Gardenia Fruit and Its Active Compound Crocin Protect Against Dexamethasone‐Induced Muscle Atrophy via Nrf2 Activation

Glucocorticoids such as dexamethasone (DEX) are commonly used clinically but can induce skeletal muscle atrophy with prolonged or high-dose exposure. Trichosanthes kirilowii Maximowicz (TK), a traditional medicinal herb, has known pharmacological effects, but its impact on muscle atrophy remains unclear. This study investigated the anti-atrophic potential of TK in DEX-induced muscle atrophy models. In C2C12 cells, TK improved cell viability and restored myotube diameter and number, downregulated atrophy markers MuRF-1 and Atrogin-1, and upregulated myogenic markers MyoD, MyoG, and MHC. TK also restored Sirtuin 1 (SIRT1) expression suppressed by DEX, and inhibition of SIRT1 with EX527 reduced TK’s beneficial effects, indicating a SIRT1–dependent mechanism. TK rescued DEX-induced reductions in phosphorylated AKT and mTOR, supporting its role in muscle anabolism. In vivo, oral TK administration attenuated DEX-induced weight loss, preserved gastrocnemius muscle mass, and improved motor performance. Molecular docking analysis revealed that key TK-derived compounds, including 5-dehydrokarounidiol, steryl glucoside, and isomultiflorenol, exhibited moderate binding affinity (− 6.3 to − 6.6 kcal/mol) toward the allosteric site of SIRT1. These findings suggest that TK protects against muscle atrophy by modulating SIRT1–related anabolic and catabolic pathways. TK may offer therapeutic potential for sarcopenia and glucocorticoid-induced muscle wasting.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-28257-2.

A simple, feasible and effective method of ultra performance liquid chromatography (UPLC) coupled with photo diode array (PDA) were established for fingerprint analysis and simultaneous quantification of three major classes of constitutions including iridoid glycosides, crocins and organic acids of fructus Gardeniae. Extraction method was optimized as 75% methanol ultrasonic extraction for 30 min. Acetonitrile-water (containing 0.2% formic acid) gradient elution on Waters Acquity BEH C18 column (50 × 2.1 mm, 1.7 μm) was used to obtain good chromatographic resolution. 24 characteristic peaks were comprised in the fingerprint common pattern. Among them, seven marked components,geniposide, shanzhiside methyl ester, geinpin, geniposidic acid, crocin I, crocin II, and chlorogenic acid, were quantified. Similarity evaluation, principal component analysis andhierachical cluster analysis were applied to demonstrate the distinction. It was concluded that chemical components of Gardenia jasminoides Eills and Gardenia jasminoides var. radicans Makino from different origins were similar. Other than determination of the contentof geniposide and crocin I, comparison of fingerprint atlas could be considered as a suitable quality control method for fructus Gardeniae. Key words: Fructus Gardeniae, chromatographic fingerprint, multi-component quantification, principal component analysis, hierachical cluster analysis.

Sarcopenia manifests as muscle atrophy and loss that is complicated with malignancy. This study explored the mechanism of extracellular vesicles (EVs) in multiple myeloma (MM) with sarcopenia.SP2/0 conditioned medium (CM) was collected to isolate SP2/0-EVs. C2C12 cells were incubated with SP2/0 CM or SP2/0-EVs. ROS, TNF-α, IL-6, MuRF1 and MyHC levels were detected by DCF-DA fluorescent probe, ELISA, and Western blot. GW4869 was used to inhibit EV secretion in SP2/0 to confirm its effect on muscle atrophy. Serum was collected from MM patients with or without sarcopenia to detect RAGE mRNA expression. SP2/0 cells were transfected with RAGE siRNA and C2C12 cells were treated with the isolated si-RAGE-EVs or/and TLR4 agonist. SP2/0 tumor-bearing mouse model was established. Healthy mice and SP2/0-tumor bearing mice were treated with SP2/0-EVs or si-RAGE-EVs.SP2/0 CM or SP2/0-EVs stimulated ROS, inflammatory responses, and myotube atrophy in C2C12 cells. GW4869 blocked EV secretion and the effects of SP2/0 CM. RAGE mRNA expression in serum EVs was increased in MM&Sarcopenia patients and RAGE knockdown in SP2/0-EVs partially nullified SP2/0-EVs' effects. SP2/0-EVs activated the TLR4/NF-κB p65 pathway by translocating RAGE. SP2/0-EVs-derived RAGE elevated ROS production, inflammation, and myotube atrophy in C2C12 cells and caused muscle loss in SP2/0 tumor-bearing mice by activating the TLR4/NF-κB p65 pathway. SP2/0-EVs partially recapitulated muscle loss in healthy mice.SP2/0-EVs-derived RAGE increased ROS production, inflammation, and myotube atrophy in MM through TLR4/NF-κB p65 pathway activation.

Doxorubicin, a conventional chemotherapeutic agent prescribed for cancer, causes skeletal muscle atrophy and adversely affects mobility and strength. Given that doxorubicin-induced muscle atrophy is attributable primarily to oxidative stress, its effects could be mitigated by antioxidant-focused therapies; however, these protective therapeutic targets remain ambiguous. The aim of this study was to demonstrate that doxorubicin triggers severe muscle atrophy via upregulation of oxidative stress (4-hydroxynonenal and malondialdehyde) and atrogenes (atrogin-1/MAFbx and muscle RING finger-1) in association with decreased expression of the antioxidant enzyme extracellular superoxide dismutase (EcSOD), in cultured C2C12myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein elevated EcSOD levels on the cellular membrane of cultured myotubes, consequently inhibiting doxorubicin-induced oxidative stress and myotube atrophy. Furthermore, doxorubicin treatment reduced interleukin-1β (IL-1β) mRNA expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment increased EcSOD protein expression on the myotube membrane. Notably, transient IL-1β treatment of cultured myotubes and local administration in mouse skeletal muscle attenuated doxorubicin-induced muscle atrophy, which was associated with increased EcSOD expression. Collectively, these findings reveal that the regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a potential therapeutic approach to counteract the muscle atrophy mediated by doxorubicin and oxidative stress. KEY POINTS: Doxorubicin, a commonly prescribed chemotherapeutic agent for patients with cancer, induces severe muscle atrophy owing to increased expression of oxidative stress; however, protective therapeutic targets are poorly understood. Doxorubicin induced muscle atrophy owing to increased expression of oxidative stress and atrogenes in association with decreased protein expression of extracellular superoxide dismutase (EcSOD) in cultured C2C12myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein increased EcSOD levels on the cellular membrane of cultured myotubes, resulting in inhibition of doxorubicin-induced oxidative stress and myotube atrophy. Doxorubicin treatment decreased interleukin-1β (IL-1β) expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment in vivo and in vitro increased EcSOD protein expression and attenuated doxorubicin-induced muscle atrophy. These findings reveal that regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a possible therapeutic approach for muscle atrophy mediated by doxorubicin and oxidative stress.

Skeletal muscle atrophy occurs when protein degradation exceeds protein synthesis and is associated with increased circulating glucocorticoid levels. Salvia plebeia R.Br. (SPR) has been used as herbal remedy for a variety of inflammatory diseases and has various biological actions such as antioxidant and anti-inflammatory activities. However, there are no reports on the effects of SPR and its bioactive components on muscle atrophy. Herein, we investigated the anti-atrophic effect of SPR and rosmarinic acid (RosA), a major compound of SPR, on dexamethasone (DEX)-induced skeletal muscle atrophy in C2C12 myotubes. Myotubes were treated with 10 μM DEX in the presence or absence of SPR or RosA at different concentrations for 24 h and subjected to immunocytochemistry, western blot, and measurements of ROS and ATP levels. SPR and RosA increased viability and inhibited protein degradation in DEX-treated C2C12 myotubes. In addition, RosA promoted the Akt/p70S6K/mTOR pathway and reduced ROS production, and apoptosis. Furthermore, the treatment of RosA significantly recovered SOD activity, autophagy activity, mitochondrial contents, and APT levels in DEX-treated myotubes. These findings suggest that SPR and RosA may provide protective effects against DEX-induced muscle atrophy and have promising potential as a nutraceutical remedy for the treatment of muscle weakness and atrophy.

Palmitate-Induced Inflammation and Myotube Atrophy in C2C12 Cells Are Prevented by the Whey Bioactive Peptide, Glycomacropeptide

Muscle atrophy, commonly triggered by glucocorticoids such as dexamethasone (DEX), involves increased protein degradation via the ubiquitin-proteasome system. Recent findings suggest that iron imbalance can also induce muscle atrophy. However, there have been no reports indicating that DEX causes intracellular iron imbalance leading to muscle atrophy. This study evaluated whether DEX causes iron imbalance-mediated muscle atrophy and whether deferoxamine (DFO), an iron chelator, can protect against DEX-induced muscle atrophy, exploring the underlying mechanisms in vitro and in vivo. Differentiated C2C12 myotubes were exposed to DEX, with or without DFO, to evaluate morphological changes, expression of muscle-specific ubiquitin ligases (atrogin-1 and MuRF1), and related signaling pathways via quantitative reverse transcription polymerase chain reaction, Western blotting, and immunocytochemistry. Intracellular iron accumulation was quantified using fluorescence imaging. Additionally, C57BL/6J mice were administered intraperitoneal injections of DEX, with or without DFO, every other day for 12 days. Muscle function was assessed by grip strength, and muscle mass and fiber size were measured histologically. DEX significantly induced muscle atrophy in C2C12 myotubes, elevating intracellular iron and upregulating atrogin-1 and MuRF1 via increased nuclear translocation of FOXO3a and expression of KLF15. DFO treatment prevented these effects by restoring the iron balance, enhancing AKT phosphorylation, inhibiting FOXO3a nuclear translocation, and reducing KLF15 expression. Consistently, animal experiments demonstrated that DFO administration effectively preserved grip strength, tibialis anterior muscle mass, and muscle fiber size in DEX-treated mice. Furthermore, DFO treatment restored insulin-like growth factor 1 and myostatin expression levels altered by DEX. DFO effectively ameliorates DEX-induced muscle atrophy by modulating the AKT/FOXO3a and KLF15 signaling pathways and restoring the intracellular iron balance. These findings highlight DFO as a potential therapeutic agent for glucocorticoid-induced muscle atrophy.

Introduction: Protein-energy waste (PEW) is a common complication in patients with chronic kidney disease (CKD), among which skeletal muscle atrophy is one of the most important clinical features of PEW. Pyroptosis is a type of proinflammatory, programmed cell death associated with skeletal muscle disease. Irisin, as a novel myokine, has attracted extensive attention for its protective role in the complications associated with CKD, but its role in muscle atrophy in CKD is unclear. Methods: Palmitic acid (PA)-induced muscular atrophy was evaluated by a reduction in C2C12 myotube diameter. Muscle atrophy model was established in male C57BL/6J mice treated with 0.2% adenine for 4 weeks and then fed a 45% high-fat diet. Blood urea nitrogen and creatinine levels, body and muscle weight, and muscle histology were assessed. The expression of carnitine palmitoyltransferase 1A (CPT1A) and pyroptosis-related protein was analysed by Western blots or immunohistochemistry. The release of IL-1β was detected by enzyme-linked immunosorbent assay. Results: In this study, we showed that PA-induced muscular atrophy manifested as a reduction in C2C12 myotube diameter. During this process, PA can also induce pyroptosis, as shown by the upregulation of NLRP3, cleaved caspase-1 and GSDMD-N expression and the increased IL-1β release and PI-positive cell rate. Inhibition of caspase-1 or NLRP3 attenuated PA-induced pyroptosis and myotube atrophy in C2C12 cells. Importantly, irisin treatment significantly ameliorated PA-induced skeletal muscle pyroptosis and atrophy. In terms of mechanism, PA upregulated CPT1A, a key enzyme of fatty acid oxidation (FAO), and irisin attenuated this effect, which was consistent with etomoxir (CPT1A inhibitor) treatment. Moreover, irisin improved skeletal muscle atrophy and pyroptosis in adenine-induced mice by regulating FAO. Conclusion: Our study firstly verifies that pyroptosis is a novel mechanism of skeletal muscle atrophy in CKD. Irisin ameliorates skeletal muscle atrophy by inhibiting FAO and pyroptosis in CKD, and irisin may be developed as a potential therapeutic agent for the treatment of muscle wasting in CKD patients.

Skeletal muscle atrophy is characterized by reduced muscle function and size. Oxidative stress contributes to muscle atrophy but can be treated with antioxidants. This study investigated the antioxidant activity of a castor oil plant leaf (Ricinus communis L.) extract (RC) and its effects on muscle atrophy. Rutin was identified as the major compound among the thirty compounds identified in RC via LC-MS/MS and was found to inhibit dexamethasone (DEX)-induced muscle atrophy and mitochondrial oxidative stress. Rutin-rich RC showed DPPH and ABTS radical scavenging activities and efficiently reduced the DEX-induced myotube atrophy and mitochondrial oxidative damage in C2C12 cells. RC supplementation prevented the loss of muscle function and muscle mass in DEX-administered mice and ameliorated DEX-induced oxidative stress via Nrf2 signaling. Taken together, both RC and rutin ameliorated muscle atrophy and helped in maintaining redox homeostasis; hence, rutin-rich RC could be a promising functional food that is beneficial for muscle health.

Fluoxetine, an antidepressant known as a selective 5-hydroxytryptamine reuptake inhibitor (SSRI), can cause side effects such as muscle atrophy with long-term use, but the mechanism is not fully understood. Geniposide (GPS) and geniposidic acid (GPSA), the main components of Gardenia jasminoides fruit, have been shown to have biological activity in disease prevention, but their role in preventing FXT-related side effects such as muscle atrophy remains unclear. The process of muscle atrophy is a complex physiological mechanism involving the balance of protein synthesis and catabolism. In this study, we hypothesized that FXT may suppress hypertrophy signaling and activate the atrophy mechanisms, resulting in proteolysis and reduced protein synthesis, while geniposide (GPS) and geniposide acid (GPSA) may be beneficial in improving muscle weakness caused by FXT. The C2C12 cell model was used to examine the expression of hypertrophy signaling (PI3K, Akt, and mTOR) and protein break signals (FOXO, MuRF-1, and MyHC). Our data indicated that FXT inhibited MyHC and promoted MuRF-1 protein expression by downregulating the signaling pathways of p-ERK1/2, p-Akt, p-mTOR, and p-FOXO, resulting in a decrease in differentiation and myotube formation in C2C12 muscle cells, which further resulted in muscle atrophy. However, GPS and GPSA can positively regulate the atrophy mechanism induced by FXT in muscle cells, thereby ameliorating the imbalance in muscle synthesis. In conclusion, GPS and GPSA have the potential to attenuate the muscle loss caused by long-term FXT administration, diseases, or the aging process.

Skeletal muscle atrophy is associated with many diseases including cancer, inflammatory diseases, neuromuscular diseases, and acute critical illness. Justicia procumbens L. has been used as a herbal remedy, but the pharmacological effect of J. procumbens on muscle atrophy has not yet been reported. Herein, we investigate the anti-atrophic effect of the n-butanol fraction of J. procumbens (JPBuFr) on dexamethasone (DEX)-induced muscle atrophy in C2C12 myotubes. The myotubes diameter, MHC positive area, ROS production, and mitochondria contents were observed under a fluorescence microscope, and various proteins related to degradation or synthesis were analyzed by western blots. JPBuFr significantly attenuated a reduction of myotube diameter, mitochondrial content, ATP level, myosin heavy chain, and myogenin expression induced by DEX. Furthermore, co-treatment of DEX and JPBuFr not only increased phosphorylation of Akt, mTOR, and p70S6K proteins but also decreased reactive oxygen species production and expression of protein degradation factors (MuRF1, Atrogin-1, FoxO3a) compared to DEX treatment. These results suggest that JPBuFr may provide potential protective effects against muscle atrophy, giving it potential for the development of anti-atrophic health functional foods.

Activation of Nrf2 signaling as a common treatment of neurodegenerative diseases.

20(S)-ginsenoside Rg3 promotes myoblast differentiation and protects against myotube atrophy via regulation of the Akt/mTOR/FoxO3 pathway

In the previous paper, thin layer and high performance liquid chromatographic methods for the determination of geniposide were reported. In this work, separation and determination of natural colors in gardenia fruit extracts were investigated.Natural colors, crocin and crocetin, and geniposide (which is a contaminant) could be simultaneously separated by reversed-phase high performance liquid chromatography (HPLC) on a Nucleosil 7 C18 column with methyl alcohol-water as the eluant (Fig. 1).The natural yellow colors in gardenia fruit could be extracted with methyl alcohol, 50v/v% aqueous methyl and ethyl alcohols or water. The color intensities (absorbances at 438nm, as crocin) of the extracts became maximum after about 24 hours. Some of the extracts showed a slight loss of colors on subsequent extraction (Fig. 2). The color intensity of the extract with methyl alcohol or 50v/v% aqueous methyl and ethyl alcohols was higher than that of the extract with water, and the ratio of geniposide/color intensity (G/C) in the former extract was 0.4, less than that in the latter extract (Table 2).The color intensity of commercial gardenia fruit extract color was expressed as E1%1cm value at 438nm, as crocin. The values obtained for eighteen samples from different companies were in the range of 4.9 to 175, and the ratios of G/C in 12 of the 18 samples were 0.4-1.6 (Table 3). These results suggested that the samples may have been manufactured by extraction with the above solvents or water.The natural colors in the extract and the samples were analyzed by HPLC. Several peaks (2-4 peaks) besides crocin were detected on the chromatograms, but crocetin was not detected (except in sample Nos. 2 and 11). In sample Nos. 2 and 11, crocin was converted into crocetin during alkaline hydrolysis [Fig. 6 (Sample 2) and Fig. 7 (Sample 2)].The peak which appeared at the retention time of about 20min on HPLC was observed on all the chromatograms (Figs. 3, 4 and 7). The color corresponding to this peak may occur together with crocin in gardenia fruit; its structure might be analogous to those of crocin and crocetin.

An herbal formula consisting of Schisandra chinensis (Turcz.) Baill, Lycium chinense Mill and Eucommia ulmoides Oliv alleviates disuse muscle atrophy in rats