Muscle Atrophy F-box Protein Research Articles

Transcutaneous carbon dioxide suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Cancer cachexia causes skeletal muscle atrophy, impacting the treatment and prognosis of patients with advanced cancer, but no treatment has yet been established to control cancer cachexia. We demonstrated that transcutaneous application of carbon dioxide (CO2) could improve local blood flow and reduce skeletal muscle atrophy in a fracture model. However, the effects of transcutaneous application of CO2 in cancer-bearing conditions are not yet known. In this study, we calculated fat-free body mass (FFM), defined as the skeletal muscle mass, and evaluated the expression of muscle atrophy markers and uncoupling protein markers as well as the cross-sectional area (CSA) to investigate whether transcutaneous application of CO2 to skeletal muscle could suppress skeletal muscle atrophy in cancer-bearing mice. Human oral squamous cell carcinoma was transplanted subcutaneously into the upper dorsal region of nude mice, and 1 week later, CO2 gas was applied to the legs twice a week for 4 weeks and FFM was calculated by bioimpedance spectroscopy. After the experiment concluded, the quadriceps were extracted, and muscle atrophy markers (muscle atrophy F-box protein (MAFbx), muscle RING-finger protein 1 (MuRF-1)) and uncoupling protein markers (uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3)) were evaluated by real-time polymerase chain reaction and immunohistochemical staining, and CSA by hematoxylin and eosin staining. The CO2-treated group exhibited significant mRNA and protein expression inhibition of the four markers. Furthermore, immunohistochemical staining showed decreased MAFbx, MuRF-1, UCP2, and UCP3 in the CO2-treated group. In fact, the CSA in hematoxylin and eosin staining and the FFM revealed significant suppression of skeletal muscle atrophy in the CO2-treated group. We suggest that transcutaneous application of CO2 to skeletal muscle suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Umami taste receptor suppresses cancer cachexia by regulating skeletal muscle atrophy in vivo and in vitro.

The umami taste receptor (TAS1R1/TAS1R3) is endogenously expressed in skeletal muscle and is involved in myogenesis; however, there is a lack of evidence about whether the expression of the umami taste receptor is involved in muscular diseases. This study aimed to elucidate the effects of the umami taste receptor and its mechanism on muscle wasting in cancer cachexia using in vivo and in vitro models. The Lewis lung carcinoma-induced cancer cachexia model was used in vivo and in vitro, and the expressions of umami taste receptor and muscle atrophy-related markers, muscle atrophy F-box protein, and muscle RING-finger protein-1 were analyzed. Results showed that TAS1R1 was significantly downregulated in vivo and in vitro under the muscle wasting condition. Moreover, overexpression of TAS1R1 in vitro in the human primary cell model protected the cells from muscle atrophy, and knockdown of TAS1R1 using siRNA exacerbated muscle atrophy. Taken together, the umami taste receptor exerts protective effects on muscle-wasting conditions by restoring dysregulated muscle atrophy in cancer cachexia. In conclusion, this result provided evidence that the umami taste receptor exerts a therapeutic anti-cancer cachexia effect by restoring muscle atrophy.

Beef peptides mitigate skeletal muscle atrophy in C2C12 myotubes through protein degradation, protein synthesis, and the oxidative stress pathway.

This study aimed to investigate the potential of beef peptides (BPs) in mitigating muscle atrophy induced by dexamethasone (DEX) with underlying three mechanisms in vitro (protein degradation, protein synthesis, and the oxidative stress pathway). Finally, the anti-atrophic effect of BPs was enhanced through purification and isolation. BPs were generated using beef loin hydrolyzed with alcalase/ProteAX/trypsin, each at a concentration of 0.67%, followed by ultrafiltration through a 3 kDa cut-off. BPs (10-100 μg mL-1) dose-dependently counteracted the DEX-induced reductions in myotube diameters, differentiation, fusion, and maturation indices (p < 0.05). Additionally, BPs significantly reduced FoxO1 protein dephosphorylation, thereby suppressing muscle-specific E3 ubiquitin ligases such as muscle RING-finger containing protein-1 and muscle atrophy F-box protein in C2C12 myotubes at concentrations exceeding 25 μg mL-1 (p < 0.05). BPs also enhanced the phosphorylation of protein synthesis markers, including mTOR, 4E-BP1, and p70S6K1, in a dose-dependent manner (p < 0.05) and increased the mRNA expression of antioxidant enzymes. Fractionated peptides derived from BPs, through size exclusion and polarity-based fractionation, also demonstrated enhanced anti-atrophic effects compared to BPs. These peptides downregulated the mRNA expression of primary muscle atrophy markers while upregulated that of antioxidant enzymes. Specifically, peptides GAGAAGAPAGGA (MW 924.5) and AFRSSTKK (MW 826.4) were identified from fractionated peptides of BPs. These findings suggest that BPs, specifically the peptide fractions GAGAAGAPAGGA and AFRSSTKK, could be a potential strategy to mitigate glucocorticoid-induced skeletal muscle atrophy by reducing the E3 ubiquitin ligase activity.

Decreased Expression of Mitochondrial Calcium Uptake Protein 1 Leads to Skeletal Muscle Dysfunction in Septic Mice

To observe the effect of sepsis on skeletal muscle function and to explore the role of skeletal muscle mitochondrial calcium uptake protein 1 (MICU1). A total of 40 specific-pathogen-free (SPF) healthy male C57BL/6J mice were randomly assigned to 4 groups, a sham operation group (Sham group, n=8), a sepsis modeling 6 h group (cecal ligation and puncture [CLP]-6 h group, n=10), a sepsis modeling 12 h group (CLP-12 h group, n=10), and a sepsis modeling 24 h group (CLP-24 h, n=12). The sepsis model was established by CLP. Mice in the Sham group only underwent laparotomic exploration of the cecum. Another 20 SPF mice were selected. The tibialis anterior muscle on one side was empty-transfected with adeno-associated virus (AAV) as controls (AAV-C), and the tibialis anterior muscle on the other side was transfected with AAV to enhance MICU1 expression (AAV-M). The mice were randomly assigned to two groups, a sham operation group (AAV-C-Sham and AAV-M-Sham, n=8) and a sepsis model 24 h group (AAV-C-CLP and AAV-M-CLP, n=12). The grip strength and compound muscle action potential (CMAP) of the tibialis anterior muscle were measured in each group at the corresponding time points. The levels of inflammatory factors, including tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), in the skeletal muscle were measured by ELISA. The morphological changes of skeletal muscle cells were observed through H&E staining. The expression levels of MICU1 and muscle atrophy-related proteins, including muscle RING-finger containing protein 1 (MuRF1) and muscle atrophy Fbox protein (MAFbx), were determined by Western blot. The expression levels of MICU1 mRNA in skeletal muscle were determined by RT-qPCR. Compared with mice in the Sham group, mice in the CLP group showed decreased body weight ( P<0.05); their grip strength decreased with the prolongation of CLP modeling time ( P<0.05); the amplitude of CMAP decreased, showing prolonged duration and latency ( P<0.05); the expression levels of inflammatory factors, including TNF-α and IL-6, in skeletal muscle increased gradually ( P<0.05); the fiber diameter and cross-sectional area of skeletal muscle decreased gradually with the prolongation of modeling time ( P<0.05); the protein expression levels of MuRF1and MAFbx proteins increased gradually ( P<0.05); the expression levels of MICU1 protein and mRNA decreased gradually ( P<0.05). There was no significant difference in all indices between AAV-M-Sham and AAV-C-Sham groups ( P>0.05). Compared with mice in the AAV-C-CLP group, mice in the AAV-M-CLP group showed increased grip strength ( P<0.05); the amplitude of CMAP increased, showing shortened duration and latency ( P <0.05); the fiber diameter and cross-sectional area of skeletal muscle increased ( P<0.05); the expression levels of MuRF1and MAFbx decreased ( P<0.05). Sepsis leads to skeletal muscle dysfunction, which is related to the decrease in mitochondrial MICU1 expression.

Cucurbitacin IIb attenuates cancer cachexia induced skeletal muscle atrophy by regulating the IL-6/STAT3/FoxO signaling pathway.

The main features of cancer cachexia include skeletal muscle atrophy, which can significantly reduce the quality of life of patients. Clinical treatment of cancer cachexia is mainly based on nutritional therapy and physical exercise; medication only improves appetite but does not reverse the symptoms of skeletal muscle wasting. In this work, we systematically studied the underlying molecular mechanisms by which cucurbitacin IIb (CuIIb) ameliorates muscle wasting in cancer cachexia both in vitro and in vivo. CuIIb significantly ameliorated the chief features of cancer cachexia in vivo, alleviating weight loss, food intake, muscle wasting, adipose tissue depletion, and organ weight reductions. In vitro, CuIIb (10 and 20 μM) dose-dependently attenuated conditioned medium (CM)-induced C2C12 myotube atrophy. Collectively, our findings demonstrated that CuIIb prevented the upregulation of the E3 ubiquitin ligase muscle atrophy Fbox protein (MAFbx), myosin heavy chain (MyHC), and myogenin (MyoG) and impacted protein synthesis and degradation. In addition, CuIIb decreased the phosphorylation of Tyr705 in STAT3 by regulating the IL-6/STAT3/FoxO pathway to reduce skeletal muscle atrophy in cancer cachexia.

Saikosaponin D alleviates cancer cachexia by directly inhibiting STAT3.

Cancer cachexia is a metabolic syndrome that is characterized by progressive loss of skeletal muscle mass, and effective therapeutics have yet to be developed. Saikosaponin D (SSD), a major bioactive component of Radix Bupleuri, exhibits antiinflammatory, anti-tumor, anti-oxidant, anti-viral, and hepatoprotective effects. In this study, we demonstrated that SSD is a promising agent for the treatment of cancer cachexia. SSD could alleviate TCM-induced myotube atrophy and inhibit the expression of E3 ubiquitin ligases muscle RING-finger containing protein-1 (MuRF1) and muscle atrophy Fbox protein (Atrogin-1/MAFbx) in vitro. Moreover, SSD suppressed the progression of cancer cachexia, with significant improvements in the loss of body weight, gastrocnemius muscle, and tibialis anterior muscle mass in vivo. Mechanism investigations demonstrated that SSD could directly bind to STAT3 and specifically inhibit its phosphorylation as well as its transcriptional activity. Overexpression of STAT3 partially abolished the inhibitory effect of SSD on myotube atrophy, indicating that the therapeutic effect of SSD was attributed to STAT3 inhibition. These findings provide novel strategies for treatment of cancer cachexia by targeting STAT3, and SSD may be a promising drug candidate for cancer cachexia.

Taurine alleviates endoplasmic reticulum stress, inflammatory cytokine expression and mitochondrial oxidative stress induced by high glucose in the muscle cells of olive flounder (Paralichthysolivaceus)

The aim of the present study was to evaluate the effects of taurine on endoplasmic reticulum stress, inflammatory cytokine expression and mitochondrial oxidative stress induced by high glucose in primary cultured muscle cells of olive flounder (Paralichthys olivaceus). Three experimental groups were designed as follows: muscle cells of olive flounder incubated with three kinds of medium containing 5 mM glucose (control), 33 mM glucose (HG) or 33 mM glucose + 10 mM taurine (HG + T), respectively. Results showed that taurine addition significantly alleviated the decreased activity of superoxide dismutase (SOD) and the ratio of reduced to oxidized glutathione (GSH/GSSG) induced by high glucose. The increase of cellular reactive oxygen species (ROS), malondialdehyde content and cell apoptosis induced by high glucose were alleviated by taurine. Besides, gene expression of glucose-regulated protein 78, PKR-like ER kinase, tumor necrosis factor-α, interleukin-6, interleukin-1β, interleukin-8, muscle atrophy F-box protein and muscle RING-finger protein 1 were significantly up-regulated in the HG group, and taurine addition decreased the expression of these genes. High glucose led to the swelling of the endoplasmic reticulum (ER). Meanwhile, the nuclear translocation of nuclear factor κB (NF-κB) and the release of cytochrome C from mitochondria induced by high glucose were suppressed by taurine addition. These results demonstrated that taurine alleviated ERS, inflammation and mitochondrial oxidative stress induced by high glucose in olive flounder muscle cells. The ROS production, NF-κB signaling pathway and mitochondria function were the main targets of the biological effects of taurine under high glucose condition.

Codonopsis lanceolata and its active component Tangshenoside I ameliorate skeletal muscle atrophy via regulating the PI3K/Akt and SIRT1/PGC-1α pathways

Background: Skeletal muscle atrophy is caused by aging, disuse, malnutrition, and several diseases. However, there are still no effective drugs or treatments for muscle atrophy. Codonopsis lanceolata (CL), a traditional medicinal plant and food, has been reported to have anti-oxidative, anti-inflammatory, anti-tumor, and anti-obesity effects.Purpose: This study aimed to investigate the efficacy and active component of CL on muscle atrophy in vitro and to confirm the effect of CL and its active component on muscle atrophy and the underlying molecular mechanisms in vivo.Study: design/Methods This study used the dexamethasone (Dex)-induced muscle atrophy C2C12 myotube model and immobilization (IM)-induced muscle atrophy C57BL/6 mice model. In vitro study, the myotube diameter was measured. In vivo study, the grip strength, muscle mass (quadriceps, gastrocnemius, and soleus) and muscle fiber cross-sectional area (CSA) was measured. Western blot analysis and qRT-PCR were performed to confirm the underlying molecular mechanismsResults:In vitro study, CL and its main component, Tangshenoside I (TSI), effectively restored C2C12 myotube diameters decreased by Dex. Surprisingly, TSI was identified as the active component responsible for the overall efficacy of CL on muscle atrophy. In vivo study, CL and TSI, dose-dependently increased grip strength, mass muscle, and muscle fiber CSA reduced by IM. In the molecular mechanism studies, CL and TSI increased muscle protein synthesis via activating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin complex 1 (mTORC1) pathway and decreased muscle protein degradation via inhibiting the muscle ring finger-1 (MuRF1) and muscle atrophy F-box protein (Atrogin-1) expressions. It also upregulated mitochondrial biogenesis via the silent information regulator 1 (SIRT1)/ peroxisome proliferator-activated receptor gamma and coactivator-1 alpha (PGC-1α) pathway.Conclusion: This study suggests that CL and its active component, TSI, can be potential drug candidates for the prevention and treatment of muscle atrophy.

Gosha-jinki-Gan (GJG) shows anti-aging effects through suppression of TNF-α production by Chikusetsusaponin V.

Frailty develops due to multiple factors, such as sarcopenia, chronic pain, and dementia. Go-sha-jinki-Gan (GJG) is a traditional Japanese herbal medicine used for age-related symptoms. We have reported that GJG improved sarcopenia, chronic pain, and central nervous system function through suppression of tumor necrosis factor-alpha (TNF-α) production. In the present study, GJG was found to reduce the production of TNF-α in the soleus muscle of senescence-accelerated mice at 12weeks and 36weeks. GJG did not change the differentiation of C2C12 cells with 2% horse serum. GJG significantly decreased the expression of Muscle atrophy F-box protein (MAFbx) induced by TNF-α in C2C12 cells on real-time PCR. TNF-α significantly decreased the expression of PGC-1α and negated the enhancing effect of GJG for the expression of PGC-1α on digital PCR. Examining 20 chemical compounds derived from GJG, cinnamaldehyde from cinnamon bark and Chikusetsusaponin V (CsV) from Achyrantes Root dose-dependently decreased the production of TNF-⍺ in RAW264.7 cells stimulated by LPS. CsV inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells. CsV showed low permeability using Caco-2 cells. However, the plasma concentration of CsV was detected from 30min to 6h and peaked at 1h in the CD1 (ICR) mice after a single dose of GJG. In 8-week-old SAMP8 mice fed 4% (w/w) GJG from one week to four weeks, the plasma CsV concentration ranged from 0.0500 to 10.0ng/mL. The evidence that CsV plays an important role in various anti-aging effects of GJG via suppression of TNF-⍺ expression is presented.

Suppressive effects of processed aconite root on dexamethasone-induced muscle ring finger protein-1 expression and its active ingredients

Processed aconite root (PA), the tuberous root of Aconitum carmichaelii prepared by autoclaving, is a crude drug used in Japanese traditional Kampo medicine and traditional Chinese medicine for the symptoms of kidney deficiency, that is related to the muscle atrophy in modern medicine. The objective of the present study is to evaluate the effectiveness of PA on muscle atrophy and to find its active ingredients using dexamethasone-induced muscle ring finger protein-1 (MuRF1) mRNA expression in murine myoblast C2C12 cells. Dexamethasone-induced MuRF1 expression was significantly suppressed by methanol-soluble part of boiling water extract of PA in a concentration-dependent manner with its IC50 value of 1.5 mg/ml. By the activity-guided fractionations of PA extract using the partition between organic solvents and its aqueous solution, the activity of PA did not transfer into the fraction containing aconitine-type diterpenoid alkaloids but into BuOH layer. Then, we found higenamine and salsolinol as the active ingredients in PA. Higenamine and salsolinol significantly suppressed dexamethasone-induced MuRF1 expression, and their IC50 values were 0.49 and 50 µM, respectively. The contents of higenamine and salsolinol in the decoctions of commercially available fourteen PA products are 0.12 and 14 µg/ml as the average values, and varied with the coefficient of variation (CV) values of 97 and 63%, respectively. Higenamine also significantly suppressed dexamethasone-induced mRNA expressions of muscle atrophy F-box protein (MAFbx)/atrogin1, casitas B-lineage lymphoma-b (Cbl-b), troponin, branched-chain amino acid aminotransferase 2 (BCAT2), and Bcl-2 binding and pro-apoptotic protein3 (Bnip3). Although the quality control of PA is regulated by the contents of diterpene alkaloids, salsolinol and higenamine can be used as the marker compounds to certificate the pharmacological activities of PA.Graphical abstract

Eicosapentaenoic acid suppresses cisplatin-induced muscle atrophy by attenuating the up-regulated gene expression of ubiquitin

Previously it was shown that cisplatin causes muscle atrophy. Under this condition, cisplatin increased the expression of atorogenes, such as muscle ring finger 1 and atrogin-1 (also known as muscle atrophy F-box protein), in mouse skeletal muscle. It was reported recently that ubiquitin (Ub) and ubiquitinated protein levels in skeletal muscle were also up-regulated in cisplatin-induced muscle atrophy, and cisplatin-induced ubiquitinated proteins were degraded by the 26S proteasome pathway. Eicosapentaenoic acid (EPA) is effective against skeletal muscle atrophy in mice. However, it is unclear how EPA suppresses the Ub–proteasome pathway. In this study, the effect of EPA on cisplatin-induced muscle atrophy in mice was examined. Mice were intraperitoneally injected with cisplatin or vehicle control once daily for 4 d. EPA or its vehicle was orally administered 30 min before cisplatin administration. Cisplatin systemic administration induced decrease in muscle mass, myofiber diameter, and increase in Ub genes and ubiquitinated proteins in mouse skeletal muscle were recovered by co-treatment with EPA. However, weight loss and up-regulated atrogenes induced by cisplatin were not changed by co-treatment with EPA in skeletal muscle. In this study, EPA attenuated cisplatin-induced muscle atrophy via down-regulation of up-regulated Ub gene expression. Although further clinical studies are needed, EPA administration can be effective in the development of muscle atrophy in cisplatin-treated patients.

Antioxidant Activity of Valeriana fauriei Protects against Dexamethasone-Induced Muscle Atrophy.

Skeletal muscle atrophy is defined as wasting or loss of muscle. Although glucocorticoids (GCs) are well-known anti-inflammatory drugs, their long-term or high-dose use induces skeletal muscle atrophy. Valeriana fauriei (VF) is used to treat restlessness, anxiety, and sleep disorders; however, its effects on skeletal muscle health have not been investigated. This study investigated whether Valeriana fauriei could ameliorate muscle atrophy. We induced muscle atrophy in vitro and in vivo, by treatment with dexamethasone (DEX), a synthetic GC. In DEX-induced myotube atrophy, Valeriana fauriei treatment increased the fusion index and decreased the expression of muscle atrophic genes such as muscle atrophy F-box (MAFbx/Atrogin-1) and muscle RING-finger protein 1 (MuRF1). In DEX-treated mice with muscle atrophy, Valeriana fauriei supplementation increased the ability to exercise, muscle weight, and cross-sectional area, whereas it inhibited myosin heavy chain isoform transition and the expression of muscle atrophy biomarkers. Valeriana fauriei treatment led to via the downregulation of muscle atrophic genes via inhibition of GC receptor translocation. Valeriana fauriei was also found to act as a reactive oxygen species (ROS) scavenger. Didrovaltrate (DI), an iridoid compound from Valeriana fauriei, was found to downregulate atrophic genes and decrease ROS in the DEX-induced myotube atrophy. Consolidated, our results indicate that Valeriana fauriei prevents DEX-induced muscle atrophy by inhibiting GC receptor translocation. Further, Valeriana fauriei acts as a ROS scavenger, and its functional compound is didrovaltrate. We suggest that Valeriana fauriei and its functional compound didrovaltrate possess therapeutic potentials against muscle atrophy.

Magnesium Lithospermate B Attenuates High-Fat Diet-Induced Muscle Atrophy in C57BL/6J Mice

Magnesium lithospermate B (MLB) is a primary hydrophilic component of Danshen, the dried root of Salvia miltiorrhiza used in traditional medicine, and its beneficial effects on obesity-associated metabolic abnormalities were reported in our previous study. The present study investigated the anti-muscle atrophy potential of MLB in mice with high-fat diet (HFD)-induced obesity. In addition to metabolic abnormalities, the HFD mice had a net loss of skeletal muscle weight and muscle fibers and high levels of muscle-specific ubiquitin E3 ligases, namely the muscle atrophy F-box (MAFbx) and muscle RING finger protein 1 (MuRF-1). MLB supplementation alleviated those health concerns. Parallel changes were revealed in high circulating tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), skeletal TNF receptor I (TNFRI), nuclear factor-kappa light chain enhancer of activated B cells (NF-κB), p65 phosphorylation, and Forkhead box protein O1 (FoxO1) as well as low skeletal phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt) phosphorylation. The study revealed that MLB prevented obesity-associated skeletal muscle atrophy, likely through the inhibition of MAFbx/MuRF-1-mediated muscular degradation. The activation of the PI3K-Akt-FoxO1 pathway and inhibition of the TNF-α/TNFRI/NF-κB pathway were assumed to be beneficial effects of MLB.

Codonopsis lanceolata ameliorates sarcopenic obesity via recovering PI3K/Akt pathway and lipid metabolism in skeletal muscle

BackgroundThe incidence of sarcopenic obesity, muscle atrophy induced by obesity, has steadily increased and is emerging as a health problem. Although the anti-obesity effect of Codonopsis lanceolata (CL) is known, its efficacy against sarcopenic obesity has not been studied. PurposeWe aimed to investigate the effect of CL on sarcopenic obesity and the changes in the related mechanisms to confirm the potential of CL as an effective natural therapeutic agent for sarcopenic obesity. MethodsC57BL/6 mice were fed a high-fat diet (HFD) for 9 weeks, and CL was administered for 6 weeks with HFD feeding. Body weight and grip strength were measured twice a week. After sacrifice, muscle fiber histological analysis, blood lipid analysis, muscle triglyceride extraction, western blot, and real-time PCR were performed. High-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry (MS) analysis and in vitro experiments using C2C12 cells were performed to verify the main and active compounds of CL. Confluent C2C12 cells were differentiated for 4 days, and then the main compound of CL was co-treated with palmitic acid for 24 h. ResultsCL reduced body weight, mass of three fat tissues (epididymal fat, mesenteric fat, and perirenal fat), adipocyte cross-sectional area (CSA), and improved insulin signaling. Simultaneously, CL improved grip strength, mass of three muscle tissues (quadriceps, gastrocnemius, and soleus), and muscle fiber CSA. These results were due to the recovery of both the phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (Akt) signaling pathway and lipid metabolisms in skeletal muscle. Lipids accumulated in skeletal muscle interrupt the PI3K/Akt pathway, but CL reduced intramyocellular triglyceride concentration by restoring gene expression of factors related to triglyceride synthesis and fatty acid oxidation. Therefore, the activated PI3K/Akt pathway enhanced muscle protein synthesis by increasing phosphorylation of ribosomal protein S6 kinase 1 and eIF4E-binding protein 1 and suppressed muscle protein degradation by decreasing expression of muscle ring finger-1 and muscle atrophy F-box protein. In addition, tangshenoside I (TS) was verified as the main compound of CL by HPLC-ESI-MS analysis, and its efficacy of inhibiting myotube atrophy and lipid accumulation in myotubes was confirmed, verifying that TS is an active compound. ConclusionCL is an effective natural material for sarcopenic obesity that suppresses muscle atrophy by inhibiting the accumulation of lipids in skeletal muscle through restoration of impaired PI3K/Akt pathway and lipid metabolism.

Metformin attenuates angiotensin II‑induced cardiomyocyte hypertrophy by upregulating the MuRF1 and MAFbx pathway

Pathological cardiac hypertrophy induced by aging and neurohumoral activation, such as angiotensin II (Ang II) activation, is an independent risk factor for heart failure. The muscle really interesting new gene-finger protein-1 (MuRF1) and muscle atrophy F-box (MAFbx) pathway has been previously reported to be an important mechanism underlying the pathogenesis of cardiac hypertrophy. Metformin is currently the first-line blood glucose-lowering agent that can be useful for the treatment of cardiovascular diseases. However, the potential role of metformin in the modulation of MuRF1 and MAFbx in cardiomyocyte hypertrophy remains poorly understood. The present study used H9c2 cells, a cardiomyocyte cell model. The surface area of cultured rat H9c2 myoblasts was measured and the expression levels of MuRF1 and MAFbx were quantified using western blot or reverse transcription-quantitative PCR. H9c2 cells were transfected with MuRF1 and MAFbx small interfering (si) RNA. The present study revealed that Ang II treatment significantly increased the cell surface area of model cardiomyocytes. Additionally, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNA and protein expression was increased following this treatment. Ang II also downregulated MuRF1 and MAFbx protein and mRNA expression. In the H9C2, treatment with metformin attenuated hypertrophic remodeling. In addition, expression of ANP and BNP was significantly reduced in metformin-treated H9C2 cells. The results indicated that metformin increased the activity of MuRF1 and MAFbx and upregulated their expression, the knockdown of which resulted in deteriorative Ang II-induced cell hypertrophy, even following treatment with metformin. Taken together, data from the present study suggest that metformin can prevent cardiac hypertrophy through the MuRF1 and MAFbx pathways.

Roles of adenosine monophosphate activated protein kinase in skeletal muscle atrophy in rats with severe scald

Objective: To investigate the expression and phosphorylation level change of adenosine monophosphate activated protein kinase (AMPK) in skeletal muscle of severely scald rats and its roles in skeletal muscle atrophy in severely scalded rats. Methods: The experimental research method was applied. Totally 100 6-week-old male Wistar rats were divided into sham injury group and scald group according to the random number table, with 50 rats in each group. After weighing the body weight, rats in scald group were inflicted with full-thickness scald of 30% total body surface area on the back, and rats in sham injury group were simulated with scald. At 6 h and on 1, 3, 5, and 7 d post injury, 10 rats in each group were taken to measure their body weights and weights of extensor digitorum longus and soleus muscle. At 6 h and on 1, 3, 5, and 7 d post injury, the tibialis anterior muscles were collected, the mRNA expressions of muscle atrophy F-box protein (MAFbx) and muscle-specific RING finger protein 1 (MuRF1) were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction; the content of adenosine monophosphate (AMP), adenosine diphosphate, and adenosine triphosphate (ATP) were detected by high performance liquid chromatography, and AMP/ATP ratio and energy charge were calculated; the protein expressions of AMPK-α and phosphorylated AMPK-α (p-AMPK-α) were detected by Western blotting, and the p-AMPK-α/AMPK-α ratio was calculated, with sample number of 4 in each time point of each group. Data were statistically analyzed with analysis of variance for factorial design and least significant difference test. Results: The body weights of rats in 2 groups before injury and at each time point post injury were close (P>0.05). At 6 h post injury, the weight of extensor digitorum longus of rats in scald group was (0.107±0.007) g, which was significantly heavier than (0.086±0.0607) g of sham injury group (P<0.01). On 3 d post injury, the weight of extensor digitorum longus of rats in scald group was (0.083±0.016) g, which was significantly lighter than (0.102±0.005) g of sham injury group (P<0.01). The weight of soleus of rats in 2 groups were close at each time point post injury (P>0.05). Compared with those of sham injury group, the mRNA expression of MAFbx in tibialis anterior muscle of rats in scald group was significantly up-regulated at 6 h post injury (P<0.01), and the mRNA expressions of MuRF1 in tibial anterior muscle of rats in scald group were significantly up-regulated at 6 h and on 1 d post injury (P<0.01). At 6 h and on 7 d post injury, compared with those of false injury group, the AMP/ATP ratios of the tibial anterior muscle of rats in scald group were significantly increased (P<0.05 or P<0.01), and energy charges of the tibial anterior muscle of rats in scald group were significantly decreased (P<0.01). At each time point post injury, the protein expressions of AMPK-α of the tibial anterior muscle of rats in 2 groups were close (P>0.05). The p-AMPK-α/AMPK-α ratios of the tibial anterior muscle of rats in scald group at 6 h and on 7 d post injury were significantly higher than those in sham injury group (P<0.05 or P<0.01). Conclusions: The decrease in energy charge and increase in AMP/ATP ratio of skeletal muscle of rats after severe scald activate AMPK. The activation of AMPK in the early stage of injury is consistent with the up-regulation of MAFbx and MuRF1 expressions and down-regulation of skeletal muscle weight. The above-mentioned changes may be one of the molecular mechanisms of skeletal muscle atrophy in rats with severe scald.

High glucose induces apoptosis, glycogen accumulation and suppresses protein synthesis in muscle cells of olive flounder Paralichthys olivaceus.

The effect and the mechanism of high glucose on fish muscle cells are not fully understood. In the present study, muscle cells of olive flounder (Paralichthys olivaceus) were treated with high glucose (33 mM) in vitro. Cells were incubated in three kinds of medium containing 5 mM glucose, 5 mM glucose and 28 mM mannitol (as an isotonic contrast) or 33 mM glucose named the Control group, the Mannitol group and the high glucose (HG) group, respectively. Results showed that high glucose increased the ADP:ATP ratio and the reactive oxygen species (ROS) level, decreased mitochondrial membrane potential (MMP), induced the release of cytochrome C (CytC) and cell apoptosis. High glucose also led to cell glycogen accumulation by increasing the glucose uptake ability and affecting the mRNA expressions of glycogen synthase and glycogen phosphorylase. Meanwhile, it activated AMP-activated protein kinase (AMPK), inhibited the activity of mammalian target of rapamycin (mTOR) signalling pathway and the expressions of myogenic regulatory factors (MRF). The expressions of myostatin-1 (mstn-1) and E3 ubiquitin ligases including muscle RING-finger protein 1 (murf-1) and muscle atrophy F-box protein (mafbx) were also increased by the high glucose treatment. No difference was found between the Mannitol group and the Control group. These results demonstrate that high glucose has the effects of inducing apoptosis, increasing glycogen accumulation and inhibiting protein synthesis on muscle cells of olive flounder. The mitochondria-mediated apoptotic signalling pathway, AMPK and mTOR pathways participated in these biological effects.

Morin attenuates dexamethasone-mediated oxidative stress and atrophy in mouse C2C12 skeletal myotubes

Glucocorticoids are the drugs most commonly used to manage inflammatory diseases. However, they are prone to inducing muscle atrophy by increasing muscle proteolysis and decreasing protein synthesis. Various studies have demonstrated that antioxidants can mitigate glucocorticoid-induced skeletal muscle atrophy. Here, we investigated the effect of a potent antioxidative natural flavonoid, morin, on the muscle atrophy and oxidative stress induced by dexamethasone (Dex) using mouse C2C12 skeletal myotubes. Dex (10 μM) enhanced the production of reactive oxygen species (ROS) in C2C12 myotubes via glucocorticoid receptor. Moreover, Dex administration reduced the diameter and expression levels of the myosin heavy chain protein in C2C12 myotubes, together with the upregulation of muscle atrophy-associated ubiquitin ligases, such as muscle atrophy F-box protein 1/atrogin-1, muscle ring finger protein-1, and casitas B-lineage lymphoma proto-oncogene-b. Dex also significantly decreased phosphorylated Foxo3a and increased total Foxo3a expression. Interestingly, Dex-induced ROS accumulation and Foxo3a expression were inhibited by morin (10 μM) pretreatment. Morin also prevented the Dex-induced reduction of myotube thickness, together with muscle protein degradation and suppression of the upregulation of atrophy-associated ubiquitin ligases. In conclusion, our results suggest that morin effectively prevents glucocorticoid-induced muscle atrophy by reducing oxidative stress.

20(s)‑ginseonside‑Rg3 modulation of AMPK/FoxO3 signaling to attenuate mitochondrial dysfunction in a dexamethasone‑injured C2C12 myotube‑based model of skeletal atrophy invitro.

Muscle atrophy, a side effect from administration of the anti-inflammatory medication dexamethasone (DEX), is preventable by concomitant administration of the major monomeric constituent of Panax ginseng C.A. Meyer, 20(S)-ginsenoside Rg3 (S-Rg3). Putative S-Rg3-associated prevention of DEX-induced muscle atrophy may involve S-Rg3 mitigation of DEX-induced mitochondrial dysfunction. In the present study, MTT assays revealed enhanced cell viability following S-Rg3 treatment of DEX-injured C2C12 myotubes. Subsequent PCR and western blotting results demonstrated S-Rg3-induced reduction of expression of muscle atrophy F-box protein (atrogin-1) and muscle RING-finger protein-1, proteins previously linked to muscle atrophy. Additionally, S-Rg3 treatment of DEX-injured myotubes led to aggregation of Rg3 monomers in cells and dose-dependent increases in cellular mitochondrial basal respiratory oxygen consumption rate and intracellular ATP levels compared with their levels in untreated DEX-injured myotubes. In addition, S-Rg3 treatment significantly reversed DEX-induced reductions of expression of key mitochondrial respiratory electron transport chain subunits of protein complexes II, III and V in DEX-injured myotube cells. Furthermore, S-Rg3 alleviation of mitochondrial dysfunction associated with DEX-induced injury of C2C12 myotubes was linked to S-Rg3-associated decreases in both forkhead box O3 (FoxO3) protein expression and phosphorylation of AMP-activated protein kinase (AMPK). Collectively, these results implicate S-Rg3 modulation of signaling within the AMPK-FoxO3 pathway as a putative mechanism underlying S-Rg3 alleviation of DEX-induced muscle atrophy.

Myostatin-1 Inhibits Cell Proliferation by Inhibiting the mTOR Signal Pathway and MRFs, and Activating the Ubiquitin-Proteasomal System in Skeletal Muscle Cells of Japanese Flounder Paralichthys olivaceus

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. The mechanisms of fish MSTN involved in muscle growth are not fully understood. In the present study, knockdown and overexpression of mstn-1 was performed in cultured Japanese flounder muscle cells to investigate the molecular function and the underlying mechanism of fish MSTN-1. Results showed that mstn-1 knockdown significantly induced cell proliferation and the mRNA expression of myogenic regulatory factors (MRFs), while overexpression of mstn-1 led to a significant decrease of cell proliferation and a suppression of the MRFs mRNA expression. The overexpression of mstn-1 also significantly increased the mRNA expression of ubiquitin–proteasomal pathway of proteolysis genes including muscle RING-finger protein 1 (murf-1) by 204.1% (p = 0.024) and muscle atrophy F-box protein (mafbx) by 165.7% (p = 0.011). However, mystn-1 overexpression inhibited the activation of mTOR signal pathway and the AKT/FoxO1 pathway through decreasing phosphorylation of AKT at Ser 473 by 56.0% (p = 0.001). Meanwhile, mystn-1 overexpression increased the dephosphorylation and nuclear localization of FoxO1 by 394.9% (p = 0.005). These results demonstrate that mstn-1 in Japanese flounder has the effects of inhibiting cell proliferation and growth, and the mTOR and AKT/FoxO1 pathways participated in these biological effects.