Abstract

   Muscle atrophy, marked by the loss of skeletal muscle mass and
   strength, presents a major health concern with diverse etiologies,
   including chronic inflammation. Effective interventions are urgently
   needed for its prevention and treatment. Although α-tocopherol, the
   most abundant form of vitamin E, is known for its antioxidant benefits
   in muscle health, γ-tocotrienol exhibits superior antioxidant and
   anti-inflammatory properties. This study investigates the protective
   effects of γ-tocotrienol against muscle atrophy and compares its
   efficacy with α-tocopherol. Muscle atrophy was induced in
   differentiated C2C12 myotubes using lipopolysaccharide (LPS), with
   vitamin E pre-treatment applied prior to LPS challenge. Myotube
   morphology, expression of atrophy-related markers, and underlying
   molecular pathways were examined through immunofluorescence, western
   blotting, and quantitative proteomics. LPS treatment induced
   significant myotube atrophy without affecting cell viability. Notably,
   γ-tocotrienol pre-treatment preserved myotube size and suppressed key
   atrophy markers, including the E3 ubiquitin ligases MuRF-1 and
   Fbxo32/Atrogin-1. Proteomic analysis quantified 5,371 proteins and
   revealed that γ-tocotrienol alleviated atrophy by enhancing
   extracellular matrix organization and attenuating oxidative stress and
   mitochondrial dysfunction. These protective effects were further
   confirmed in vivo, where γ-tocotrienol administration preserved muscle
   strength, suppressed pro-inflammatory signaling, and restored
   mitochondrial biogenesis in LPS-treated mice. Collectively, these
   findings demonstrate that γ-tocotrienol offers superior protection
   against muscle atrophy compared to α-tocopherol, highlighting its
   therapeutic potential for individuals at risk of muscle wasting.

   Keywords: Muscle atrophy, γ-Tocotrienol, Vitamin E, Proteomics,
   Mitochondrial oxidative stress

Graphical abstract

   [43]Image 1
   [44]Open in a new tab

Highlights

     * •
       γ-Tocotrienol exhibits superior muscle-protective effects than
       α-tocopherol in LPS-induced atrophy.
     * •
       Pre-treatment of γ-tocotrienol preserves myotube morphology and
       suppresses MuRF-1 and Atrogin-1 expression.
     * •
       Proteome analysis reveals γ-tocotrienol enhances ECM organization,
       reduce oxidative stress, and protects mitochondria.
     * •
       In vivo administration of γ-tocotrienol restores muscle strength,
       promotes mitochondrial biogenesis, and reduces inflammation.
     * •
       γ-Tocotrienol as a promising therapy for inflammation-induced
       muscle loss, offering advantages over α-tocopherol.

1. Introduction

   Muscle atrophy, characterized by a reduction in muscle mass and
   strength, represents a significant health challenge with diverse
   etiologies [[45]1]. Various pathological conditions—including metabolic
   syndromes such as diabetes, cancer, and sclerosis—can disrupt skeletal
   muscle homeostasis and trigger atrophic processes [[46]2,[47]3]. These
   disorders are frequently associated with chronic inflammation,
   mitochondrial dysfunction, apoptosis, and dysregulated protein
   turnover. The inflammatory mechanisms underlying skeletal muscle
   atrophy are highly complex, necessitating molecular-level insights to
   develop effective preventive and therapeutic strategies. Although
   exercise remains the most effective intervention for both preventing
   and reversing muscle atrophy [[48]4], its applicability is often
   limited in patients with physical impairments or chronic illnesses
   [[49]2]. Therefore, identifying alternative pharmacological or
   nutritional approaches is essential to address the multifactorial
   nature of muscle wasting.

   Lipopolysaccharide (LPS) is a widely used agent for inducing muscle
   atrophy in vitro and mimics the molecular and physiological features of
   muscle loss observed in inflammation-related diseases [[50][5],
   [51][6], [52][7], [53][8]]. Elevated circulating endotoxins, including
   LPS, have been reported in various conditions such as obesity [[54]9],
   insulin resistance [[55]10], type 2 diabetes [[56]11], non-alcoholic
   fatty liver disease [[57]12], cancer [[58]13], and other chronic
   inflammatory disorders [[59]14], all of which are associated with
   skeletal muscle wasting.

   As a component of the outer membrane of Gram-negative bacteria, LPS
   acts as a potent pro-inflammatory stimulus by binding to toll-like
   receptor 4 (TLR4), thereby triggering the production of
   pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α),
   interleukin-6 (IL-6), and interleukin-1β (IL-1β), as well as generating
   reactive oxygen species (ROS). LPS activates NADPH oxidases and impairs
   mitochondrial function, leading to excessive ROS production. These ROS
   further amplify inflammation and activate transcription factors such as
   NF-κB and FOXO. The FOXO family (including FOXO1, FOXO3, and FOXO4)
   directly induces the expression of the muscle-specific E3 ubiquitin
   ligases MuRF1 and Atrogin-1 (Fbxo32), key mediators of
   proteasome-dependent protein degradation during muscle atrophy
   [[60]15].

   This study aims to investigate the protective effects of γ-tocotrienol,
   a potent isoform of vitamin E, against LPS-induced muscle atrophy using
   C2C12 murine myotubes. While α-tocopherol, the predominant form of
   vitamin E, is well-known for its antioxidant benefits in muscle health
   [[61]16], γ-tocotrienol has demonstrated superior antioxidant and
   anti-inflammatory activities [[62]17,[63]18]. Structurally,
   tocotrienols possess an unsaturated isoprenoid side chain that
   facilitates better membrane penetration and distribution within lipid
   bilayers. This unique property contributes to their wide-ranging health
   benefits, including improved glycemic control and insulin sensitivity
   in diabetic rats [[64]19], favorable modulation of lipid profiles
   [[65]20], and cardioprotective effects [[66]21]. Although several
   studies have reported the muscle-preserving potential of tocotrienols,
   most employed tocotrienol-rich fractions (TRFs) derived from palm oil
   [[67]22], without directly comparing the effects of γ-tocotrienol to
   α-tocopherol. For example, recent findings showed that TRF
   supplementation mitigated hyperglycemia-induced muscle atrophy and
   insulin resistance in diabetic mice [[68]19], and enhances myogenic
   differentiation in myoblasts [[69]23]. These collective findings
   suggest that γ-tocotrienol may exert more potent protective effects on
   skeletal muscle than α-tocopherol.

   In the present study, we systematically compared the effects of
   γ-tocotrienol and α-tocopherol on LPS-induced muscle atrophy using mass
   spectrometry-based quantitative proteomics. We further examined the
   molecular pathways involved in muscle protein turnover in vivo and in
   vitro to elucidate the mechanisms by which γ-tocotrienol exerts its
   protective actions against inflammation-driven muscle wasting.

2. Materials and methods

2.1. Cell culture and treatment

   C2C12 murine myoblast cell line was obtained from Bioresource
   Collection and Research Center (BCRC), Hsinchu, Taiwan). Cells was
   cultured in high-glucose DMEM (Gibco, Grand Island, NY, USA)
   supplemented with 10 % FBS (Gibco) at 37 °C with 5 % CO[2]. Culture
   medium were changed to differentiation medium (DM, DMEM supplemented
   with 2 % HS (Gibco)) upon reaching 80 % confluency, and replaced every
   2 days throughout the differentiation process. After 6 days of
   differentiation, multinuclear myotubes were formed. C2C12 myotubes were
   pretreated with different doses of γ-tocotrienol (0.2, 1, 5 μM) (Cayman
   Chemical, Ann Arbor, MI, USA) or α-tocopherol (Cayman Chemical) for 6 h
   before treated with LPS (Chemscene, Monmouth Junction, NJ, USA) for
   48 h to induce muscle atrophy. All experiments were conducted using
   C2C12 cells at passages between 5 and 10.

2.2. Cell viability assay

   Cells were plated in a 96-well culture plate at a density of
   1 × 10^4 cells per well, induced to differentiation for 6 days, and
   pre-treated with various concentrations of γ-tocotrienol for 6 h prior
   to 48-h LPS administration. Cell viability was determined by CCK8 assay
   (Dojindo Laboratories, Mashiki, Japan). 10 μL of the CCK-8 reagent was
   added directly into the wells and incubated in the dark for 30 min.
   Absorbance at 450 nm wavelength was read with an ELISA microplate
   reader (Molecular Devices, San Jose, CA, USA).

2.3. Immunofluorescence staining

   Differentiated myotubes were fixed by 4 % paraformaldehyde
   (Sigma‐Aldrich, Burlington, MA, USA), for 10 min at room temperature,
   permeabilized by 0.2 % Triton X-100 (Sigma-Aldrich) in PBS for 5 min,
   and then blocked with blocking buffer (BlockPRO, Taiwan) for 1 h at
   room temperature. Myotubes were incubated with Recombinant Alexa Fluor®
   488 Anti-TOMM20 antibody (Abcam, Cambridge, UK), anti-NFκB p65 (Cell
   signalling Technology, Danvers, MA, USA), anti-Myh1 (Abclonal) or
   anti-Myh4 (DHSB, Iowa City, IA, USA) diluted in blocking buffer
   overnight at 4 °C, followed by incubation with Alexa Fluor 562-goat
   anti-mouse IgG and Alexa Fluor 488-goat anti-rabbit IgG antibody
   (Thermo Fisher Scientific (Boston, MA, USA)). Cells were visualized
   with a fluorescence microscope (Axio Observer D1, Carl Zeiss AG,
   Oberkochen, Germany), and the diameter of myotubes was measured by
   ImageJ software (NIH, Bethesda, MD, USA). The nuclear fusion index
   (NFI) were measured using MyoCount software version 1.3 [[70]24].

2.4. Nuclear protein fractionation

   Nuclear fraction isolation was performed using the REAP method as
   described previously [[71]25]. Briefly, collected cells were
   centrifuged down and resuspended in 900 μL of ice-cold 0.1 % NP40 in
   PBS, and triturated 5 times using a P1000 pipette tip. The lysate was
   then centrifuged on a tabletop microcentrifuge (GeneReach, Taiwan), and
   the supernatant was collected as the cytosol fraction. The remaining
   pellet was then wash with 0.1 % NP40 in PBS twice and the nuclear
   pellet was resuspended in RIPA buffer.

2.5. Proteomics analysis

   Cells lysis and protein extraction were performed using PTS buffer
   [[72]26]. The proteins were treated with 10 mM TCEP (Sigma-Aldrich) at
   37 °C for 30 min to disrupt disulfide bonds, and alkylated by 25 mM CAA
   (Sigma-Aldrich) at 37 °C for 30 min. Protein digestion into peptides
   was achieved by incubating with ABC (Sigma-Aldrich), Lys-C
   endopeptidase (FUJIFILM Wako Chemicals USA, Richmond, VA, USA), and
   trypsin (w:w = 1:100) (Promega) for 3 h and 16 h respectively. After
   digestion, the reaction was quenched with 10 % TFA (Thermo Fisher
   Scientific) and EtOAc (Sigma-Aldrich) was added for detergent
   extraction. The mixture was vigorously shaken for 2 min, followed by
   centrifugation at 17000×g for 2 min to remove the organic layer
   containing EtOAc and detergent. The remaining EtOAc was removed using
   SpinVac (Thermo Fisher Scientific) and the sample was desalted with
   SDB-XC StageTip [[73]27]. Peptide concentration was quantified
   according to the absorbance at 214 nm with a SpectraMax ABS Plus
   Absorbance ELISA Microplate Reader. For TMT labeling and sample
   fractionation, equal amount of samples were dissolved in 200 mM HEPES
   pH 8.5 (Thermo Fisher Scientific) and labeled with TMTpro™ 18 plex
   Label Reagent Set (Thermo Fisher Scientific) according to the manual.
   Pooled sample was fractionated under alkaline condition, followed by
   desalted with C18 StageTip.

   Samples were reconstituted 2 % ACN (Thermo Fisher Scientific) with
   0.1 % FA (Thermo Fisher Scientific). Analysis was performed using an
   Ultimate system 3000 nanoLC system (Thermo Fisher Scientific, Bremen,
   Germany) coupled with an Orbitrap Fusion Lumos Tribrid quadrupole-ion
   trap-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose,
   CA). The column used was a 75 μm ID, 25 cm length C18 Acclaim PepMap
   NanoLC column (Thermo Fisher Scientific, San Jose, CA) packed with 2 μm
   particles with a pore of 100 Å. Mobile phase A (0.1 % FA in Milli-Q
   water) and B (0.1 % FA in 80 % ACN) were used as the LC buffer. The
   initial mobile phase composition was set to 98 % A and 2 % B, with a
   gradient increase to 40 % B over 100 min, separating the peptides at a
   flow rate of 300 nL/min in 50 min. Mass spectrometer parameters were
   set as follows: Data-dependent mode with Full-MS (mass accuracy of
   <5 ppm and a resolution of 120,000 at m/z 200, AGC target 5e5, maximum
   injection time of 50 ms); HCD-MS/MS of the most intense ions in 3 s.
   MS/MS (resolution of 15,000) within a 1.4 Da isolation window at a
   normalized collision energy of 32. AGC value 5e4 was set for MS/MS
   analysis with previously selected ions dynamically excluded for 60 s.
   Max injection time of MS/MS was set at 50 ms.

2.6. Peptide identification and analysis of differential expressed proteins

   The raw MS data was processed using MaxQuant software version 2.0.3.0
   for peptide and protein identification and quantification. The
   Andromeda search engine [[74]28] was employed to search MS2 data
   against the mouse reference protein database UniProt (downloaded on
   December 24, 2023). Subsequent processing and analysis of the
   identified proteins file were performed using Perseus 1.6.15.0
   [[75]29]. Decoy hits and contaminations were removed, and protein data
   was log2 transformed and normalized. Differentially expressed proteins
   (DEPs) were identified using Significance Analysis of Microarrays (SAM)
   with an S0 factor applied to a two-sample t-test and false discovery
   rate (FDR) of less than 0.05. DEPs were visualized in heatmaps and
   volcano plots. Subsequently, STRING and Metascape was utilized to
   obtain protein-protein interaction networks [[76]30], and functional
   pathway enrichment analysis was performed using Enrichr [[77]31] and
   GSEA [[78]32].

2.7. Measurement of intracellular reactive oxygen species (ROS)

   C2C12 cells were seeded and differentiated in 6-well plates containing
   coverslips, treated with γ-tocotrienol for 6 h and then stimulated with
   LPS (1 μg/mL) for 48 h. To detect intracellular ROS, H[2]-DCFDA
   fluorescence staining (Thermo Fisher Scientific) was performed on live
   C2C12 myotubes. To monitor the generation of mitochondrial ROS, cells
   were stained with 50 nM MitoTracker™ Red CMXRos (Thermo Fisher
   Scientific, Boston, MA, USA) for 30 min in the dark. After washing
   three times with PBS, cells were fixed with 4 % paraformaldehyde for
   15 min and proceed to fluorescence immunostaining. Coverslips were
   mounted using a mounting medium containing 4,6-diamidino-2-phenylindole
   (DAPI, Biotium, CA, USA). Fluorescent images were captured by a Axio
   Observer D1 microscope (ZEISS, Oberkochen, Germany) and processed in
   ZEN Imaging Software (ZEISS).

2.8. Mitochondrial membrane potential (ΔΨm) measurement

   C2C12 cells were seeded and differentiated in 6-well plates containing
   coverslips, treated with γ-tocotrienol for 6 h and then stimulated with
   LPS (1 μg/mL) for 48 h. The fluorescent cationic dye JC-1 (Thermo
   Fisher Scientific) was used to assess mitochondrial membrane potential.
   Fluorescent images were captured by a Axio Observer D1 microscope
   (ZEISS) and processed in ZEN Imaging Software (ZEISS). Changes in the
   JC-1 fluorescence intensity ratio (red: 540/570 nm to green:
   495/520 nm) were calculated based on signals recorded within a fixed
   exposure time.

2.9. Mitochondrial oxygen consumption rate analysis

   Seahorse XFe24 analyzer (XFe24, Agilent Technologies, CA) was employed
   to evaluate the mitochondrial oxygen consumption rate (OCR) of C2C12
   myotubes. C2C12 myoblasts were seeded at equal density in Seahorse
   XFe24 microplates, cultured to full confluence, and subsequently
   differentiated into myotubes to ensure consistent differentiation and
   minimize variability from post-assay normalization. Myotubes were
   treated with either γ-tocotrienol or α-tocopherol (2 μM) for 6 h,
   followed by exposure to LPS (1 μg/mL) for 48 h. Before conducting the
   assay, the sensors were hydrated overnight at 37 °C in a non-CO2
   incubator using Agilent Seahorse XF calibrant. The culture medium was
   then replaced with Agilent Seahorse XF basal medium, and the plates
   were incubated at 37 °C in an incubator without CO[2] for 1 h.
   Mitochondrial function was assessed using the Seahorse XF Cell Mito
   Stress Test, with measurements taken at baseline and after the
   sequential addition of oligomycin (1 μM), FCCP (3 μM), and a
   combination of antimycin A and rotenone (1 μM).

2.10. Animal model of LPS-induced muscle atrophy

   All animal experiments were conducted in accordance with the guidelines
   and regulations approved by the Institutional Animal Care and Use
   Committee (IACUC) of the Laboratory Animal Center at National Defense
   Medical University (IACUC-25-076). Six-week-old male C57BL/6 mice were
   purchased from the National Laboratory Animal Center (Taipei, Taiwan)
   and housed under controlled conditions with a 12-h light/dark cycle, at
   25 °C and 65 % relative humidity. Male mice were used to ensure
   consistent experimental conditions and to minimize potential
   confounding effects of sex steroids on metabolism and atrophy
   progression [[79]33,[80]34].

   After one-week acclimation, supplementation was administered daily by
   oral gavage for six weeks, starting four weeks before LPS exposure.
   Mice were randomly divided into six experimental groups (n = 8 per
   group): Control, LPS-only, and four treatment groups receiving LPS with
   prior supplementation of either γ-tocotrienol or α-tocopherol at
   20 mg/kg or 200 mg/kg body weight. The dosages were selected based on
   previous studies [[81]35].

   Beginning at week 11, muscle atrophy was induced by intraperitoneal
   injection of LPS (1 mg/kg body weight), administered once every five
   days for a total of three injections. Body weight was monitored twice
   weekly throughout the study. Mice were sacrificed 14 days after the
   initial LPS injection, and the gastrocnemius muscles were collected for
   subsequent histological and biochemical analyses.

2.11. Hematoxylin-eosin (H&E) staining and muscle fiber cross-sectional area
analysis

   Gastrocnemius muscle samples were fixed, sectioned, and stained with
   hematoxylin and eosin (H&E) by Catching Micro-Tech Co., Ltd. (Taipei,
   Taiwan). Stained tissue sections were imaged at 40× magnification using
   a ZEISS Axioscan 7 Slide Scanner (Zeiss). The cross-sectional area
   (CSA) of individual muscle fibers was quantified using ImageJ software.
   At least 100 fibers per sample were measured to ensure statistical
   robustness.

2.12. Immunohistochemical staining

   Paraffin-embedded tissue sections were deparaffinized, rehydrated, and
   subjected to blocking using a blocking buffer for 1 h at room
   temperature. The sections were then incubated overnight at 4 °C with
   primary antibodies against PGC-1α and SIRT1 (1:500 dilution). After
   washing, the sections were incubated for 1 h at room temperature with
   fluorescently labeled secondary antibodies: Alexa Fluor 562-conjugated
   goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit
   IgG. Immunofluorescent signals were visualized using a multiphoton
   laser scanning confocal microscope (LSM980, Zeiss).

2.13. Grip strength test

   Muscle strength was evaluated using a grip strength meter (SH‐III‐20,
   China). Mice were gently placed on a stainless-steel grid and allowed
   to grasp it with all four limbs while being held by the tail. The grid
   was then pulled horizontally at a constant speed until the mouse
   released its grip, and the peak force was recorded. Each mouse
   underwent four consecutive trials, and the average force from these
   trials was calculated as the representative grip strength. Measurements
   were performed both before and after LPS treatment to assess
   treatment-related changes in muscle strength.

2.14. Enzyme-linked immunosorbent assay (ELISA)

   Serum interleukin-6 (IL-6) levels were measured using a commercial
   ELISA kit (BioLegend, CA, USA; Cat. No. 431304), following the
   manufacturer's instructions. Each sample was analyzed in at least two
   technical replicates to ensure accuracy. Absorbance was read at 450 nm
   using a microplate reader (Molecular Devices, USA), and cytokine
   concentrations were calculated based on standard curves generated from
   known IL-6 concentrations.

2.15. Western blot

   Cell pellets or mice tissues were sonicated in RIPA buffer with
   phosphatase/protease inhibitor (MedChemexpress LLC, Middlesex, NJ,
   USA). The lysates were centrifuged at 17,000×g for 20 min at 4 °C.
   Protein concentration was determined by BCA protein assay (T-Pro
   biotechnology, New Taipei City, Taiwan) using different concentrations
   of BSA (BioShop Canada Inc., Burlington, ON, Canada) as standard, and
   equal amounts of protein samples were separated by 10–15 % SDS-PAGE and
   transferred to a PVDF membrane (Millipore, Burlington, MA, USA). The
   PVDF membranes were blocked in 5 % skimmed milk in TBS containing 0.1 %
   Tween 20 for 1 h at room temperature and then incubated with primary
   antibodies diluted in blocking buffer at 4 °C overnight with the
   following concentration: MuRF-1 (1:1000), Fbxo32 (1:2000), Myh1
   (1:2000), Sod2 (1:1000) (Abclonal, Woburn, MA, USA), NFκB p65 (1:1000),
   Sirt1 (1:1000), p-Akt (1:1000), Akt (1:1000) (Cell signalling
   Technology), Lamin B1 (1:1000), Pgc-1α (1:1000) (Genetex, Irvine, CA,
   USA), OXPHOS (1:2000) and α-Tubulin (1:5000) (Abcam). Membranes were
   then washed with TBST, and incubated with anti-mouse (Abcam) and
   anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody
   (Abcam) for 1h in TBST. An ECL chemiluminescent kit (T-Pro
   biotechnology) was used to visualize the protein bands, and the
   chemical luminescence of membranes was detected using an eBlot Touch
   Imager (eBlot Photoelectric Technology, Shanghai, China). The relative
   band intensity was quantified using ImageJ software. The data were
   expressed as fold change compared with the untreated group (n > 3).

2.16. Statistical analysis

   All data are performed with at least 3 independent experiment of
   biological replicates. Data are presented as mean ± standard deviation
   (SD). Statistical significance between treatments was analyzed using
   one-way analysis of variance (ANOVA) followed by Tukey's multiple
   comparisons test, unless specified otherwise. All analyses were
   conducted using GraphPad Prism 8.0. A p-value of less than 0.05 was
   considered statistically significant.

3. Results

3.1. LPS effectively induced myotube atrophy in differentiated C2C12 cells

   To model inflammation-induced muscle atrophy, C2C12 cells were
   differentiated in 96-well plates and treated with various
   concentrations of LPS for 48 h. Cell viability analysis indicated that
   only the highest dose of LPS exhibited cytotoxic effects ([82]Fig. 1A).
   In LPS-treated groups, significant reductions in myotube diameter and
   size were observed, accompanied by marked morphological changes. Even
   at non-cytotoxic doses, LPS-treated myotubes exhibited fragmented and
   shrunken morphologies, indicating that LPS-induced atrophy occurs
   independently of cell death ([83]Fig. 1B).

Fig. 1.

   [84]Fig. 1
   [85]Open in a new tab

   LPS-induced myotube atrophy and the protective effects of γ-tocotrienol
   in C2C12 cells. (A) Cell viability of C2C12 myotubes treated with
   various concentrations of LPS for 48 h, as measured by the CCK-8 assay.
   (B) Phase-contrast images of myotubes treated with LPS, showing
   reductions in diameter and size. Scale bar = 200 μm. (C) Cell viability
   of C2C12 myotubes treated with γ-tocotrienol at different
   concentrations for 48 h, measured by the CCK-8 assay. (D) Fluorescent
   images of myotubes pretreated with γ-tocotrienol and exposed to LPS,
   stained for nuclei (blue, DAPI) and Myh1 (green). Scale bar = 100 μm.
   (E) Western blot analysis of Myh1, MuRF-1, and Atrogin-1 protein
   expression in LPS-treated myotubes, with α-tubulin as the internal
   control. Data are presented as mean ± SD from three independent
   experiments.

   To evaluate the protective effects of γ-tocotrienol against LPS-induced
   muscle atrophy, myotubes differentiated for 5 days were pretreated with
   γ-tocotrienol at various concentrations before LPS exposure.
   γ-Tocotrienol demonstrated excellent tolerance in myotubes, even at
   high concentrations ([86]Fig. 1C). Pretreatment with γ-tocotrienol
   significantly preserved the morphological integrity of myotubes and
   prevented LPS-induced atrophy ([87]Fig. 1D). To confirm the occurrence
   of myotube atrophy in LPS-treated C2C12 cells, we analyzed the
   expression of the differentiation marker Myh1 (MyHC-IIx/d) and the
   atrophy markers MuRF-1 and Atrogin-1. LPS treatment led to a
   significant reduction in Myh1 expression, coupled with an upregulation
   of MuRF-1 and Atrogin-1, corroborating the observed morphological
   changes ([88]Fig. 1E). These findings validate that LPS effectively
   induces muscle atrophy in differentiated C2C12 cells and demonstrate
   the protective effects of γ-tocotrienol against this process.

3.2. γ-tocotrienol alleviates LPS-induced muscle atrophy

   To further assess the protective effects of γ-tocotrienol on
   LPS-induced myotube atrophy, differentiated C2C12 myotubes were
   pretreated with varying concentrations of γ-tocotrienol prior to LPS
   exposure. Specifically, 0.2 μM and 2 μM of γ-tocotrienol were added to
   the culture medium 6 h before a 48-h LPS treatment. Myotubes pretreated
   with γ-tocotrienol showed a dose-dependent reduction in susceptibility
   to LPS-induced damage, with the 2 μM concentration demonstrating a more
   pronounced protective effect compared to 0.2 μM. This was evidenced by
   improved cell morphology and the preservation of myotube diameter
   ([89]Fig. 2A and B). In contrast, myotubes exposed to LPS alone
   exhibited significant disruptions in myogenesis, characterized by
   reduced myotube diameter and structural disintegration ([90]Fig. 2B).

Fig. 2.

   [91]Fig. 2
   [92]Open in a new tab

   Protective effects of γ-tocotrienol against LPS-induced myotube atrophy
   in C2C12 cells. C2C12 cells (5000 cells/well) were differentiated in
   DMEM supplemented with 2 % horse serum (HS) and pretreated with various
   concentrations of γ-tocotrienol or vehicle on day 5
   post-differentiation for 6 h, followed by treatment with 1 μg/mL LPS or
   vehicle for 48 h. (A) Phase-contrast images of C2C12 myotubes under
   different treatments. Scale bar = 100 μm. (B) Quantification of myotube
   diameter using ImageJ software. (C) Western blot analysis of the
   differentiation marker (Myh1) and atrophy markers (MuRF-1 and
   Fbxo32/Atrogin-1) under different treatment conditions, with α-tubulin
   as the loading control. (D) Statistical analysis of protein expression.
   Data are presented as mean ± SD from three independent experiments.
   ∗p < 0.05, ∗∗p < 0.01 compared to the control group; #p < 0.05,
   ##p < 0.01 compared to the LPS-treated group.

   Western blot analysis supported these morphological findings, revealing
   increased Myh1 expression in γ-tocotrienol-treated groups and a lack of
   significant upregulation of atrophy markers MuRF-1 and Atrogin-1
   compared to the LPS-only group ([93]Fig. 2C and D). These results
   suggest that γ-tocotrienol effectively mitigates LPS-induced myotube
   atrophy in C2C12 cells, potentially by modulating proteasome-dependent
   protein degradation pathways.

3.3. γ-tocotrienol inhibits LPS-induced nuclear translocation of NF-κB

   Lipopolysaccharide (LPS) triggers an inflammatory response by
   activating nuclear factor kappa-light-chain-enhancer of activated B
   cells (NF-κB) and facilitating its translocation into the nucleus
   [[94]33]. To determine whether γ-tocotrienol mitigates myotube atrophy
   via NF-κB pathway modulation, differentiated myotubes were pretreated
   with γ-tocotrienol prior to LPS exposure. NF-κB p65 levels were
   assessed using immunoblotting and immunocytochemistry (ICC). LPS
   treatment significantly increased NF-κB p65 protein expression, while
   γ-tocotrienol pretreatment restored p65 levels in a dose-dependent
   manner ([95]Fig. 3A and B). ICC analysis revealed that LPS-treated
   myotubes exhibited increased NF-κB p65 expression with pronounced
   nuclear localization. In contrast, γ-tocotrienol pretreatment
   significantly inhibited NF-κB p65 nuclear translocation ([96]Fig. 3C).
   Additionally, γ-tocotrienol preserved myotube diameter and nuclear
   fusion index (NFI), calculated as the ratio of nuclei in multinucleated
   myotubes to total nuclei [[97]34] ([98]Fig. 3D).

Fig. 3.

   [99]Fig. 3
   [100]Open in a new tab

   Effects of γ-tocotrienol on NF- κB activation. (A) Western blot
   analysis of NF-κB p65 protein levels in myotubes pretreated with
   γ-tocotrienol at different concentrations prior to LPS exposure.
   α-Tubulin was used as the loading control. (B) Quantification of NF-κB
   p65 protein expression relative to α-tubulin. Data are presented as
   mean ± SD from three independent experiments. (C) Immunocytochemistry
   (ICC) analysis of NF-κB p65 (green) and Myh4 (red) in myotubes. DAPI
   (blue) was used to stain nuclei. Scale bar = 100 μm. (D) Quantification
   of myotube diameter and nuclear fusion index (NFI), calculated as the
   ratio of nuclei in myocytes with two or more nuclei to the total number
   of nuclei. Data are presented as mean ± SD. (E) Western blot analysis
   of NF-κB p65 protein levels in nuclear and cytosolic fractions of
   myotubes. Lamin B1 was used as the nuclear loading control, and
   α-tubulin as the cytosolic loading control. ∗p < 0.05, ∗∗p < 0.01
   compared to the control group; #p < 0.05; ##p < 0.01; ###p < 0.001
   compared to the LPS-treated group.

   Nuclear and cytosolic fractionation corroborated these findings,
   demonstrating elevated nuclear NF-κB p65 levels following LPS
   treatment, which were attenuated in γ-tocotrienol-pretreated cells
   ([101]Fig. 3E). These results indicate that γ-tocotrienol inhibits
   NF-κB activation and nuclear translocation, contributing to its
   protective effects against LPS-induced myotube atrophy.

3.4. Comparative protective effects of γ-tocotrienol and α-tocopherol against
LPS-induced myotube atrophy

   The protective effects of γ-tocotrienol and α-tocopherol on LPS-induced
   myotube atrophy were compared by pretreating C2C12 myotubes with each
   compound before LPS exposure. Morphological analysis revealed that both
   compounds mitigated LPS-induced atrophy, but γ-tocotrienol exhibited
   superior preservation of structural integrity ([102]Fig. 4A). Western
   blot analysis revealed that γ-tocotrienol significantly reduced the
   upregulation of atrophy markers, including Fbxo32/Atrogin-1, and
   maintained higher levels of Myh1, a differentiation marker, compared to
   α-tocopherol ([103]Fig. 4B and C). These findings highlight
   γ-tocotrienol's greater efficacy in protecting against both
   morphological and molecular hallmarks of LPS-induced myotube atrophy,
   suggesting its potential as a more effective therapeutic agent.

Fig. 4.

   [104]Fig. 4
   [105]Open in a new tab

   Differential effects of γ-tocotrienol and α-tocopherol on LPS-induced
   myotube atrophy (A) Effects of γ-tocotrienol and α-tocopherol on
   LPS-induced morphological changes in C2C12 myotubes. Scale
   bar = 100 μm. (B) Western blot analysis of atrophy-related protein
   expression, with α-tubulin as the loading control. (C) Quantification
   of protein expression relative to α-tubulin. Data are presented as
   mean ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01
   compared to the control group; #p < 0.05, ##p < 0.01 compared to the
   LPS-treated group.

3.5. Proteomic insights into γ-Tocotrienol's protective mechanisms

   To elucidate the molecular mechanisms underlying the protective effects
   of γ-tocotrienol and α-tocopherol against LPS-induced myotube atrophy,
   an isobaric label-based quantitative proteomics analysis was conducted.
   A total of 4,003 quantifiable protein groups were identified from
   23,902 peptides. Principal component analysis (PCA) revealed four
   distinct clusters, with γ-tocotrienol-treated myotubes exhibiting a
   protein expression profile more closely resembling the control group
   compared to α-tocopherol-treated myotubes ([106]Fig. 5A).

Fig. 5.

   [107]Fig. 5
   [108]Open in a new tab

   Proteomic and functional enrichment analysis of differentially
   expressed proteins in C2C12 myotubes treated with LPS and
   γ-tocotrienol. (A) Principal component analysis (PCA) of proteomic
   profiles under different treatments. (B) Heatmap and volcano plot of
   differentially expressed proteins identified by ANOVA (FDR = 0.01). (C)
   Overlap analysis of DEPs between the LPS-only group and the
   LPS + γ-tocotrienol group. (D) Protein-protein interaction (PPI)
   network of overlapping DEPs generated using STRING. Proteins
   downregulated in the LPS + γ-tocotrienol group are marked in blue,
   upregulated proteins in red, and mitochondria-related proteins are
   highlighted in a green box. (E) Functional enrichment analysis of
   upregulated (orange) and downregulated (purple-blue) proteins in the
   γ-tocotrienol vs. LPS group, using Enrichr with the Wikipathway 2023
   database.

   LPS treatment induced significant changes in protein expression, with
   172 proteins upregulated and 133 proteins downregulated compared to the
   vehicle control ([109]Table S1). Pathway enrichment analysis revealed
   that upregulated proteins were predominantly associated with
   pro-inflammatory responses, including C3, P2RX4, HMOX1, and NFKB2
   ([110]Table S3). In contrast, downregulated proteins were linked to
   extracellular matrix (ECM) organization, such as COL1A1 and ITGB1
   ([111]Table S4).

   γ-Tocotrienol pretreatment mitigated these effects, with 23 proteins
   upregulated and 49 downregulated compared to the LPS-only group.
   Notably, 27 proteins overlapped with those upregulated by LPS
   ([112]Fig. 5B, [113]Table S2, [114]Table S6). γ-Tocotrienol reversed
   LPS-induced proteomic changes by reducing the expression of oxidative
   stress-related proteins and enhancing those associated with ECM
   organization and energy metabolism ([115]Fig. 5E, [116]Table S5).
   Approximately one-third of the proteins upregulated by LPS were
   restored to normal levels following γ-tocotrienol pretreatment
   ([117]Fig. 5B).

   Protein-protein interaction and functional enrichment analyses revealed
   that γ-tocotrienol primarily restored proteins involved in
   mitochondrial functions, metal ion homeostasis, structural molecule
   activity, and myofibril assembly ([118]Fig. 5D). Notably, the
   downregulation of superoxide dismutase 2 (Sod2) expression in
   γ-tocotrienol-treated myotubes suggested reduced oxidative stress
   ([119]Fig. 5D, [120]Table S2).

   These findings highlight γ-tocotrienol's ability to modulate critical
   cellular pathways, particularly those related to oxidative stress and
   ECM integrity, thereby protecting against LPS-induced myotube atrophy.

3.6. γ-tocotrienol protects against LPS-induced mitochondrial dysfunction and
oxidative stress

   Gene set enrichment analysis (GSEA) revealed that pathways associated
   with mitochondrial fusion and apoptotic mitochondrial changes were
   significantly enriched among downregulated proteins in
   γ-tocotrienol-pretreated C2C12 myotubes, indicating a protective effect
   on mitochondrial integrity in response to LPS exposure ([121]Fig. 6A).
   JC-1 and MitoTracker staining further confirmed this protective effect,
   as γ-tocotrienol-pretreated myotubes maintained mitochondrial integrity
   and membrane potential despite LPS-induced damage ([122]Fig. 6B,
   [123]Fig. S1A). Immunostaining for translocase of outer mitochondrial
   membrane 20 (TOMM20) corroborated these findings, demonstrating that
   γ-tocotrienol mitigated LPS-induced reductions in mitochondrial mass
   and metabolic activity, thereby preventing mitochondrial damage-related
   myotube atrophy ([124]Fig. 6C).

Fig. 6.

   [125]Fig. 6
   [126]Open in a new tab

   γ-Tocotrienol protects C2C12 myotubes from atrophy by maintaining
   mitochondrial integrity. (A) Gene Set Enrichment Analysis (GSEA) of
   mitochondria-related pathways and the corresponding protein expression
   levels in each gene set. (B) Representative fluorescence images of JC-1
   dye staining for mitochondrial membrane potential. (C) Representative
   fluorescence images of TOMM20 staining for mitochondrial mass. Scale
   bar = 100 μm. Data are presented as mean ± SD from three independent
   experiments. ∗∗p < 0.01 compared to the control group; ##p < 0.05
   compared to the LPS-treated group.

   Given the established antioxidant properties of vitamin E isoforms,
   mitochondrial reactive oxygen species (mtROS) and total ROS levels were
   assessed. LPS treatment significantly elevated mtROS in C2C12 myotubes;
   however, γ-tocotrienol pretreatment effectively prevented mtROS
   accumulation, with effects comparable to those observed in
   α-tocopherol-treated cells ([127]Fig. 7A). A similar trend was observed
   for total ROS levels, where γ-tocotrienol significantly attenuated
   LPS-induced ROS accumulation ([128]Fig. S1B).

Fig. 7.

   [129]Fig. 7
   [130]Open in a new tab

   γ-Tocotrienol prevents LPS-induced mitochondrial oxidative stress and
   preserves mitochondrial respiration in C2C12 myotubes. (A)
   Mitochondrial superoxide levels were measured using MitoSOX staining
   and fluorescence microscopy. Representative images show (top) nuclear
   staining with DAPI, (middle) MitoSOX fluorescence, and (bottom) merged
   images of MitoSOX and DAPI. (B) Oxygen consumption rate (OCR) was
   measured using a Seahorse XFe24 analyzer. Sequential injections of
   oligomycin (1 μM), FCCP (3 μM), and rotenone/antimycin-A (1 μM) were
   added at the indicated time points (dashed lines). (C) Quantification
   of basal OCR, maximal respiration, non-mitochondrial oxygen
   consumption, ATP production OCR, coupling efficiency, and spare
   respiratory capacity. Data are presented as mean ± SEM from three
   independent experiments (n = 3). ∗p < 0.05, ∗∗p < 0.01 compared to the
   control group; #p < 0.05, ##p < 0.01 compared to the LPS-treated group.

   To further evaluate the impact of γ-tocotrienol on mitochondrial
   respiratory function, the oxygen consumption rate (OCR) was analyzed.
   LPS exposure led to a marked reduction in basal OCR, maximal
   respiration, and ATP production in C2C12 myotubes. Pretreatment with
   γ-tocotrienol or α-tocopherol effectively mitigated these LPS-induced
   reductions in OCR parameters ([131]Fig. 7B and C). Interestingly, while
   coupling efficiency and spare respiratory capacity were unaffected,
   non-mitochondrial oxygen consumption increased in myotubes pretreated
   with γ-tocotrienol or α-tocopherol.

3.7. γ-tocotrienol inhibits LPS‐induced muscle loss and muscle strength in
vivo

   To evaluate whether the in vitro findings could be replicated in an in
   vivo context, an animal study was performed using male C57BL/6 mice
   treated with LPS ([132]Fig. 8A). Mice were pretreated with different
   doses of γ-tocotrienol and α-tocopherol for four weeks prior to LPS
   administration. Following LPS injection, all groups except the
   untreated control exhibited a significant drop in body weight. However,
   body weight gradually recovered over the subsequent two weeks, with the
   LPS-only group showing the greatest weight loss at the study endpoint
   ([133]Fig. 8B). Pretreatment with either γ-tocotrienol or α-tocopherol
   effectively prevented LPS-induced muscle strength loss, as assessed by
   grip strength normalized to body weight ([134]Fig. 8C). Serum IL-6
   levels were also significantly reduced in the pretreated groups
   compared to the LPS-only group, displaying a dose-dependent effect
   ([135]Fig. 8D). Histological analysis revealed a reduction in muscle
   fiber cross-sectional area in the LPS group, whereas γ-tocotrienol
   pretreatment displayed slightly better protection than α-tocopherol,
   particularly in the high dose treatment ([136]Fig. 8E and F).

Fig. 8.

   [137]Fig. 8
   [138]Open in a new tab

   γ-tocotrienol effectively prevent LPS-induced myotube atrophy in vivo.
   (A) Experiment setting of the in vivo study using LPS-induced atrophy
   model (n = 8). (B) Percentage body weight changes across the 6-week
   study. (C) Left: Measurement of grip strength test before and after LPS
   administration; Right: Grip strength normalized to (D) Comparison of
   serum IL-6 levels between groups. (E) Raw figure and H&E-stained tissue
   section of the gastrocnemius muscle. Scale Bar: 100 μm. (F)
   Quantification of cross sectional area of muscle fibre using imageJ.
   (G) Relative expression of atrophy-related protein was quantified using
   Western Blot. Data represented the mean ± SD (n = 5–8). ∗p < 0.05,
   ∗∗p < 0.01 vs. control group (vehicle only); #p < 0.05, ##p < 0.01 vs.
   LPS-treated group.

3.8. γ-tocotrienol protects muscle degradation via Sirt1/Pgc-1α axis

   To further characterize LPS-induced muscle atrophy, the expression of
   the E3 ubiquitin ligases MuRF-1 and Atrogin-1 was analyzed in
   gastrocnemius muscle tissue. Consistent with in vitro results, LPS
   significantly upregulated both proteins, while pretreatment with
   γ-tocotrienol or α-tocopherol attenuated this increase ([139]Fig. 8G).
   The expression of NF-κB p65 and Foxo3a was also examined to explore the
   inflammatory response. LPS treatment significantly increased NF-κB p65
   expression, which was reduced by both forms of vitamin E, with
   γ-tocotrienol showing a slightly better inhibitory effect than
   α-tocopherol ([140]Fig. 9A). A similar result was also observed with
   Foxo3a, where LPS induced a significant upregulation, while
   pretreatment with γ-tocotrienol suppressed Foxo3a expression more
   effectively compared to α-tocopherol. As proteomics study found the
   protection effect of γ-tocotrienol were primarily mediated through
   mitochondrial maintenance, the expressions of the mitochondrial
   biogenesis marker Pgc-1α and its upstream regulator Sirt1 were
   investigated. Both proteins were notably upregulated only in the
   γ-tocotrienol-treated groups, with minimal increases observed in mice
   treated with α-tocopherol ([141]Fig. 9B). While Sirt1 expression was
   elevated in both γ-tocotrienol treatment groups, increased Pgc-1α
   expression was observed only in the higher-dose group.

Fig. 9.

   [142]Fig. 9
   [143]Open in a new tab

   γ-tocotrienol treatment protects LPS‐induced inflammatory response and
   mitochondrial dysregulation in muscle. Western blot analysis showing
   the protein expression of (A) SIRT1 and PGC-1α, (B) FOXO3a and NF-κB,
   (C) SOD2 and mitochondrial complex protein expression in response to
   γ-tocotrienol or α-tocopherol treatment in LPS-treated mice
   gastrocnemius muscle. α-tubulin was used as an internal control. (D)
   Immunohistological staining using Pgc-1α (green) and Sirt1 (red)
   antibodies on the harvested gastrocnemius muscle tissue section, with
   DAPI (blue) counterstaining. Scale Bar: 50 μm. Values are expressed as
   the mean ± SD (n = 5–8). ∗p < 0.05, ∗∗p < 0.01 vs. control group
   (vehicle only); #p < 0.05, ##p < 0.01 vs. LPS-treated group.

3.9. Mitochondrial damage protection by inhibition of mitochondrial ROS

   Additionally, the expression of Sod2 and mitochondrial oxidative
   phosphorylation (OXPHOS) proteins was also assessed. Western blot
   analysis found increased γ-tocotrienol pre-treated mice expressed a
   higher level of Sod2 expression compared to other groups. Additionally,
   the dysregulation in mitochondria caused by LPS was observed with
   diminishing levels of the OXPHOS respiratory complexes, while both
   treatment group protects the muscle from the impaired mitochondrial
   function, γ-tocotrienol has shown a more significant protection
   compared to α-tocopherol ([144]Fig. 9C). Immunohistological staining
   was also performed on harvested gastrocnemius muscle tissue sections.
   Both Pgc-1α and Sirt1 expression were reduced in the LPS-treated group.
   This decrease was prevented by pre-supplementation with either
   γ-tocotrienol or α-tocopherol. However, only the γ-tocotrienol group
   showed a significant increase in muscle fiber cross-sectional area.
   These results indicated that γ-tocotrienol is better in protecting
   cells from damaged mitochondrial function and muscle atrophy induced by
   LPS.

   These findings highlight γ-tocotrienol's ability to preserve
   mitochondrial function, reduce oxidative stress, and sustain cellular
   energy metabolism under LPS-induced stress, further supporting its role
   as a protective agent against inflammation-associated mitochondrial
   dysfunction and myotube atrophy.

4. Discussion

   Lipopolysaccharide (LPS) induces muscle atrophy through multiple
   mechanisms, primarily by triggering pro-inflammatory responses. LPS
   binds to toll-like receptors (TLRs), activating downstream signaling
   pathways such as NF-κB and MAPK, which drive the transcription of
   pro-inflammatory cytokines [[145]36]. These inflammatory responses
   impair muscle function and contribute to muscle atrophy [[146]37]. In
   this study, well-differentiated myotubes were treated with LPS to
   induce muscle atrophy, providing a model to examine the protective
   effects of γ-tocotrienol.

   Previous research has demonstrated that different isoforms of vitamin
   E, such as α-tocopherol and γ-tocotrienol, exhibit distinct effects on
   muscle health. While both α-tocopherol and γ-tocotrienol are potent
   antioxidants, γ-tocotrienol uniquely suppresses COX- and 5-LOX-mediated
   eicosanoids to reduce inflammation—an effect not observed with
   α-tocopherol [[147]18]. Tocotrienols, including γ-tocotrienol, have
   shown superior efficacy in reducing oxidative stress and inflammation
   in muscular dystrophy models, enhancing muscle stem cell function, and
   decreasing fibro-adipogenic progenitors [[148]38]. Furthermore,
   γ-tocotrienol has demonstrated greater potency than tocopherols in
   lowering cholesterol levels, reducing inflammatory cytokines, and
   mitigating oxidative stress markers, suggesting its potential to
   improve muscle health and overall well-being [[149]20].

   Proteomic analysis in this study revealed that γ-tocotrienol modulates
   key molecular pathways associated with muscle atrophy. It suppressed
   the expression of pro-atrophy markers such as E3 ubiquitin ligases
   MuRF-1 (Trim63) and Fbxo32 (Atrogin-1), while enhancing muscle fiber
   markers like myosin heavy chain 1 (Myh1). γ-Tocotrienol also
   downregulated oxidative stress response pathways and upregulated
   proteins involved in calcium transport regulation and extracellular
   matrix organization, further contributing to its protective effects.
   These findings highlight γ-tocotrienol as a promising therapeutic
   strategy for muscle atrophy, necessitating further research to
   elucidate the distinct mechanisms of α-tocopherol and γ-tocotrienol in
   muscle health.

   The measurement of oxygen consumption rate (OCR) revealed that
   γ-tocotrienol effectively mitigates LPS-induced mitochondrial
   dysfunction, with results comparable to the well-established
   antioxidant α-tocopherol. Interestingly, non-mitochondrial oxygen
   consumption was elevated in both γ-tocotrienol- and
   α-tocopherol-treated groups, potentially compensating for mitochondrial
   deficits. Although ATP production OCR levels were preserved, the
   reduced maximal OCR observed in γ-tocotrienol-treated groups suggests a
   protective metabolic shift that prioritizes ATP synthesis over maximal
   respiratory capacity [[150]39]. This higher non-mitochondrial OCR may
   reflect a broader shift in metabolic pathways, highlighting the need
   for further studies on the interaction between mitochondrial and
   non-mitochondrial respiration in γ-tocotrienol-mediated protection.

   LPS is known to disrupt zinc homeostasis by upregulating the expression
   of the metal transporter Slc39a14 (Zip14) through acute inflammatory
   responses [[151]40]. Prior studies have linked increased Slc39a14
   expression to muscle wasting [[152]41], and our findings are consistent
   with this observation. Proteomic analysis revealed significant
   upregulation of Slc39a14 in LPS-treated C2C12 myotubes, a phenomenon
   that was partially prevented by γ-tocotrienol pretreatment. qPCR
   analysis corroborated these findings, showing concurrent changes in
   metallothionein 1 (Mt1) expression across treatments ([153]Fig. S2).
   The disruption of zinc homeostasis is strongly associated with muscle
   atrophy, as evidenced by studies reporting Slc39a14 upregulation in
   cachectic muscles of mice with metastatic cancer [[154]42]. Notably,
   the upregulation of Slc39a14 in muscle cells has been proposed as a
   direct driver of muscle atrophy, rather than a secondary effect of
   muscle wasting.

   The animal study supports the protective effect of γ-tocotrienol
   against LPS-induced muscle atrophy. While both γ-tocotrienol and
   α-tocopherol were effective in preserving muscle strength and
   mitigating systemic inflammation, as evidenced by improved grip
   strength and reduced serum IL-6 levels, γ-tocotrienol demonstrated a
   more robust protective effect compared to α-tocopherol, particularly in
   preserving muscle fiber morphology and suppressing the expression of
   atrophy-related proteins MuRF-1 and Atrogin-1. While Western blot
   analysis showed that Akt activation did not significantly differ
   between groups ([155]Fig. S6), Akt phosphorylation is known to be
   transient, and previous studies have suggested that LPS-induced muscle
   atrophy in vivo may occur independently of the Akt pathway [[156]37].
   Therefore, the enhanced protective effect of γ-tocotrienol against
   muscle atrophy was examined through alternative mechanisms,
   particularly the key regulators of mitochondrial biogenesis, Sirt1 and
   Pgc-1α. Indeed, γ-tocotrienol significantly upregulated these two
   proteins, suggesting that its protective role is closely linked to
   mitochondrial preservation These results are consistent with the in
   vitro findings and proteomic data, suggesting that γ-tocotrienol
   confers muscle-protective effects in part through maintenance of
   mitochondrial function.

   Sirt1 and Pgc-1α are known to regulate mitochondrial biogenesis and
   overall muscle function. In skeletal muscle, the Sirt1/Pgc-1α axis
   contributes to the maintenance of mitochondrial content and function
   and also protects muscle fibers from oxidative stress and atrophy
   [[157]43]. One previous study has shown that a tocotrienol-rich
   fraction can indeed upregulate this pathway to ameliorate skeletal
   muscle damage in diabetic mice [[158]19]. Vitamin E has also been shown
   to upregulate Pgc-1α under normal conditions [[159]44]. Although
   α-tocopherol did restore the affected Pgc-1α expression to some extent
   following LPS treatment, the present study demonstrated that
   γ-tocotrienol induced a significantly higher upregulation.
   Immunohistostaining also observed that LPS treatment reduced the
   expression of Sirt1 and Pgc-1α in gastrocnemius muscle, while
   pre-supplementation with either γ-tocotrienol or α-tocopherol was able
   to prevent this decline, suggesting that both compounds exert
   protective effects against LPS-induced downregulation of mitochondrial
   regulatory proteins, which is consistent with the in vitro findings.
   However, only γ-tocotrienol supplementation resulted in a significant
   increase in muscle fiber cross-sectional area. This might indicate that
   while both compounds can preserve molecular markers of mitochondrial
   function, γ-tocotrienol can provide better myoprotective or anabolic
   effects which is weaker in α-tocopherol treatment. The improvement in
   muscle fiber size observed with γ-tocotrienol may be related to
   enhanced mitochondrial biogenesis mediated by the enhanced Sirt1/Pgc-1α
   axis, leading to improved energy metabolism and muscle maintenance
   under inflammatory stress.

   In the animal study, Sod2 was markedly downregulated by LPS, while
   γ-tocotrienol pretreatment significantly prevented this suppression.
   Interestingly, proteomic data in differentiated C2C12 myotubes shown a
   reduction in Sod2 following γ-tocotrienol. This may due to the
   differences of cell culture and animal model, where in systemic
   inflammation plays an important role in producing excessive ROS
   [[160]45]. The elevated Sod2 expression suggested that γ-tocotrienol
   supplementation could respond to mitochondrial ROS in vivo more
   effectively compared to the relatively homogenous cell culture
   environment. This has shown the importance to consider the
   physiological complexity of whole organisms when studying antioxidant
   responses of γ-tocotrienol.

   One of the limitations of the present study is the lack of discussion
   on the deacetylation activity of Sirt1. While the study focused on the
   protein expression levels of Sirt1 and Pgc-1α, it is well established
   that Sirt1 modulates cellular function through the deacetylation of key
   transcriptional regulators, including Pgc-1α and NF-κB [[161]46].
   Deacetylation of Pgc-1α enhances its transcriptional activity and
   promotes mitochondrial biogenesis, while deacetylation of the p65
   subunit of NF-κB reduces its pro-inflammatory effect [[162]47]. Future
   work measuring acetylation levels to study the post-translational
   modifications of these proteins may provide a more complete
   understanding of how γ-tocotrienol modulates these signaling cascades
   to protect against muscle atrophy.

   Another important consideration in the application of γ-tocotrienol is
   its bioavailability, which is known to be affected by α-tocopherol.
   Studies have shown that α-tocopherol, commonly included in supplements,
   can reduce plasma concentrations of γ-tocotrienol, thereby impairing
   its efficacy [[163]48]. When supplemented without tocopherols,
   δ-tocotrienol demonstrates superior bioavailability and can be
   metabolically converted into other tocotrienols and tocopherols,
   enhancing its potential to combat age-related and chronic disorders
   [[164]49]. Besides, other research indicates that α-tocopherol exhibits
   significantly higher oral bioavailability than γ-tocotrienol due to
   differences in intestinal permeability [[165]50], whereas tocotrienols
   are more readily taken up into cells compared to tocopherols [[166]51].
   In the present study, γ-tocotrienol demonstrated potent biological
   activity despite its faster clearance in cell culture studies,
   suggesting that its efficacy is not solely dependent on intracellular
   concentration ([167]Fig. S5). The in vivo study clearly demonstrated
   biological efficacy, even though LC-MS/MS analysis found lower
   intracellular concentrations of γ-tocotrienol in C2C12 cells after 48 h
   of treatment. This may be due to its preferential accumulation in
   specific tissues or its ability to modulate key cellular pathways at
   lower concentrations [[168]52]. These observations underscore the
   importance of considering both bioavailability and tissue-specific
   efficacy when evaluating tocotrienol-based interventions and highlight
   the need for optimized formulations of vitamin E isoforms. Lastly, this
   study was limited to a male-only cohort, which restricts the
   generalizability of the findings. Previous research has demonstrated
   that sex-specific differences influence vitamin E metabolism
   [[169]33,[170]53] and susceptibility to muscle atrophy
   [[171]34,[172]54]. Therefore, while the present results highlight the
   therapeutic potential of γ-tocotrienol, future studies incorporating
   both male and female subjects are warranted to account for sex-specific
   biological differences and to ensure broader translational relevance.

5. Conclusion

   Overall, this study demonstrated that γ-tocotrienol effectively
   prevents the onset of myotube atrophy induced by LPS in vitro and in
   vivo. LPS treatment of C2C12 myotubes and male mice led to
   characteristic atrophy symptoms, including reduced myofiber diameter
   and muscle strength. Notably, pre-treatment with γ-tocotrienol, in
   contrast to α-tocopherol, significantly inhibited the LPS-induced
   upregulation of the inflammatory cytokine NF-κB and atrophy-associated
   proteins MuRF-1 and Atrogin-1, while increasing the expression of
   mitochondrial biogenesis markers Sirt1 and Pgc-1α. Additionally,
   γ-tocotrienol effectively reduced ROS accumulation and the expression
   of Slc39a14, mitigating oxidative stress and protecting against
   mitochondrial damage. These findings provide evidence that
   γ-tocotrienol is a promising bioactive compound with muscle-protective
   properties, supporting its potential use as a dietary intervention to
   prevent inflammation-induced muscle wasting.

CRediT authorship contribution statement

   Jun Yi Chong: Writing – original draft, Methodology, Investigation.
   Tsui-Chin Huang: Methodology, Investigation, Formal analysis.
   Sheng-Ming Chueh: Methodology, Formal analysis. Cheng-Yi Ma:
   Methodology. Tzu-Ting Kuo: Methodology. Jia-Jun He: Formal analysis.
   Yii-Jwu Lo: Formal analysis. Kuan-Chieh Peng: Formal analysis. Mohamed
   Ali: Resources. Hsin-Yi Chang: Writing – review & editing, Supervision,
   Resources, Project administration, Funding acquisition,
   Conceptualization. Shih-Min Hsia: Writing – review & editing,
   Supervision, Resources, Project administration, Conceptualization.

Ethical approval

   Not applicable.

Declaration of competing interest

   The authors have declared no conflict of interest.

Acknowledgments