Antioxidant Apigenin Relieves Age-Related Muscle Atrophy by Inhibiting Oxidative Stress and Hyperactive Mitophagy and Apoptosis in Skeletal Muscle of Mice

Abstract

Skeletal muscle atrophy in the aged causes loss in muscle mass and functions. Naturally occurring antioxidant flavonoid apigenin is able to ameliorate obesity- and denervation-induced muscle atrophies, but its effects on age-related muscle atrophy remain unknown. We hypothesized that apigenin can relieve muscle atrophy in aged mice, probably through special effects on reactive oxygen species and enzymes with antioxidant functions. For the male mice of the study, apigenin showed significant dose-dependent effects in relieving aging-related muscle atrophy according to results of frailty index as indicator of frailty associated with aging, grip strength, and running distance. Apigenin also improved myofiber size and morphological features and increased mitochondria number and volume, as manifested by succinate dehydrogenase staining and transmission electron microscopy. Our tests also suggested that apigenin promoted activities of enzymes such as superoxide dismutase and glutathione peroxidase for antioxidation and those for aerobic respiration such as mitochondrial respiratory enzyme complexes I, II, and IV, increased ATP, and enhanced expression of genes such as peroxisome proliferator-activated receptor-γ coactivator 1α, mitochondrial transcription factor A, nuclear respiratory factor-1, and ATP5B involved in mitochondrial biogenesis. The data also suggested that apigenin inhibited Bcl-2/adenovirus E1B 19kD-interacting protein 3 and DNA fragmentation as indicators of mitophagy and apoptosis in aged mice with skeletal muscle atrophy. Together, the results suggest that apigenin relieves age-related skeletal muscle atrophy through reducing oxidative stress and inhibiting hyperactive autophagy and apoptosis.

Keywords: Apigenin; Apoptosis; Mitophagy; Reactive oxygen species; Skeletal muscle atrophy.

Figure 1.

Apigenin treatment relieved frailty and improved muscle function in aged mice. (A) Mean frailty index (FI) scores were evaluated in young mice and (B) in old mice treated either with apigenin (AP) at dose of 25 mg·kg⁻¹·day⁻¹ (Old + AP25, low dose), 50 mg·kg⁻¹·day⁻¹ (Old + AP50, middle dose), or 100 mg·kg⁻¹·day⁻¹ (Old + AP100, high dose), or without treatment as Old control mice. The muscle function was detected by (C) grip strength and (D) running distance. Data are presented as the mean ± SD (n = 8–10). ^†p < .05, versus 16 months (in the Old group); *p < .05, versus the Old group (25 months).

Figure 2.

Apigenin attenuated muscle atrophy in aged mice. (A) Weights of tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL) muscles were normalized to tibia length. (B) Relative weights of TA, EDL, and SOL normalized to body weight. (C) Succinate dehydrogenase (SDH) staining was performed on 10-μm-thick sections from TA muscles frozen in liquid nitrogen-chilled isopentane. Scale bar: 50 μm. (D) Average fiber size (cross-sectional area, CSA) of the SDH-stained TA muscles. (E) Muscle fiber frequency distribution of the SDH-stained TA muscle. (F) Numbers of types I (slow oxidative), IIa (fast oxidative glycolytic), and IIb (fast twitch glycolytic) muscle fibers. Data are presented as the mean ± SD (n = 8–10). *p < .05, versus the Old group (25 months).

Figure 3.

Apigenin improved mitochondrial function and promoted mitochondrial biogenesis in skeletal muscle of aged mice. (A) Activities of mitochondrial complexes I, II, and IV. (B) Oxygen consumption rate (OCR). (C) Mitochondrial membrane potential (MMP) Δψm. (D) ATP content. (E) Transmission electron microscopy micrographs (magnification: 12 000) of tibialis anterior (TA) muscles. White arrows indicate mitochondria, black arrows the Z-line, and brackets the I-bands. Scale bar: 2 μm. Quantitative analysis of mitochondrial (F) size, (G) number, and (H) volume density. (I) The relative mitochondrial DNA (mtDNA) copy number. (J) Representative images and quantification (fold change from Young) of (K) PGC-1α, (L) TFAM, (M) NRF-1, and (N) ATP5B were shown. Data are presented as the mean ± SD (n = 8–10). *p < .05, versus the Old group (25 months).

Figure 4.

Apigenin suppressed oxidative stress and enhanced antioxidant capacity in skeletal muscle of aged mice. (A) Mitochondrial O₂^•− (mt O₂^•−) content. (B) H₂O₂ levels. (C) Malondialdehyde (MDA) levels. (D) Carbonyl protein content. Activities of (E) glutathione peroxidase (GPx) and (F) superoxide dismutase (SOD). (G) Total antioxidative capability (T-AOC). Data are presented as the mean ± SD (n = 8–10). *p < .05, versus the Old group (25 months).

Figure 5.

Apigenin inhibited autophagy and mitochondria-dependent apoptosis in skeletal muscle of aged mice. (A) Representative images and quantification (fold change from Young) were shown for (B) BNIP3, (C) p62, and (D) LC3-II/I. (E) Representative images and quantification (fold change from Young) of cytochrome C in (F) cytosol and (G) mitochondria fractions were shown, respectively. (H) The DNA fragmentation. Activities of (I) PARP (fold change from Young), and (J) caspase 3. Data are presented as the mean ± SD (n = 8–10). *p < .05, versus the Old group (25 months).