Nobiletin Improves D-Galactose-Induced Aging Mice Skeletal Muscle Atrophy by Regulating Protein Homeostasis

Abstract

Sarcopenia, a decrease in skeletal muscle mass and function caused by aging, impairs mobility, raises the risk of fractures, diabetes, and other illnesses, and severely affects a senior's quality of life. Nobiletin (Nob), polymethoxyl flavonoid, has various biological effects, such as anti-diabetic, anti-atherogenic, anti-inflammatory, anti-oxidative, and anti-tumor properties. In this investigation, we hypothesized that Nob potentially regulates protein homeostasis to prevent and treat sarcopenia. To investigate whether Nob could block skeletal muscle atrophy and elucidate its underlying molecular mechanism, we used the D-galactose-induced (D-gal-induced) C57BL/6J mice for 10 weeks to establish a skeletal muscle atrophy model. The findings demonstrated that Nob increased body weight, hindlimb muscle mass, lean mass and improved the function of skeletal muscle in D-gal-induced aging mice. Nob improved myofiber sizes and increased skeletal muscle main proteins composition in D-gal-induced aging mice. Notably, Nob activated mTOR/Akt signaling to increase protein synthesis and inhibited FOXO3a-MAFbx/MuRF1 pathway and inflammatory cytokines, thereby reducing protein degradation in D-gal-induced aging mice. In conclusion, Nob attenuated D-gal-induced skeletal muscle atrophy. It is a promising candidate for preventing and treating age-associated atrophy of skeletal muscles.

Keywords: aging; nobiletin; protein homeostasis; sarcopenia.

Figure 1

Nob increased skeletal muscle mass in D-gal-induced aging mice. (A,B) Body weight (BW) (A) and BW gain (B) in the CK, D-gal, and D-gal + Nob groups, n = 10. (C,D) The weight of gastrocnemius (Gas) and Gas to BW in the CK, D-gal, and D-gal + Nob groups, n = 10. (E,F) The weight of soleus (Sol) and Sol to BW in the CK, D-gal, and D-gal + Nob groups, n = 10. (G,H) The weight of lean and lean to BW in the CK, D-gal, and D-gal + Nob groups, n = 8. (I,J) Running time and grip strength in the CK, D-gal, and D-gal + Nob groups, n = 6. Significant differences between treatment groups are represented by the lowercase letters a and b (p < 0.05).

Figure 2

Nob improved skeletal muscle atrophy in D-gal-induced aging mice. (A) The number of myofibers in the CK, D-gal, and D-gal + Nob groups, n = 5. (B)The myofiber size (mean cross-section area) in the CK, D-gal, and D-gal + Nob groups, n = 5. (C) Representative images of hematoxylin and eosin (HE) staining of myofiber in the CK, D-gal, and D-gal + Nob groups (Magnification ×20), n = 5. (D) Representative images of myofiber ultrastructure in the CK, D-gal, and D-gal + Nob groups. Scale bar, 2 μm, n = 3. Significant differences between treatment groups are represented by the lowercase letters a, b, and c (p < 0.05).

Figure 3

Nob improved skeletal muscle composition in D-gal-induced aging mice. (A,B) SDS-PAGE images and relative abundance of myofibrillar protein in the CK, D-gal and D-gal + Nob groups, n = 3. (C) mRNA of ACTA1, TMP1, TNNC1, TNNC2, TNNT1, TNNT3, TNNI1, TNNI2, MYH1, MYH2, MYH4, and MYH7 in the CK, D-gal and D-gal + Nob groups, n = 3. Significant differences between treatment groups are represented by the lowercase letters a, b, and c (p < 0.05).

Figure 4

Nob promoted skeletal muscle protein synthesis in D-gal-induced aging mice. Western blot analysis of Akt, p-S473-Akt, mTOR, p-Ser2448-mTOR, p70 S6K, p-p70 S6K and GAPDH in the CK, D-gal and D-gal + Nob groups, n = 3. Significant differences between treatment groups are represented by the lowercase letters a, b, and c (p < 0.05).

Figure 5

Nob-inhibited skeletal muscle protein degradation in D-gal-induced aging mice. (A) Western blot analysis of FoxO 3a, p-FoxO 3a, MAFbx, MuRF1, and GAPDH in the CK, D-gal and D-gal + Nob groups, n = 3. (B) Inflammation markers including LBP, TNF-α, and 1L-6 in the CK, D-gal and D-gal + Nob groups, n = 8. Significant differences between treatment groups are represented by the lowercase letters a, b, and c (p < 0.05).

Figure 6

Schematic diagram of Nob-mediated prevention of skeletal muscle atrophy. Protein kinase B (AKT); Rapamycin (mTOR); Ribosomal protein S6 kinase beta-1 (S6K1), also called P 70 S6 kinase (P 70 S6K), Nuclear factor-kappa B (NF-ĸB), Foxhead box O3 (FOXO3), Muscle atrophy F-box (MAFbx), Muscle RING Finger 1 (MuRF1).