Grape seed extract prevents skeletal muscle wasting in interleukin 10 knockout mice
© Wang et al.; licensee BioMed Central Ltd. 2014
Received: 4 November 2013
Accepted: 13 May 2014
Published: 20 May 2014
Muscle wasting is frequently a result of cancers, AIDS, chronic diseases and aging, which often links to muscle inflammation. Although grape seed extract (GSE) has been widely used as a human dietary supplement for health promotion and disease prevention primarily due to its anti-oxidative and anti-inflammative effects, it is unknown whether GSE affects muscle wasting. The objective is to test the effects of GSE supplementation on inflammation and muscle wasting in interleukin (IL)-10 knockout mice, a recently developed model for human frailty.
Male IL-10 knockout (IL10KO) C57BL/6 mice at 6 weeks of age were assigned to either 0% or 0.1% GSE (in drinking water) groups (n = 10) for 12 weeks, when skeletal muscle was sampled for analyses. Wild-type C57BL/6 male mice were used as controls.
Tibialis anterior muscle weight and fiber size of IL10KO mice were much lower than wild-type mice. IL10KO enhanced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and inflammasome formation when compared to wild-type mice. Phosphorylation of anabolic signaling was inhibited, whereas muscle specific ubiquitin ligase, AMP-activated protein kinase (AMPK) and apoptotic signaling were up-regulated in IL10KO mice. GSE supplementation effectively rectified these adverse changes in IL10KO muscle, which provide an explanation for the enhanced muscle mass, reduced protein degradation and apoptosis in GSE supplemented mice compared to IL10KO mice without supplementation.
GSE supplementation effectively prevents muscle wasting in IL10KO mice, showing that GSE can be used as an auxiliary treatment for muscle loss associated with chronic inflammation and frailty.
KeywordsApoptosis Atrophy Grape seed extract IL-10 Inflammasome Inflammation Skeletal muscle Wasting
Muscle wasting is frequently a consequence of cancers, AIDS, immobilization and fasting . During ageing, there is a gradual loss of muscle mass and a diminished capacity to reverse that loss, resulting in weakness and frailty [2, 3]. Currently, there are few options to prevent or slow down muscle wasting and, thus, there are compelling reasons to develop new medicines or nutritional remedies that can maintain skeletal muscle mass .
Muscle wasting is frequently associated with chronic inflammation [4, 5]. Polyphenolic compounds are known for their anti-oxidative and anti-inflammatory effects, and have preventive or therapeutic effects on a number of metabolic diseases including obesity, diabetes, hypercholesterolemia, cardiovascular diseases and cancer [6–12]. Resveratrol, the best studied polyphenol, improves mitochondrial function, muscle strength and endurance capacity by activating silent mating type information regulation 2 homolog 1 (SIRT1) and AMP-activated protein kinase (AMPK) [13, 14]. However, up to now, the role of polyphenolic compounds in inflammation and muscle wasting has not been defined. Grape seed extract (GSE) is a by-product of the winery and grape juice industry, which is rich in polyphenolic compounds . Consistently, GSE is known for its anti-oxidative and anti-inflammatory effects [16, 17], and alleviates oxidative stress in skeletal muscle , which prompted us to examine the role of GSE in preventing muscle wasting.
Interleukin 10 knockout (IL10KO) mice is a recently proposed model for studying low-grade inflammation, multisystemic decline and frailty . IL10KO mice show accelerated muscle loss and weakness , and also chronic inflammation, ideal for assessing inflammation associated muscle wasting and frailty [19, 21]. Using this mouse model, the objective of this study is to test the effectiveness of GSE in preventing muscle loss in IL10KO mice and further explore underlying mechanisms.
Animals and diets
All animal procedures were approved by the Washington State University Animal Care and Use Committee. Wild-type (WT) C57BL/6 and homozygous IL-10 deficient mice (B6.129P2-Il10tm1Cgn/) were initially purchased from Jackson Lab (Bar Harbor, ME, USA) and then bred under pathogen-free (SPF) conditions in the Experimental Animal Laboratory Building at Washington State University. Mice had free access to food (a standard rodent diet) and drinking water. IL10KO female mice at 6 weeks of age were randomly assigned into 2 groups (n = 10 for each group), receiving either 0 or 0.1% GSE (g/ml in drinking water, equal to ~0.2 mg/g body weight/day) for 12 weeks; WT female mice aged 6 weeks were used as controls. Water was changed daily to avoid the possible oxidation of functional compounds in GSE. There was no difference for the amount of water and diet consumed among these groups. Similar dosages of GSE have been used in previous studies [22, 23]. GSE used in this study is a commercial GSE product (Gravinol-S) purchased from OptiPure Chemco Industries Inc. (Los Angeles, CA). Per company product specification sheet, it contains a minimum 95% flavonols, of which 82% are oligomeric proanthocyanidins (OPCs), and 12% being the highly active monomeric OPCs. The composition of GSE was further analyzed by mass spectrometry in our lab and the major components include catechin monomer 7.3%, dimer 35.8%, trimer 38.6%, tetramer 12.8%, pentamer 5.4%, and trace amount of hexamer.
Mice were anaesthetized by fluorine inhalation before euthanization by cervical dislocation. Intact Tibialis anterior was isolated from hind legs, weighed before fixing for paraffin embedding. Gastrocnemius muscle was isolated and frozen in liquid nitrogen and then stored under -80°C until analyses.
Antibodies and chemicals
Antibodies against nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) p65 (#4764), phospho-p65 (#3033), Akt (4691), phospho-Akt (#Ser473), AMPKα (#6707), phospho-AMPKα (#4188), mammalian target of rapamycin (mTOR) (#2983), phospho-mTOR (#5536) were purchased from Cell Signaling (Danvers, MA). NACHT, LRR and PYD domains-containing protein 3 (NLRP3) antibody (PA1665) was purchased from Boster Biological Technology (Fremont, CA). IRDye 800CW goat anti-rabbit secondary antibody and IRDye 680 goat anti-mouse secondary antibody were bought from LI-COR Biosciences (Lincoln, NE). Caspase-1 Fluorometric Assay Kit (#K110-100) was purchased from Bio Vision (Milpitas, CA). Apoptosis Kit TACS® XL DAB (diaminobenzidine) Kit (#4810-60-K) was purchased from R&D system (Minneapolis, MN).
Immunoblotting analyses were conducted according to the procedures previously described . Membranes were visualized by Odyssey infrared imaging system (LI-COR Biosciences). Density of bands was quantified and then normalized according to the β-tubulin content.
Quantitative real time PCR
Total mRNA was extracted from Gastrocenemius muscle using Trizol reagent (Invitrogen, Carlsbad, CA), treated with deoxyribonuclease, and reverse transcribed into cDNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Real time-PCR was performed on a CFX ConnectTM Real-Time PCR detection system (Bio-Rad) using SYBR Green RT-PCR kit from Bio-Rad. The following cycle parameters were used: 34 three-step cycles of 95°C, 20 sec; 55°C, 20 sec; and 72°C, 20 sec. Primer sequences and their respective PCR fragment lengths were as follows: IL-1β (77 bp), forward 5′- TCGCTCAGGGTCACAAGAAA-3′ and reverse 5′-CATCAGAGGCAAGGAGGAAAAC-3′ ; IL-18 (89 bp), forward 5′- ATGCTTTCTGGACTCCTGCCTGCT-3′ and reverse 5′- GGCGGCTTTCTTTGTCCTGATGCT-3′; tumor necrosis factor (TNF)α (67 bp), forward 5′- TGGGACAGTGACCTGGACTGT-3′ and reverse 5′- TTCGGAAAGCCCATTTGAGT-3′ ; 18S (110 bp) forward 5′-TGCTGTCCCTGTATGCCTCT-3′, and reverse 5′-TGTAGCCACGCTCGGTCA-3′. After amplification, a melting curve (0.01°C/sec) was used to confirm product purity, and agarose gel electrophoresis was performed to confirm that only a single product of the right size was amplified. Relative mRNA content was normalized to 18S rRNA content.
Histochemical staining and image analysis
Muscle tissue sections (5 μm) were deparaffinized, rehydrated, and used for Masson’s trichrome staining , which stains muscle fibers red, nuclei black, and collagen blue. Muscle fiber sizes were measured using the ImageJ software (National Institute of Health, Baltimore, MD) and at least 400 muscle fibers per animal were measured (8 images per section and 5 sections at 50 μm interval per mice). To measure the apoptotic level of skeletal muscle cells, 8 images per section and 2 sections per mice were stained by Apoptosis Kit. Normal cells were stained blue and apoptotic cells were black. All images were analyzed at 200 × magnification.
All data were analyzed using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC), pairwise comparison was performed using fisher’s LSD procedure. Arcsine transformation was applied on percentage data before analysis. Mean values and standard errors of the mean were reported. P < 0.05 was considered significant.
Results and discussion
The maturation and secretion of IL-1β and IL-18 are tightly regulated by a diverse class of cytosolic complexes known as the inflammasome, which is associated with inflammation . Upon activation, NLRP3 aggregates with cytosolic oligomers with apoptosis-associated speck-like protein (ASC) to form inflammasome , which then triggers activation of caspase-1. Caspase-1, in turn cleaves pro-IL-1β and pro-IL-18 to produce mature IL-1β, and IL-18 . Here, we found that GSE reduced the contents of NLRP3, pro-caspase-1 and cleaved caspase-1 in IL10KO mice (Figure 5E); consistently, the activity of caspase-1 was also reduced in GSE muscle (Figure 5B). Therefore, GSE inhibited inflammation and the activation of inflammasome in the skeletal muscle of IL10KO mice, which is likely associated with the anti-oxidative capacity of GSE because reactive oxygen species induces the activation of NLRP3 inflammasome and inflammation .
Protein kinase B
Apoptosis-associated speck-like protein
Grape seed extract
Muscle atrophy F box
Nuclear factor kappa-light-chain-enhancer of activated B cells
Tumor necrosis factor α
AMP-activated protein kinase α
The mechanistic target of rapamycin.
This activity was funded, in part, with an Emerging Research Issues Internal Competitive Grant from the Agricultural Research Center at Washington State University, College of Agricultural, Human, and Natural Resource Sciences.
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