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. 2022 Oct 8;23(19):11963.
doi: 10.3390/ijms231911963.

Nobiletin Prevents D-Galactose-Induced C2C12 Cell Aging by Improving Mitochondrial Function

Hui-Hui Wang  1 , Ya-Nan Sun  1   2 , Tai-Qi Qu  2 , Xue-Qin Sang  1 , Li-Mian Zhou  2 , Yi-Xuan Li  1   2 , Fa-Zheng Ren  1
Affiliations

Nobiletin Prevents D-Galactose-Induced C2C12 Cell Aging by Improving Mitochondrial Function

Hui-Hui Wang et al. Int J Mol Sci. .

Abstract

Age-associated loss of skeletal muscle mass and function is one of the main causes of the loss of independence and physical incapacitation in the geriatric population. This study used the D-galactose-induced C2C12 myoblast aging model to explore whether nobiletin (Nob) could delay skeletal muscle aging and determine the associated mechanism. The results showed that Nob intervention improved mitochondrial function, increased ATP production, reduced reactive oxygen species (ROS) production, inhibited inflammation, and prevented apoptosis as well as aging. In addition, Nob improved autophagy function, removed misfolded proteins and damaged organelles, cleared ROS, reduced mitochondrial damage, and improved skeletal muscle atrophy. Moreover, our results illustrated that Nob can not only enhance mitochondrial function, but can also enhance autophagy function and the protein synthesis pathway to inhibit skeletal muscle atrophy. Therefore, Nob may be a potential candidate for the prevention and treatment of age-related muscle decline.

Keywords: C2C12 myoblast; ROS; aging; mitochondrial function; nobiletin.

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Conflict of interest statement

The authors have declared no conflict of interest.

Figures

Figure 1
Figure 1
Nob improved D-gal-induced atrophy of skeletal muscle. (A) Immunostaining of myotube myosin heavy chain (MHC) in CK and D-gal-induced differentiated C2C12 cells treated for 5 days with DMSO or Nob. (B) Quantification of the myotube area in CK and D-gal-induced C2C12 myoblasts treated for 5 days with DMSO or Nob. (C,D) Western blot analysis of myogenin, p-S473-Akt, Akt, p-p70 S6K, p70 S6K, ubiquitin, MAFbx, MuRF1, and GAPDH in CK and D-gal-induced differentiated C2C12 cells treated for 5 days with DMSO or Nob. Different lowercase letters represent significant differences between different treatment groups (p < 0.05).
Figure 2
Figure 2
Nob decreased the expression of senescence markers in D-gal-induced senescent cells. (A,B) Representative images of SA-β-gal staining in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Scale bar, 200 μm. (C,D) Immunostaining of P16 in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Representative images are shown. Scale bar, 25 μm. (E) mRNA of P53 and P21 in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. (F) Western blot analysis of P16, P53, P21, and GAPDH in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Different lowercase letters represent significant differences between different treatment groups (p < 0.05).
Figure 3
Figure 3
Nob decreased levels of ROS and inflammation in D-gal-induced senescent cells. (A,B) Quantification of ROS levels by laser confocal microscopy in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Representative images are shown. Scale bar, 25 μm. (C) Quantification of ROS levels by flow cytometry in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. (D) Western blot analysis of Nrf2, p-P65, P65, and GAPDH in CK and D-gal-induced C2C12 myoblasts treated for 48 h with DMSO or Nob. Different lowercase letters represent significant differences between different treatment groups (p < 0.05).
Figure 4
Figure 4
Nob improved mitochondrial function and prevented apoptosis in D-gal-induced senescent cells. (A) Oxygen consumption rate (OCR), (B) basal respiration, (C) ATP production, (D) maximum respiration, and (E) spare respiratory capacity were measured by the XF Cell Mito Stress Test Kit in CK and D-gal-induced C2C12 myoblasts treated with DMSO or Nob. (F) Analysis of fluorescence intensity of JC-1 monomer (green) by flow cytometry in CK and D-gal-induced C2C12 myoblasts treated with DMSO or Nob. (G,H) Western blot analysis of BAX and Bcl-2 in CK and D-gal-induced C2C12 myoblasts treated with DMSO or Nob. Different lowercase letters represent significant differences between different treatment groups (p < 0.05).
Figure 5
Figure 5
Nob improved autophagy in D-gal-induced senescent cells. Western blot analysis of p-T172-AMPK, AMPK, LC3 II/I, LAMP2, and GAPDH in CK and D-gal-induced C2C12 myoblasts treated with DMSO or Nob. Different lowercase letters represent significant differences between different treatment groups (p < 0.05).
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