Grape seed extract prevents skeletal muscle wasting in interleukin 10 knockout mice

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1

GSE prevented the Tibialis anterior muscle weight loss and the reduction in muscle fiber diameters in IL10 knockout mice. (A) Body weight of mice aged 6 weeks and 18 weeks. (B)Tibialis anterior muscle weight. (C) Average muscle fiber diameter. (D) Trichrome staining of muscle. (E) Muscle fiber size distribution. (Bars with different letters differ significantly, P < 0.05; n = 10; mean ± SE).

Figure 2

GSE reduced ubiquitin ligase and caspase-3 expression in IL10 knockout mice. (A) Atrogin-1/Mafbx content by immunoblotting. (B) Pro-caspase 3 and activated caspase 3 contents by immunoblotting. (Bars with different letters differ significantly, P < 0.05; n = 10; mean ± SE).

Figure 3

GSE reduced apoptotic nuclear number in IL10 knockout mice.In situ staining of nuclei undergoing apoptosis (Arrows and circles point to nuclei stained black, which were undergoing apoptosis, and boxes point to nuclei stained blue, which were normal). (Bars with different letters differ significantly, P < 0.05; n = 10; mean ± SE).

Figure 4

GSE activated Akt, mTOR and AMPK signaling in IL10 knockout mice. (A) Representative immunoblots of total Akt, AMPKα, mTOR and their phosphorylated forms. (B) Quantitative data of total and phosphorylated Akt (C) Quantitative data of total and phosphorylated AMPKα (D) Quantitative data of total and phosphorylated mTOR (Bars with different letters differ significantly, P < 0.05; n = 10; mean ± SE).

Figure 5

GSE reduced inflammation and inflammasome activation in IL10 knockout mice. (A) mRNA expression of inflammatory cytokines. (B) Caspase 1 activity (C, D) NF-κB p65 phosphorylation by immunoblotting. (E, F) Pro-caspase 1 and activated caspase 1 contents by immunoblotting. (*P < 0.05; n = 10; mean ± SE). (Bars with different letters differ significantly, P < 0.05; n = 10; mean ± SE).

Figure 6

Mechanisms protecting muscle wasting due to GSE supplementation in IL10 knockout mice.