Hdac3-deficiency increases senescence-associated distention of satellite DNA and telomere-associated foci in osteoprogenitor cells.

Histone deacetylase 3 (Hdac3) is an epigenetic regulator of gene expression and interacts with skeletal transcription factors such as Runx2. We previously reported that conditional deletion of Hdac3 in Osterix-Cre recombinase-expressing osteoprogenitor cells (Hdac3 CKOOsx) caused osteopenia and increased marrow adiposity, both hallmarks of skeletal aging. We also showed that Runx2+ cells within osteogenic cultures of Hdac3-depleted bone marrow stromal cells (BMSCs) contain lipid droplets (LDs). Cellular senescence, a nonproliferative metabolically active state, is associated with increased marrow adiposity, bone loss, and aging. In this study, we sought to determine if Hdac3 depleted Runx2+ pre-osteoblasts from young mice exhibit chromatin changes associated with early cellular senescence and how these events correlate with the appearance of LDs. We first confirmed that BMSCs from Hdac3 CKOOsx mice have more Runx2 + LD+ cells compared with controls under osteogenic conditions. We then measured senescence-associated distention of satellite (SADS) DNA and telomere-associated foci (TAFs) in Hdac3 CKOOsx and control BMSCs. In situ, Runx2+ cells contained more SADS per nuclei in Hdac3 CKOOsx femora than in controls. Runx2+ BMSCs from Hdac3 CKOOsx mice also contained more SADS and TAFs per nuclei than Runx2+ cells from age-matched control mice in vitro. SADs and TAFs were present at similar levels in Runx2 + LD+ cells and Runx2 + LD- cells from Hdac3 CKOOsx mice. Hdac inhibitors also increased the number of SADS in Runx2 + LD+ and Runx2 + LD- WT BMSCs. Senolytics reduced viable cell numbers in Hdac3 CKOOsx BMSC cultures. These data demonstrate that the depletion of Hdac3 in osteochondral progenitor cells triggers LD formation and early events in cellular senescence in Runx2+ BMSCs through mutually exclusive mechanisms.

Histone deacetylase 3 (Hdac3) is an enzyme within cells that binds factors in cell nuclei such as Runx2 to regulate the expression of genes and control cellular functions. Deleting Hdac3 in cells responsible for bone formation causes bone loss and increases fat in the bone marrow, both hallmarks of skeletal aging. We observed that Hdac3-deletion causes Runx2+ bone marrow stromal cells to store fats in lipid droplets (LD) even though the cultures were stimulated to become bone cells. Here, we investigated whether these Runx2 + LD+ cells exhibit signs of cellular senescence, which is a zombie-like state associated with increased marrow fat, bone loss, and aging. We found that Hdac3-depleted Runx2+ cells showed chromatin changes linked to early cellular senescence alongside the formation of LDs. These findings suggest that Hdac3 plays a crucial role in preventing skeletal aging via regulating both LD formation and cellular senescence in osteochondral progenitor cells.

Pulling rank with RNA: RANKL promotes the association of PGC-1ß/RNA complexes with NCoR/HDAC3 to activate gene expression in osteoclasts.

In this issue, Abe et al1 report a novel mechanism by which RANKL stimulates osteoclast differentiation and bone resorption through non-coding RNAs that bind PGC-1ß and convert the NCoR/HDAC3 co-repressor complex into a co-activator of AP-1- and NFκB-regulated genes.

Exercise training attenuates pulmonary inflammation and mitochondrial dysfunction in a mouse model of high-fat high-carbohydrate-induced NAFLD.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) can lead to pulmonary dysfunction that is associated with pulmonary inflammation. Moreover, little is known regarding the therapeutic role of exercise training on pulmonary pathophysiology in NAFLD. The present study aimed to investigate the effect of exercise training on high-fat high-carbohydrate (HFHC)-induced pulmonary dysfunction in C57BL/6 mice. METHODS: Male C57BL/6 mice (N = 40) were fed a standard Chow (n = 20) or an HFHC (n = 20) diet for 15 weeks. After 8 weeks of dietary treatment, they were further assigned to 4 subgroups for the remaining 7 weeks: Chow (n = 10), Chow plus exercise (Chow+EX, n = 10), HFHC (n = 10), or HFHC plus exercise (HFHC+EX, n = 10). Both Chow+EX and HFHC+EX mice were subjected to treadmill running. RESULTS: Chronic exposure to the HFHC diet resulted in obesity with hepatic steatosis, impaired glucose tolerance, and elevated liver enzymes. The HFHC significantly increased fibrotic area (p < 0.001), increased the mRNA expression of TNF-α (4.1-fold, p < 0.001), IL-1ß (5.0-fold, p < 0.001), col1a1 (8.1-fold, p < 0.001), and Timp1 (6.0-fold, p < 0.001) in the lung tissue. In addition, the HFHC significantly altered mitochondrial function (p < 0.05) along with decreased Mfn1 protein levels (1.8-fold, p < 0.01) and increased Fis1 protein levels (1.9-fold, p < 0.001). However, aerobic exercise training significantly attenuated these pathophysiologies in the lungs in terms of ameliorating inflammatory and fibrogenic effects by enhancing mitochondrial function in lung tissue (p < 0.001). CONCLUSIONS: The current findings suggest that exercise training has a beneficial effect against pulmonary abnormalities in HFHC-induced NAFLD through improved mitochondrial function.

Maintenance of NAD+ Homeostasis in Skeletal Muscle during Aging and Exercise.

Nicotinamide adenine dinucleotide (NAD) is a versatile chemical compound serving as a coenzyme in metabolic pathways and as a substrate to support the enzymatic functions of sirtuins (SIRTs), poly (ADP-ribose) polymerase-1 (PARP-1), and cyclic ADP ribose hydrolase (CD38). Under normal physiological conditions, NAD+ consumption is matched by its synthesis primarily via the salvage pathway catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). However, aging and muscular contraction enhance NAD+ utilization, whereas NAD+ replenishment is limited by cellular sources of NAD+ precursors and/or enzyme expression. This paper will briefly review NAD+ metabolic functions, its roles in regulating cell signaling, mechanisms of its degradation and biosynthesis, and major challenges to maintaining its cellular level in skeletal muscle. The effects of aging, physical exercise, and dietary supplementation on NAD+ homeostasis will be highlighted based on recent literature.

Protective effects of extra virgin olive oil and exercise training on rat skeletal muscle against high-fat diet feeding.

A diet high in saturated fat leads to skeletal muscle deteriorations including insulin resistance, mitochondrial dysfunction and muscle fiber atrophy. Consumption of long-chain polyunsaturated fatty acids and exercise have shown promise in ameliorating high-fat diet (HFD)-induced oxidative stress and inflammation. However, the impact of extra virgin olive oil (EVOO) on mitochondrial homeostasis in muscle is largely unknown. This study aimed to investigate whether 12 wks of EVOO feeding alone and in conjunction with endurance training could protect against metabolic and mitochondrial dysfunction rat muscle with HFD. Female Sprague-Dawley rats were divided into 4 groups fed a control diet (C), HFD, EVOO diet, and EVOO diet with training (EVOO+T). Mitochondrial enzyme activity and protein content decreased with HFD compared to C, but were restored with EVOO and EVOO+T. EVOO+T elevated muscle cytochrome c and PGC-1α levels. HFD increased muscle proteolytic markers and protein ubiquitination, whereas these effects were not seen in EVOO and EVOO+T. HFD suppressed mitochondrial fusion protein level while increasing fission protein levels, but were restored with EVOO and EVOO+T. Mitophagy marker PINK1 content decreased with HFD, but was unchanged in EVOO and EVOO+T. EVOO+T upregulated autophagy markers, along with decreased phosphorylated/dephosphorylated FoxO3 ratio. Antioxidants enzyme levels were upregulated by EVOO and EVOO+T, and EVOO+T reduced HFD-induced lipid peroxidation. In conclusion, HFD impaired muscle oxidative capacity, promoted protein ubiquitination and mitochondrial fission, and upregulated autophagy markers. Replacement of HFD with EVOO corrected the observed adverse effects, while exercise training in conjunction with EVOO provided additional protection to the muscle.

Oxidative stress: an evolving definition.

Thirty-five years ago, Sies and colleagues insightfully described the universal phenomenon that the generation of reactive oxygen species could modify macromolecules in living organisms, resulting in a wide range of measurable damage. They used the term "oxidative stress" to define the loss of the balance between oxidants and antioxidants in favor of the former. After decades of research, it became increasingly clear that cells are not simply passive receivers of oxidative modification but can act dynamically to resist and adapt to oxidants. Furthermore, many redox-sensitive pathways have been identified wherein certain oxidants (mainly hydrogen peroxide and nitric oxide) are used as messenger molecules to transduce the signals required for these adaptations. Since the turn of the century, redox signaling has developed into a vibrant multidisciplinary field of biology. To reflect the evolution of the study in this field, the definition of oxidative stress is postulated to define a state in which the pro-oxidative processes overwhelm cellular antioxidant defense due to the disruption of redox signaling and adaptation.

The role of mitochondria in redox signaling of muscle homeostasis.

In the past, contraction-induced production of reactive oxygen species (ROS) has been implicated in oxidative stress to skeletal muscle. As research advances, clear evidence has revealed a more complete role of ROS under both physiologic and pathologic conditions. Central to the role of ROS is the redox signaling pathways that control exercise-induced major physiologic and cellular responses and adaptations, such as mitochondrial biogenesis, mitophagy, mitochondrial morphologic dynamics, antioxidant defense, and inflammation. The current review focuses on how muscle contraction and immobilization may activate or inhibit redox signalings and their impact on muscle mitochondrial homeostasis and physiologic implications.

Aging alters acetylation status in skeletal and cardiac muscles.

During aging, organs such as skeletal muscle and heart require sufficient NAD+ both as a coenzyme for oxidative-reductive electron transfer and as a substrate for multiple signaling pathways. Sirtuins (SIRTs), a family of NAD+-dependent deacetylase, play an important role in regulating mitochondrial homeostasis and antioxidant defense by deacetylating transcription factors and enzymes such as PGC-1α, p65, GCN5, and SOD2. However, age-related DNA damage and increased SASP activate PARP-1 and CD38, the enzymes competing with SIRTs for NAD+. Thus, it is important to know how aging alters intracellular NAD+ status and NAD+-depending enzyme expression in muscles. In this study, we report that the acetylation level of muscle protein pool, as well as major SIRTs target proteins (PGC-1α, GCN5, p65, and SOD2), was significantly increased in hindlimb and cardiac muscles of 24-month old mice compared with their 6-month old counterparts, despite the fact that most members of the SIRT family were upregulated with aging. Aging increased the protein content of PARP-1 and CD38, whereas decreased NAD+ levels in both skeletal and heart muscles. Aged muscles demonstrated clear signs of mitochondrial dysfunction, oxidative stress, and inflammation. Taken together, our data suggest that despite the upregulation of SIRTs, aged muscles suffered from NAD+ deficit partly due to the competition of elevated CD38 and PARP-1. The enhanced acetylation of several key proteins involved in broad cellular functions may contribute to the age-related muscle deterioration.

Muscle Disuse Atrophy Caused by Discord of Intracellular Signaling.

Significance: Regular contractile activity plays a critical role in maintaining skeletal muscle morphological integrity and physiological function. If the muscle is forced to stop contraction, such as during limb immobilization (IM), the IGF/Akt/mTOR signaling pathway that normally stimulates protein synthesis and inhibits proteolysis will be suppressed, whereas the FoxO-controlled catabolic pathways such as ubiquitin-proteolysis and autophagy/mitophagy will be activated and dominate, resulting in muscle fiber atrophy. Recent Advances: Mitochondria occupy a central position in the regulation of both protein synthesis and degradation through several redox-sensitive pathways, including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial fusion and fission proteins, mitophagy, and sirtuins. Prolonged IM downregulates PGC-1α due to AMPK (5'-AMP-activated protein kinase) and FoxO activation, thus decreasing mitochondrial biogenesis and causing oxidative damage. Decrease of mitochondrial inner membrane potential and increase of mitochondrial fission can trigger cascades of mitophagy leading to loss of mitochondrial homeostasis (mitostasis), inflammation, and apoptosis. The phenotypic outcomes of these disorders are compromised muscle function and fiber atrophy. Critical Issues: Given the molecular mechanism of the pathogenesis, it is imperative that the integrity of intracellular signaling be restored to prevent the deterioration. So far, overexpression of PGC-1α via transgene and in vivo DNA transfection has been found to be effective in ameliorating mitostasis and reduces IM-induced muscle atrophy. Nutritional supplementation of select amino acids and phytochemicals also provides mechanistic and practical insights into the prevention of muscle disuse atrophy. Future Directions: In light of the importance of mitochondria in regulating the various critical signaling pathways, future work should focus on exploring new epigenetic strategies to restore mitostasis and redox balance.

Would a right shift of the oxy-hemoglobin dissociation curve improve exercise capacity in patients with heart failure?

Exercise intolerance is a hallmark of symptoms in patients with heart failure. In addition to reduced cardiac output, a series of impairments in pulmonary and vascular systems leads to decreases in oxygen delivery and availability in locomotor muscles. This contributes to exercise intolerance in heart failure. The oxy-hemoglobin dissociation curve is essentially a graph illustrating the relationship between the partial pressure of oxygen (PO2, X-axis) and oxygen saturation (SaO2, Y-axis) of hemoglobin. The rightward shift of the curve indicates that hemoglobin's affinity for oxygen decreases and in turn, it may allow the release of more oxygen to tissues. In the present study, we discuss the pathophysiological impairment, which causes exercise intolerance in heart failure patients and suggest a strategy to improve exercise capacity without altering cardiac output via modulating the oxy-hemoglobin dissociation curve.

The impact of pulsed electromagnetic field therapy on blood pressure and circulating nitric oxide levels: a double blind, randomized study in subjects with metabolic syndrome.

Purpose: Regulation of blood pressure (BP) is important in reducing the risk for cardiovascular disease. There is growing interest in non-pharmacological methods to treat BP including a novel approach using pulsed electromagnetic field therapy (PEMF). PEMF therapy has been proposed to impact physiological function at the cellular and tissue level and one possible mechanism is through an impact on endothelial function and nitric oxide (NO) related pathways. The focus of the present study was to evaluate the effect of PEMF on BP and NO in subjects with mild to moderate metabolic syndrome.Materials and methods: For 12 weeks, 23 subjects underwent PEMF therapy and 21 subjects underwent sham therapy. BP was measured at rest and near the end of submaximal exercise pre- and 12 week post-therapy. Additionally, plasma NO was measured at similar time points.Results: The PEMF demonstrated an increase in NO after therapy (p = .04) but SHAM did not (p = .37). For resting BP, there were no differences in systolic BP (SBP), diastolic BP (DBP) or mean arterial pressure (MAP) between groups (p > .05). During exercise, PEMF had a reduction in peak SBP (p = .04), but not SHAM (p = .57). PEMF demonstrated significant relationships between baseline SBP and change in SBP following therapy (r = -0.71, p < .01) and between MAP and change in MAP following therapy (r = -0.60, p < .01), but no such relationships were found in SHAM. Subjects with resting hypertension (SBP ≥140 mmHg) in PEMF (n = 11) had significant reductions in SBP, DBP and MAP when compared to SHAM with hypertension (n = 9) (p < .05). In this sub-group analysis, PEMF demonstrated lowered peak SBP (p = .04) at a given exercise load (p = .40) but SHAM did not (p > .05).Conclusion: PEMF may increase plasma NO availability and improve BP at rest and during exercise. However, this beneficial effect appears to be more pronounced in subjects with existing hypertension.

Mitochondrial dysregulation and muscle disuse atrophy.

It is well established that mitochondria play a critical role in the metabolic and physiological adaptation of skeletal muscle to enhanced contractile activity. Several redox-sensitive signaling pathways such as PGC-1α, AMPK, IGF/Akt/mTOR, SIRT, NFκB, and FoxO are involved with extensive crosstalk to regulate vital cellular functions such as mitochondrial biogenesis, mitochondrial fusion and fission dynamics, autophagy/mitophagy, and apoptosis under altered demand and stress. However, when muscles cease contraction, such as during immobilization and denervation, mitochondria undergo a series of detrimental changes characterized by downregulation of PGC-1α and antioxidant defense, increased ROS generation, activated FoxO, NFκB, and inflammation, enhanced ubiquitination, and finally mitophagy and apoptotic cascades. The phenotypic outcome of the discord of mitochondrial homeostasis is elevated proteolysis and muscle atrophy. The demonstration that PGC-1α overexpression via transgene or in vivo DNA transfection can restore mitochondrial homeostasis and reverse myocyte atrophy supports the "mitostasis theory of muscle atrophy".

PGC-1α Overexpression via Local In Vivo Transfection in Mouse Skeletal Muscle.

The overexpression of a specific protein is a common method for investigating the specific biological function of the substance and the mechanism of action. In vivo electrotransfer has been confirmed to be one of the most reliable, efficient and cost-effective way to overexpress a protein in a select biological tissue. Typically, this technique involves a physical injection of plasmid DNA followed by electric pulses across the injection site. Here, we introduce this method that we used to transfect green fluorescent protein (GFP)-tagged PGC-1α plasmid DNA into mouse tibialis anterior (TA) muscle, which attained high transfection efficiency with no muscle damage. To quantify the transfection efficiency, we also demonstrate the visualization of plasmid DNA transfected fibers via immunohistochemical staining on muscle cross sections.

Avenanthramides attenuate inflammation and atrophy in muscle cells.

BACKGROUND: Chronic inflammation is an important etiologic mechanism for muscle atrophy. Oat-derived phytochemical avenanthramides (AVAs) have been shown to suppress inflammatory responses in human clinical studies and in several cell lines in vitro, but their role in skeletal muscle is unclear. The aim of this study was to investigate whether AVA treatment can prevent tumor necrosis factor (TNF)-α-induced muscle fiber atrophy in C2C12 cells. METHODS: We treated 70% confluent cells for 24 h with AVA. Then, TNF-α was added to cell-cultured medium. Subsequently, cells were harvested at different time points. The cells were examined using various biochemical techniques for measuring protein, messenger RNA levels, nuclear binding activity, and viability. Fluorescence microscope was used for analysis of the myotube morphology. RESULTS: Cells treated with TNF-α significantly increased nuclear factor κB activation, indicated by a marked decrease of IκB (p < 0.05) and a 6.6-fold increase in p65-DNA binding (p < 0.01); however, 30 µmol of AVA-A, -B, and -C treatment reduced the binding by 33%, 18%, and 19% (p < 0.01), respectively, compared with cells treated with TNF-α without AVA. The interleukin-6 level increased by 2.5 fold (p < 0.01) with TNF-α, but decreased by 24%, 32%, and 28% (p < 0.01), respectively, with AVA-A, -B, and -C. The interleukin-1ß level also showed a 47% increase with TNF-α (p < 0.01), whereas this increment was abolished in all AVA-treated cells. Reactive oxygen species production was 1.3-fold higher in the TNF-α-treated group (p < 0.01) but not in the TNF-α + AVAs groups. Messenger RNA levels of muscle-specific E3 ubiquitin ligase atrogin-1 increased 23% in TNF-α vs. control (p < 0.05) but was decreased by 46%, 34%, and 53% (p < 0.01), respectively, with treatment of AVA-A, -B, and -C. Moreover, TNF-α treatment increased the muscle RING finger 1 messenger RNA level by 76% (p < 0.01); this change was abolished by AVAs. Cells treated with TNF-α demonstrated a reduced proliferation compared with control cells (p < 0.01), but this effect was not seen in TNF-α + AVAs cells. The diameter of the C2C12 myotube decreased by 28% (p < 0.01) with TNF-α, whereas it showed no change when AVAs were included in the cell media. CONCLUSION: These results indicated that AVAs can reduce proinflammatory cytokine and reactive oxygen species production and ameliorate TNF-α-induced myotube atrophy in muscle cells.

Data on in vivo PGC-1alpha overexpression model via local transfection in aged mouse muscle.

The data presented in this article are related to the research paper entitled "Intensified mitophagy in skeletal muscle with aging is downregulated by PGC-1alpha overexpression in vivo" (Yeo et al., 2019). The data explained the surgical procedure of in vivo local transfection by electroporation method in aged mouse tibialis anterior muscle, and plasmid DNA preparation and verification protocol. The data also showed the transfection efficiency levels of GFP or GFP-tagged PGC-1alpha through immunohistochemistry method for frozen muscle cross-sections.

Intensified mitophagy in skeletal muscle with aging is downregulated by PGC-1alpha overexpression in vivo.

Mitochondrial dysfunction plays an important role in the etiology of age-related muscle atrophy known as sarcopenia. PGC-1α is positioned at the center of crosstalk in regulating mitochondrial quality control, but its role in mitophagy in aged skeletal muscle is currently unclear. The present study investigated the effects of aging and PGC-1α overexpression via in vivo DNA transfection on key mitophagy protein markers, as well as mitochondrial dynamics related proteins, metabolic function and antioxidant capacity in mouse muscle. C57BL/6J mice at the age of 2 mo (young, Y; N = 14) and 24 mo (old, O; N = 14) were transfected in vivo with either PGC-1α DNA (OE, N = 7) or GFP (N = 7) into the tibialis anterior (TA) muscle followed by electroporation. PINK1 and Parkin protein contents were 3.6 and 1.4-fold higher (P < 0.01), whereas mitochondrial ubiquitination (Ub) increased 1.5-fold (P < 0.05), in O vs. Y mice. PGC-1 OE suppressed PINK and Parkin protein levels by 50-60% (P < 0.01), and decreased Ub by 20% (P < 0.05) in old mice. Aging significantly increased the protein content of LC3II (30%, P < 0.05), p62 (42%, P < 0.05), RheB (5.5-fold, P < 0.01), Beclin-1 (3-fold, P < 0.01) and Mfn2 (~4-fold, P < 0.01) in the TA muscle. However, these age-related increases in mitophagy markers were attenuated by PGC-1α OE. Furthermore, aging dramatically increased Fis-1 protein content by 14-fold (P < 0.01), along with a severe reduction of citrate synthase activity (64%, P < 0.01) and cytochrome c oxidase subunit IV (COXIV) protein content (85%, P < 0.01). PGC-1α OE mitigated the age effects on Fis-1 and Drp-1 (P < 0.05). Moreover, PGC-1α OE enhanced mitochondrial oxidative function and antioxidant enzyme activities, and decreased lipid peroxidation and inner membrane damage found in old mice (P < 0.01). In summary, our data demonstrate that mitophagy protein expression in skeletal muscle was enhanced at old age driven possibly by increased mitochondrial dysfunction, damage, and fission. PGC-1α OE was effective in ameliorating mitochondrial deficits but did not restore muscle fiber atrophy.

Cellular mechanism of immobilization-induced muscle atrophy: A mini review.

It is well-established that regular contraction maintains morphological and functional integrity of skeletal muscle, whereas rigorous exercise training can upregulate muscle metabolic and contractile function. However, when muscles stop contraction, such as during immobilization (IM) and denervation, withdrawal of IGF/Akt/mTOR signaling allows FoxO-controlled protein degradation pathways to dominate. Mitochondria play an important role in regulating both protein synthesis and degradation via several redox sensitive signaling pathways such as mitochondrial biogenesis, fusion and fission dynamics, ubiquitin-proteolysis, autophagy/mitophagy, and apoptosis. During prolonged IM, downregulation of PGC-1α and increased mitochondrial oxidative damage facilitate fission protein and inflammatory cytokine production and activate mitophagic process, leading to a vicious cycle of protein degradation. This "mitostasis theory of muscle atrophy" is the opposite pathway of hormesis, which defines enhanced muscle function with contractile overload. The demonstration that PGC-1α overexpression via transgene or in vivo DNA transfection can successfully restore mitochondrial homeostasis and reverse myocyte atrophy supports such a proposition. Understanding the mechanism governing mitostasis can be instrumental to the treatment of muscle atrophy associated with bedrest, cancer cachexia and sarcopenia.

Data on the mode of binding between avenanthramides and IKKß domains in a docking model.

The data presented in this article are related to the research paper entitled "Anti-inflammatory effect of avenanthramides via NF-κB pathways in C2C12 skeletal muscle cells." (Kang et al., in press) [1] This article includes experimental procedures used to analyze the mode of binding between and IkB kinase (IKKß) and avenanthramides which are a group of phenolic alkaloids found in oats. The protein-ligand docking and the computer simulation method of molecular dynamics (MD) for studying the physical interactions of molecules were performed.

Anti-inflammatory effect of avenanthramides via NF-κB pathways in C2C12 skeletal muscle cells.

Avenanthramides (Avns), the polyphenol compounds found only in oats, have been shown to exhibit anti-inflammatory effects mainly by inhibiting nuclear factor (NF)-κB activation in select cell lines. However, the molecular mechanism by which Avns regulate the NF-κB pathway is still unclear. The purpose of this study was to investigate (1) the molecular mechanism by which three main fractions of Avns (AvnA, AvnB and AvnC) interact with IκB Kinase ß (IKKß); and (2) whether this interaction results in reduced inflammatory responses in skeletal muscle cells. The protein-ligand docking and molecular dynamics simulation studies suggest that Avns acted as an allosteric inhibitor for modulating IKKß's affinity for the NF-κB complex. Thus, Avns reduced IKKß kinase activity in response to tert-butyl hydroperoxide (tBHP) stimulation and attenuated tBHP-induced TNFα and IL-1ß mRNA expression. Furthermore, the three-fold increases in cyclooxygenase-2 (COX-2) protein and luciferase activity with tBHP treatment were reduced by 50% with Avns (P < .01), along with decreased prostaglandin E2 levels (P < .01). These data indicate that Avns are potent inhibitors of NFκB-mediated inflammatory response due to the downregulation of IKKß activity in C2C12 cells.