result in disability, poor life quality and mortality. Studies have demonstrated that dietary C₄₀ carotenoids have the potential to prevent muscle atrophy (Semba et al., 2007; Yoshihara et al., 2019). For instance, astaxanthin intake attenuated the rate of disuse muscle atrophy by inhibiting oxidative stress and proteolysis via major proteolytic pathways (Shibaguchi et al., 2016). Also, fucoxantinol, one of the metabolites of fucoxanthin, attenuated oxidative stress-induced atrophy and loss of myotubes (Yoshikawa et al., 2020). However, whether C₅₀ carotenoids have similar protective effects remained unclear. In this study, we tested the protective effect of bacterioruberin extract (BRE) from ‘Hfx. marinum’ against lipopolysaccharide (LPS)-induced skeletal muscle atrophy in C2C12 myotubes.

EXPERIMENTAL PROCEDURES

Bacterioruberin extract

A colony of ‘Hfx. marinum’ MBLA0078 (= KCTC 4290 = JCM 34171) was selected and placed into 20 mL of DB characterization medium no. 2 (DBCM2), as described previously by Burns et al. (2010). It was pre-cultivated at 37°C and shaken at 180 rpm until the absorbance at 600 nm was 0.6, which was then used as the inoculum at 1% (v/v). A 2-L Erlenmeyer flask containing 500 mL DBCM2 medium was inoculated and cultured for 72 h at 37°C, at 180 rpm. The harvested culture broth, whose final cell density (OD₆₀₀) was 0.96 ± 0.06, was centrifuged at 12,300 x g for 5 min to obtain the cell pellet. Then, the cell pellet was suspended in acetone:methanol (7:3, v/v) and stirred to extract the pigments until the cell pellet showed a white appearance. This suspension was then centrifuged at 18,500 x g for 10 min and the supernatant was collected, evaporated and stored at −20°C for future use. BRE was dissolved in methanol for antioxidant assays, and in DMSO for DNA nicking and cellular assays.

Analyses of pigments by thin-layer chromatography

The BRE obtained was analysed by thin-layer chromatography (TLC), as described by Strand et al. (1997). A TLC plate of silica Merck 5553 was utilized with a development liquid composed of acetone and n-heptane (50:50, v/v). Multiple bands appeared on the resulting TLC plate, and each pigment band was scraped off separately and dissolved in 200 μL methanol. Each resulting solution was filtered through a 0.22 μm syringe filter, adjusted to a volume of 1 mL with methanol, placed in a quartz cuvette and scanned at a wavelength of 300–600 nm to obtain UV–Vis absorbance spectra of each individual pigment band using a Shimadzu UV-1280 spectrophotometer. Thereafter, the crude carotenoid and sorted extracts were analysed by high-performance liquid chromatography (HPLC).

High-performance liquid chromatography (HPLC)

The sorted extracts and crude carotenoids were analysed by the YL9100 HPLC system equipped with a YL9160 photodiode array (PDA) detector (Youngin Chromass, Anyang, Korea). Chromatographic analysis was carried out on a reverse-phase Syncronis C18 (Thermo Scientific, Milano, Italy) column (250 mm × 4.6 mm, 5 μm). The mobile phase was isocratic eluent using 100% methanol at an elution flow rate of 1.0 mL/min, with an injection volume of 20 μL. The eluent was scanned at 490 nm and online spectra were registered between 300 and 600 nm. The carotenoids were confirmed based on their elution order, UV–visible spectra characteristics and previously reported literature about the carotenoid profile of halophilic archaeon.

Ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) and quantification of carotenoids

UPLC–MS analysis was performed using an UltiMate 3000 System (Thermo Scientific, Waltham, MA, USA) with a Thermo Scientific ISQ EM Single Quad MS System with atmospheric pressure chemical ionization (APCI). BRE analysis was carried out using methanol as the mobile phase at a flow rate of 1 mL/min, and the injection volume was 20 μL. Separation was performed on a Syncronis C18 column. Chromatograms were recorded at 490 nm. Ionization was generated by an atmosphere-pressure chemical ionization source. The APCI vaporizer temperature was set at 450°C, the capillary voltage at 13 V, the discharge current at 5 μA and the tube lens offset at −15 V, as described by Squillaci et al. (2017). The scanning of positively ionized fragments was monitored at 300 to 1000 m/z. Due to the lack of commercially available standards for BR and its derivatives, their concentrations were expressed as μg astaxanthin equivalents against dried cell weight (μg astaxanthin equivalent/g dry cell weight) using pure astaxanthin, which is a representative marine carotenoid (Lizama et al., 2021; Taniguchi et al., 2017). The maximum absorbance in the astaxanthin spectrum (A_472nm) was utilized for quantification. A standard curve was calculated with astaxanthin, covering eight points, with standard solutions from 0.06 to 7.8 μg/mL, and an R² value of 0.9957.

Antioxidant assays

To compare the antioxidant activity of BRE with other antioxidants and carotenoids (i.e. lycopene, β-carotene, astaxanthin, BHT and ascorbic acid), ABTS, DPPH and ferric reducing antioxidant power (FRAP) assays were conducted according to previously study described by Kim et al. (2023). All reactions were conducted at room temperature in the dark unless otherwise specified. In the DPPH assay, 20 μL of sample solutions were added to 180 μL of 2 mM DPPH methanolic solution. The mixtures were shaken vigorously and left to stand for 30 min. Then the absorbance was read at 517 nm. The reduction in absorbance at 517 nm was measured using pure methanol as a blank to evaluate the radical scavenging capacity of each antioxidant sample.

In the ABTS assay, a solution of 7 mM ABTS dissolved in distilled water was mixed with 2.45 mM potassium persulfate in a 1:1 (v/v) ratio and left to react in the dark at 4°C for 14–16 h to generate the radical cation ABTS^*+. This ABTS^*+ solution was diluted with absolute ethanol until the mixture reached moderate absorbance of 0.7 ± 0.02 at 734 nm. Subsequently, 180 μL of the ABTS^*+ solution was mixed with 20 μL of sample solutions and allowed to react for 7 min. The decrease in absorbance at 734 nm was measured to calculate the antioxidant activity of the samples, quantified as a percentage of inhibition of the oxidation of ABTS^*+.

In the FRAP assay, a fresh solution containing TPTZ-Fe³⁺ was prepared, composed of 10 mM TPTZ, 20 mM FeCl₃·6H₂O and 0.3 M acetate buffer at pH 3.6, mixed in a ratio of 1:1:10 (v/v/v). After mixing 1 mL of the FRAP reagent with 100 μL of a BRE solution, the assay reacted for 30 min. The change in absorbance (ΔA_593nm) was calculated for each sample and compared to the ΔA_593nm of a FeSO₄·7H₂O standard solution tested in parallel. The antioxidant capacity of the BRE was expressed as Trolox equivalent antioxidant capacity (TEAC) and EC₅₀ (the concentration of carotenoids, expressed as μg/mL, needed to scavenge 50% of the radicals). Each independent experiment was performed with more than three different concentrations of the antioxidant, in triplicate.

DNA nicking assay

DNA nicking assay was used to evaluate the ability of BRE to protect DNA from hydroxyl radicals generated by Fenton's reagent (Kitts et al., 2000). The pUC19 plasmid DNA (500 μg/μL) was incubated with the Fenton reagent using 10 μL of 5% H₂O₂ (v/v), 10 μL of 100 μM FeCl₂ and 5 μL of 100 mM phosphate buffer (pH 7.4) at 37°C for 5 min and the assay was stopped by adding 5 × loading buffer for agarose gel. The electrophoresis of the reacted solution was performed at room temperature on 0.8% (w/v) agarose gels with neogreen for 30 min at 100 mV. Antioxidant capacity was calculated based on diminishing the degree of supercoiled DNA in electroporation. Gel imaging was determined using a ChemiDoc MP imager (Bio-Rad, Hercules, CA, USA), and band densitometry was estimated by ImageJ software (NIH; National Institutes of Health, Frederick, MD, USA). Each independent assay was performed with more than three different concentrations of antioxidants, in triplication.

C2C12 cell culture and differentiation

The C2C12 mouse skeletal muscle cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and 1% P/S in 100 mm cell culture dishes and incubated in 5% CO₂ at 37°C. The medium was replaced every 2 days. To induce myogenic differentiation, C2C12 myoblasts were seeded (2.5 × 10⁵ cells/well) into six-well culture plates. When the cells reached approximately 90–100% confluence, the medium was replaced with differentiation medium (DMEM containing 2% FBS and 1% P/S) for 4 days, with medium change every 2 days. To investigate the protective effects of BRE against LPS-induced damage, differentiated C2C12 myotubes were stimulated with LPS (1 μg/mL), with or without BRE (dissolved in DMSO), for 48 h. The maximum dose of BRE (100 μg/mL) was selected based on cell viability test. All cell experiments were performed in the dark to prevent any light-induced degradation of BRE.

Giemsa staining and visualization

The morphology of matured C2C12 myotubes was observed by Giemsa staining. After the indicated treatments, C2C12 myotubes were washed with PBS. After fixing in absolute methanol for 10 min, they were incubated with Giemsa staining solution for 30 min at room temperature. Subsequently, the myotubes were washed with distilled water and visualized using a BioTek Cytation 5 Image Reader (Agilent Technologies, Santa Clara, CA, USA).

Measurement of myotube number and diameter

The diameter of the C2C12 myotubes was determined using Image J software. The diameters of at least 100 myotubes were measured from at least five random fields and the diameter per myotube was expressed as the mean of three measurements. Results were expressed as percentages of the diameter in the control.

Measurement of intracellular reactive oxygen species (ROS) production

The intracellular ROS content was measured using 5,6-carboxy-2′,7′-dichlorofluorescein diacetate (DCF-DA). Following treatment, the cells were washed with PBS and incubated with 20 μM of DCFH-DA solution for 30 min in the dark at 37°C. After incubation, the cells were washed three times with HBSS, and stained cells were photographed using a BioTek Cytation 5 Image reader in at least five randomly selected fields from each well. The DCF-DA fluorescence intensity was analysed using Image J software.

RNA isolation, reverse transcription and qRT-PCR

Total RNA was isolated using TRIzol reagent, according to the manufacturer's instructions. The RNA (1 μg) was reverse transcribed into cDNA using Smart Gene Compact cDNA Synthesis Kit using a GeneAmp PCR System 9700 (PerkinElmer, Inc., Waltham, MA, USA). The qRT-PCR was performed with the SYBR Green Q-PCR Master Mix in a QuantStudio™ 1 Real-Time PCR Instrument (Thermo Fisher Scientific Inc., Rockford, IL, USA). The primers for real-time PCR were manufactured by Macrogen Co., (Seoul, South Korea), and the sequences are listed in Table S1. All mRNA levels were normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal controls. Gene expression was calculated by the 2^−ΔΔCT method.

Western blotting

Protein expression level was determined by Western blot analysis. The C2C12 myotubes were lysed with lysis buffer, according to the manufacturer's instructions. Protein concentration was determined using the BCA protein assay kit. Protein samples (30 μg) were separated by 8% SDS-PAGE and transferred onto PVDF membranes at 25 V for 1 h. The membranes were blocked with blocking solution for 1 h at room temperature and then incubated with primary antibodies (diluted 1:500–1000) at 4°C overnight. Subsequently, the membranes were washed three times using Tris-Buffered Saline-Tween 20 detergent (TBS-T, 0.1% Tween-20) and then blotted with the respective IgG HRP-linked secondary antibodies (diluted 1:10000 in TBS-T) for 1 h at room temperature. The membranes were then washed three times with TBS-T. The protein band signals were detected using ECL solution and visualized using the ImageQuant LAS 500. Image J software was used for the quantification of the protein band. α-Tubulin was used as an internal control.

Statistical analysis

The results were expressed as the mean ± standard deviation (SD), using at least three independent replications. One-way analysis of variance (ANOVA) was conducted to determine significant differences between groups using GraphPad Prism software, version 9.0 (GraphPad Software Inc., San Diego, CA, USA).

RESULTS

Identification of carotenoids in BRE

The separation and identification of carotenoids from the BRE of ‘Hfx. marinum’ MBLA0078 was carried out using TLC, followed by HPLC with a PDA detector and UPLC–MS analyses. Figure 1A illustrates the results of TLC analysis, revealing four distinct bands with varying colours. A re-extraction process of each band was performed using methanol through prep-TLC, and the UV spectrum was determined as shown in Figure 1B. The UV-Vis spectra results revealed the typical absorption spectrum peaks of C₅₀ carotenoids, including BR, monoanhydrobacterioruberin (MABR) and bisanhydrobacterioruberin (BABR) for Bands 1, 2 and 4, respectively, and the absorption spectrum peak of the C₄₅ carotenoid 2-isopentenyl-3,4-dehydrorhodopin (IDR) for Band 3 (Fang et al., 2010). The chromatographic profiles at 490 nm for extracts from individual bands were determined (Figure 1C). The presence of multiple peaks suggests the existence of multiple pigments within each band, comprising various isomeric forms of BR and its derivatives. Peaks 1, 7 and 11 had a similar maximum absorption spectrum pattern (Figure S1). Based on the retention time, spectrum pattern and TLC results, these peaks were identified as all-trans-BR, all-trans-MABR and all-trans-BABR (Table S2), which are all C₅₀ carotenoids. The major peak of band 3, eluted at 12.5 min (peak 8), had a maximum absorption spectrum of 483 nm. This spectrum also encompassed peaks at 457 and 517 nm. It was determined as IDR, a C₄₅ carotenoid. The difference in the number of carbons composing their carotene backbones is attributed to their difference in the absorption pattern (Britton et al., 2004).

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The UPLC chromatogram of BRE showed a very similar pattern to that of the aforementioned HPLC chromatogram, except for a slight difference in the retention time (Figure 2). Mass spectrometric analysis revealed that peaks 1–6 possessed a similar pseudo-molecular ion at m/z 742.1, consistent with BR and its isomers (Table 1). Peaks 7 and 11 showed MS ions at m/z 724.1 and 706.1, corresponding to MABR and BABR, respectively (Dina et al., 2017; Mandelli et al., 2012; Squillaci et al., 2017). Since the mass spectrometric analyses could not distinguish between BR isomers, their structures were assigned based on UV/Vis spectra analysis, spectral fine structure (% III/II), cis peak intensity (% AB/AII) (Ke et al., 1970; Zechmeister & Polgar, 1944) and comparison with previously reported data (Kelly et al., 1992; Mandelli et al., 2012; Serino et al., 2023; Squillaci et al., 2017). The presence of cis isomers of BR was demonstrated by a hypochromic shift of 3–11 nm in the farthest absorption maximum of the corresponding all-trans compound. The double cis isomer 5-cis, 9′-cis-BR, was identified by using the data from previous studies (Serino et al., 2023, Squillaci et al., 2017). Three geometric cis isomers with a single double bond were assigned as 9-cis-BR, 13-cis-BR and 15-cis-BR by comparing the absorption intensity of the cis peak, which increases in the three-finger shape when the double bond is closer to the centre of the molecule (Figure S2 and Table 1) (Zechmeister & Polgar, 1944). Given the above information, the structure of each carotenoid is shown in Figure S3. Quantitative analysis revealed that the major carotenoid in ‘Hfx. marinum’ MBLA0078 was all-trans-BR (751.19 ± 1.05 μg astaxanthin equivalent/g dry cell weight) out of the total carotenoids (987.75 ± 77.25 μg astaxanthin equivalent/g dry cell weight, Table 1 and Table S3).

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TABLE 1 Identification of bacterioruberin and derivatives produced by ‘Hfx. marinum’ MBLA0078 from UPLC-MS analysis.

Peak	Carotenoid (Tentative identification)	R.T. (min)a	Cis λbmax (nm)	λcmax (nm)	% III/IId	%AB/AIIe	Area (%)f	[M + H]+ (m/z)
1	All-trans-bacterioruberin	6.0	388	466, 493, 526	58.62	10.93	75.9	742.1
2	5-cis, 9′-cis-Bacterioruberin	6.4	388	459, 483, 514	33.65	15.82	1.0	742.1
3	Bacterioruberin isomer	7.1	–	453, 484, 522	2.41	–	1.2	742.1
4	9-cis-Bacterioruberin	7.9	386	460, 486, 519	49.44	28.32	1.6	742.1
5	13-cis-Bacterioruberin	8.9	386	460, 486, 518	38.02	82.76	7.0	742.1
6	15-cis-Bacterioruberin	9.4	385	464, 490, 522	28.83	105.61	0.9	742.1
7	All-trans-monoanhydrobacterioruberin	10.4	387	466, 493, 526	51.66	11.60	3.9	724.1
8	2-Isopentenyl-3,4-dehydrorhodopin	12.6	375	457, 483, 517	35.91	8.91	3.6	–
9	–	13.5	345	428, 453, 483	82.57	11.14	2.1	–
10	–	14.9	386	459, 486, 517	31.04	76.15	1.2	–
11	All-trans-bisanhydrobacterioruberin	20.5	386	468, 492, 526	52.22	13.66	0.4	706.1
12	–	25.0	389	455, 482, 512	1.06	21.25	1.2	–

Antioxidant capacity of BRE

ABTS, FRAP and DPPH assays were adopted to evaluate total antioxidant capacity of BRE (Table 2). In the ABTS assay, BRE exhibited a Trolox equivalent antioxidant capacity (TEAC) value of 2.3, surpassing that of other tested standard compounds, including Trolox (1.00), ascorbic acid (1.0), butylated hydroxytoluene (BHT) (0.98), β-carotene (0.9), lycopene (1.1) and astaxanthin (0.3). The TEAC value for BRE in the FRAP assay was 4.62 μg TEAC/mL, significantly higher than that of other compounds tested, such as Trolox (1.00), ascorbic acid (0.8), BHT (0.2), β-carotene (1.90) and astaxanthin (0.2). Additionally, results from the DPPH assay indicated that BRE from MBLA0078 was highly efficient at scavenging free radicals. In summary, the ABTS, FRAP and DPPH results collectively suggest that the antioxidant activity of BRE was approximately 2.0–8.0 times, 2.4–23.1 times and 1.4–3.0 times higher, respectively, than that of Trolox, ascorbic acid, BHT, β-carotene, lycopene and astaxanthin.

TABLE 2 Antioxidant activity of BRE extracted from strain MBLA0078.

Antioxidant	ABTS (TEAC)	DPPH (TEAC)	FRAP (TEAC)
μg/mL	EC50	μg/mL	EC50	μg/mL
Trolox	1	2.2	1	8.4	1
MBLA0078 (BRE)	2.3 ± 0.11	0.55	1.92 ± 0.11	3.5	4.62 ± 0.02
β-Carotene	0.9 ± 0.04	2.44	0.63 ± 0.03	11.3	1.90 ± 0.01
Lycopene	1.1 ± 0.2	1.72	–	–	–
Astaxanthin	0.3 ± 0.04	15.2	1.41 ± 0.22	4.72	0.2 ± 0.02
Ascorbic acid	1.0 ± 0.01	1.95	0.66 ± 0.12	8.56	0.8 ± 0.1
BHT	0.98 ± 0.1	3.81	0.76 ± 0.08	11.2	0.2 ± 0.01

DNA nicking protection activity of the BRE

The DNA nicking result showed that DNA relaxation was decreased by the addition of BRE (Figure 3A). Specifically, supercoiled form of DNA was calculated as 57.6%, 67.9%, 47.1% and 33.3% with BRE (0.4 μg/mL), lycopene (1.6 μg/mL), β-carotene (1.6 μg/mL) and astaxanthin (1.6 μg/mL), respectively, compared to the positive control. Neither β-carotene, lycopene nor astaxanthin was as effective as BRE (Figure 3B).

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Effect of BRE on LPS-induced C2C12 myotube atrophy

Carotenoids possessing strong antioxidant properties are known to have the ability to delay muscle atrophy (Semba et al., 2007; Yoshihara et al., 2019). Since BRE showed a strong antioxidant property, we first investigated the protective effects of BRE on muscle atrophy in LPS-induced C2C12 myotubes. As shown in Figure 4A, BRE showed no toxicity on C2C12 up to 100 μg/mL. The myotube number and diameter of the C2C12 myotubes were significantly decreased by LPS while they were significantly recovered by BRE at 25, 50 and 100 μg/mL (Figure 4B,C). Especially, the diameter of the LPS-treated myotubes decreased by 37.94% compared to the control (LPS-untreated myotubes). In contrast, 100 μg/mL of BRE alleviated muscle atrophy by 41.51% compared to the LPS-treated myotubes.

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The muscle-specific E3 ubiquitin ligases such as muscle atrophy F-box (MAFbx; also known as atrogin-1) and muscle RING-finger 1 (MuRF1) are potent biomarkers of muscle protein degradation that are highly upregulated by the transcription factor forkhead box O type 3 (FoxO3) (Tia et al., 2018). As shown in Figure 4D, the mRNA expression of FoxO3a, MAFbx and MuRF1 was markedly elevated by LPS, and this increase was effectively attenuated by BRE.

Effects of BRE on protein synthesis-related pathways

Protein kinase B (also called Akt) is a crucial regulator of skeletal muscle protein synthesis, by activating mammalian target of the rapamycin (mTOR) and its downstream targets, including 70-kDa ribosomal protein S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). We investigated the effects of BRE on the Akt/mTOR pathway by Western blot assay. As shown in Figure 5A,B, LPS decreased the phosphorylation levels of Akt, mTOR and p70S6K, but BRE restored these changes. Especially, BRE-supplemented samples showed even higher phosphorylation levels than those of the non-atrophic samples. Although Akt downstream mediator, phosphorylated 4E-BP1, was not altered by LPS, BRE upregulated it compared to the control and LPS-treated myotubes.

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Effects of BRE on the expression of pro-inflammatory cytokines

As inflammation is known to be a main factor in skeletal muscle atrophy (Ji et al., 2022), the gene expression of pro-inflammatory cytokines (interleukin (IL)-6, IL-1β and tumour necrosis factor-α (TNF-α)) and inflammatory mediators (inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2)) was evaluated in LPS-induced C2C12 myotube atrophy. The mRNA expression of pro-inflammatory cytokines and inflammatory mediators significantly increased in LPS-treated myotubes, whereas their expression was attenuated by BRE (Figure 6).

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Effect of BRE on ROS production and antioxidant enzymes in LPS-treated C2C12 myotubes

To examine whether BRE could be a radical scavenger against LPS-induced ROS production, intracellular ROS levels and the expression of antioxidant enzymes were measured in LPS-induced C2C12 myotube atrophy. As shown in Figure 7, BRE remarkably reduced LPS-induced ROS production to a level similar to the control group. LPS significantly increased the mRNA expression of NADPH oxidase 2 (Nox-2), an oxidative stress marker, while BRE suppressed the LPS-induced Nox2 mRNA expression (Figure 7B). In the case of nuclear factor erythroid-2-related factor 2 (Nrf2), which induces the expression of antioxidant genes, its mRNA expression increased by LPS but showed a similar tendency to the control when treated with BRE (Figure 7C). LPS significantly increased the mRNA expression of antioxidant enzymes, such as heme oxygenase-1 (Hmox1), glutathione peroxidase-1 (Gpx1), superoxide dismutase 1 (Sod1) and catalase (Cat). In contrast, BRE further increased the mRNA expression of Hmox1 and Gpx1. However, the mRNA expression of Sod1 was not altered by BRE.

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DISCUSSION

The present study analysed the chemical composition and antioxidant potential of carotenoids extracted from ‘Hfx. marinum’ strain MBLA0078. Among the detected carotenoids, all-trans-BR emerged as the primary C₅₀ carotenoid (751.19 μg astaxanthin equivalent/g) followed by 13-cis-BR (70.44 μg astaxanthin equivalent/g) and all-trans-MABR (36.91 μg astaxanthin equivalent/g). The presence of these compounds aligns with existing literature, as BR is widely recognized as the most abundant and consistently observed pigment in halophilic archaea (Lizama et al., 2021; Serino et al., 2023). BR precursors such as tetrahydrobisanhydrobacterioruberin (TH-BABR) and trisanhydrobacterioruberin (TABR) have been reported in certain haloarchaea species (de la Vega et al., 2016; Giani et al., 2024; Serino et al., 2023; Squillaci et al., 2017; Yang et al., 2015). However, this study did not detect precursors of BR, such as lycopene and TABR. These results indicate a variation in carotenoid composition depending on the strain (Sahli et al., 2022). Further experiments are needed to analyse this, such as different extraction methods using the antioxidant BHT and specific organic solvents, analysis techniques or culture conditions (Serino et al., 2023).

The superior antioxidant ability of C₅₀ carotenoids compared to the more ubiquitously occurring C₄₀ carotenoids has been recognized (Flores et al., 2020). This study confirms that the antioxidant activity of C₅₀ carotenoid from ‘Hfx. marinum’ surpasses that of representative C₄₀ carotenoids, including β-carotene, lycopene and astaxanthin. Notably, these C₅₀ carotenoids also show a higher antioxidant capacity than ascorbic acid, Trolox and butylated hydroxytoluene, which are standard chemicals used for quantitative antioxidant analysis. Carotenoid extracts from several haloarchaea strains have demonstrated interesting scavenging activities in a dose-dependent manner. Similar to the observed antioxidant potency of BRE from ‘Hfx. marinum’ MBLA0078, carotenoid extracts from various haloarchaeal species, including Hfx. volcanii, Hfx. mediterranei, Halogranum rubrum, Halopelagius inordinatus, Halogeometricum rufum and Halorhabdus utahensis, have exhibited elevated antioxidant efficacy exceeding conventional antioxidants (Giani et al., 2022; Hou & Cui, 2018; Serino et al., 2023). Additionally, BR and its derivatives exhibit strong intracellular antioxidant activities (Abbes et al., 2013; Hwang et al., 2024). This remarkable antioxidant property of BR and its derivatives is related to their larger number of conjugated double bonds and the presence of hydroxyl groups (Albrecht et al., 2000). The DNA nicking assay further demonstrated the potent protective effect of BRE against hydroxyl free radicals-induced DNA damage.

Oxidative stress and inflammation are two common and interrelated mechanisms in the process of muscle atrophy (Gomez-Cabrera et al., 2020). Carotenoids with strong antioxidant and anti-inflammatory activity are appealing natural resources for preventing muscle atrophy. Several natural C₄₀ carotenoids, such as β-carotene, astaxanthin and fucoxanthin, have been reported to exhibit preventive effects against muscle atrophy and show promise as therapeutic agents (Ogawa et al., 2013; Shibaguchi et al., 2016; Yoshikawa et al., 2021). Although BR, C₅₀ carotenoid derived from haloarchaea, has exhibited certain biological properties, including antimicrobial, anticancer and antiviral activities (Fariq et al., 2019; Giani et al., 2023; Hegazy et al., 2020), its potential protective effects against muscle atrophy had remained unclear. In the present study, BRE ameliorates LPS-induced myotube diameter reduction and myotube number reduction. MuRF1 and MAFbx/atrogin-1, both muscle-specific E3 ubiquitin ligases, undergo transcriptional upregulation in skeletal muscle under conditions that induce atrophy, establishing them as markers of muscle atrophy (Bodine & Baehr, 2014). The FoxO3a is a major mediator of protein breakdown by activating the transcription of MAFbx and MuRF1 (Tia et al., 2018). BRE effectively inhibited the transcription of FoxO3a, MuRF1 and MAFbx induced by LPS. Overall, these data demonstrate the ability of BRE to counter muscle atrophy in LPS-treated C2C12 myotubes.

Skeletal muscle mass depends on protein turnover, which is determined by the relative balance between protein synthesis and breakdown (Sartori et al., 2021). Muscle atrophy occurs when protein breakdown exceeds protein synthesis. Treatments that prevent the activation of proteolysis or increase protein synthesis offer considerable promise to combat this debilitating process (Cohen et al., 2015). One main pathway responsible for protein synthesis and the regulation of skeletal muscle mass is the highly conserved Akt/mTOR signalling pathway (Bodine et al., 2001; Zanchi & Lancha, 2008). Targets of mTOR such as 4E-BP1 and S6K1 facilitate ribosomal biogenesis and protein translation. When 4E-BP1 is dephosphorylated, it can bind with 4E, which blocks the initiation of mRNA translation (Azar et al., 2008). When p70S6K is phosphorylated, it stimulates the phosphorylation of S6, which promotes the production of proteins involved in cell growth and proliferation. Additionally, Akt can attenuate protein degradation by inhibiting FoxO family (Gomez-Cabrera et al., 2020). Thus, activation of the Akt/mTOR pathway can protect against muscle atrophy.

BRE attenuated the LPS-induced inhibition of Akt/mTOR signalling. The depressed phosphorylation level of Akt, mTOR and p70S6K, induced by LPS, was ameliorated by BRE. At concentrations of 50 and 100 μg/mL, BRE even elevated their phosphorylation levels higher than those observed in the non-atrophic control samples. Although BRE elevated the phosphorylation of 4E-BP1, LPS treatment did not alter its level. This may be due to the time-sensitive nature of 4E-BP1 phosphorylation. The phosphorylation of 4E-BP1 tends to be more transient in comparison to other mTOR-related factors (Cao et al., 2021).

Inflammation can trigger skeletal muscle atrophy by changing the metabolic state of cells (Ji et al., 2022). For instance, the activation of NF-κB in skeletal muscle results in degradation of specific muscle proteins, triggers inflammation and fibrosis and impedes the regeneration of myofibers (Li et al., 2008). Besides FoxO3, NF-κB is another transcription factor that promotes the transcription of MAFbx and MuRF1. Inflammatory signals, such as cytokines (e.g. IL-6, IL-1β and TNF-α), can activate catabolic pathways, promoting the breakdown of muscle proteins (Thoma & Lightfoot, 2018). In this study, LPS increased the transcription of inflammatory mediators in C2C12 myotubes. In contrast, BRE reduced their mRNA levels. The anti-inflammatory effect of BRE may contribute to its anti-atrophy effect.

Elevated levels of ROS and disturbance in redox signalling play a significant role in the promotion of muscle atrophy (Gomez-Cabrera et al., 2020). Excessive ROS contributes to the induction of E3 ubiquitin ligases by stimulating the nuclear translocation of NF-κB in C2C12 myotubes (Powers et al., 2007). The increase in ROS formation in atrophic C2C12 myotubes by LPS was significantly reduced by co-treatment with BRE. The expression of Nox2, which is involved in ROS production, also showed a similar trend. ROS are controlled through the production of various NRF2-regulated antioxidant enzymes, such as HMOX1, GPX1, SOD and CAT. Here, we found that BRE inhibited the LPS-induced transcription of Nrf2. Furthermore, BRE exhibited a greater increase in the transcription levels of Hmox1 and Gpx1 compared to those observed in the non-atrophic control samples. The results showed that BRE decreased the production of inflammatory mediators and strongly inhibited LPS-induced ROS production while enhancing cellular antioxidant capacity. This aligns with the recent findings by Avila-Romen et al., which reported that a BR-rich extract from a Haloarcula species inhibits LPS-induced inflammation and ROS production in macrophages (Avila-Roman et al., 2023).

The results suggest that BR promotes protein synthesis in skeletal muscle via the Akt/mTOR pathway, indicating that anti-atrophy activity of BRE is related to its protein degradation/synthesis modulation, anti-inflammation and antioxidant effects.

CONCLUSIONS

In the present study, the primary carotenoids in ‘Hfx. marinum’ MBLA0078 have been identified as C₅₀ BR and its derivatives. They exhibit exceptional antioxidant capacities and offer protection against skeletal muscle atrophy in LPS-treated C2C12 myotubes. The anti-atrophy effect is achieved by activating the Akt/mTOR signalling pathway to enhance protein synthesis and alleviate protein breakdown. Furthermore, BRE significantly decreased the LPS-stimulated pro-inflammatory mediators and intracellular ROS production while enhancing the antioxidant defence. These findings suggest that BR could be a promising candidate for the treatment of muscle atrophy.

AUTHOR CONTRIBUTIONS

Hyeju Lee: Methodology; formal analysis; investigation; data curation; writing – original draft. Eui-Sang Cho: Investigation; writing – original draft; methodology; formal analysis; data curation. Chi Young Hwang: Methodology; data curation; writing – review and editing. Lei Cao: Visualization; writing – review and editing. Mi-Bo Kim: Visualization; writing – review and editing. Sang Gil Lee: Conceptualization; resources; supervision; funding acquisition; writing – review and editing. Myung-Ji Seo: Conceptualization; funding acquisition; writing – review and editing; supervision; resources.

ACKNOWLEDGEMENTS

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MIST) (Grant No. 2019R1A2C1006038, 2022R1F1A1062699 and 2022R1C1C1011770), and the “Regional Innovation Strategy (RIS)” through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2023RIS-007). The funding sources had no impact or role in the study design or conduct; collection, analysis and interpretation of the data; preparation, review or approval of the manuscript; and decision to submit the manuscript for publication.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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