• Koji Nonaka, Junichi Akiyama, Yoshiyuki Yoshikawa, Satsuki Une, and Kenichi Ito.
• Faculty of Health Sciences, Naragakuen University, Nara, Nara 631-8524, Japan.
• Medicina (Kaunas). 2020 Sep 4; 56 (9).

AbstractBackground and Objective. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proinflammatory cytokines in microglial cells and monocytes. However, it is unclear whether 1,25(OH)2D3 inhibits proinflammatory cytokines in muscle cells. This study was conducted to investigate whether 1,25(OH)2D3 inhibits the production of proinflammatory cytokines, resulting in inhibition of the protein expression of E3 ubiquitin ligases and muscle protein loss. Materials and Methods. C2C12 myoblasts were proliferated in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum, and myoblasts were differentiated into myotubes in DMEM containing 2% horse serum. Myotubes were treated with 1,25(OH)2D3 for 24 h, followed by lipopolysaccharide (LPS) stimulation for 48 h. Results. Interleukin (IL)-6 protein concentrations were higher in the culture supernatant following LPS stimulation compared to that without LPS stimulation (p < 0.001). However, the IL-6 concentration was significantly lower in C2C12 myotubes following 1,25(OH)2D3 treatment than in C2C12 myotubes without 1,25(OH)2D3 treatment (p < 0.001). The myosin heavy chain (MHC), muscle atrophy F-box, and muscle ring-finger protein-1 protein levels did not significantly differ (P = 0.324, 0.552, and 0.352, respectively). We could not compare tumor necrosis factor α (TNFα) protein levels because they were below the limit of detection of our assay in many supernatant samples, including in LPS-stimulated samples. Conclusions. 1,25(OH)2D3 inhibited increases in IL-6 protein concentrations in muscle cells stimulated by LPS, suggesting that 1,25(OH)2D3 inhibits inflammation in muscle cells. The findings suggest that 1,25(OH)2D3 can prevent or improve sarcopenia, which is associated with IL-6. The TNFα protein content could not be measured, and MHC was not decreased despite LPS stimulation of C2C12 myotubes. Further studies are needed to examine the effects of higher doses of LPS stimulation on muscle cells and use more sensitive methods for measuring TNFα protein to investigate the preventive effects of 1,25(OH)2D3 on increased TNFα and muscle proteolysis.

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