Abstract
Background 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.
Highlights
Vitamin D is well known to be involved in maintaining calcium homeostasis and bone metabolism.Vitamin D is supplied to the body via food intake and is synthesized in the skin following exposure to sun rays
To verify the protein production of proinflammatory cytokines, IL-6 and tumor necrosis factor α (TNFα) protein concentrations in the culture supernatant of C2C12 myotubes were measured by ELISA
Not compare the TNFα protein levels because they were below the limit of detection of our assay
Summary
Vitamin D is well known to be involved in maintaining calcium homeostasis and bone metabolism.Vitamin D is supplied to the body via food intake and is synthesized in the skin following exposure to sun rays. Vitamin D is hydroxylated by 25-hydroxylase and converted to 25-hydroxyvitamin. 1,25-dihydroxyvitamin D (1,25(OH) D), which is the active form of vitamin D, in the kidney [1]. Vitamin D may be involved in reducing inflammation [3]. In a systematic review and meta-analysis, vitamin D supplementation was described to reduce rheumatoid disease activity [4]. Objective: 1,25-dihydroxyvitamin D3 (1,25(OH) D3 ) inhibits proinflammatory cytokines in microglial cells and monocytes. It is unclear whether 1,25(OH) D3 inhibits proinflammatory cytokines in muscle cells. Myotubes were treated with 1,25(OH) D3 for 24 h, followed by lipopolysaccharide (LPS) stimulation for 48 h. The IL-6 concentration was significantly lower in C2C12 myotubes following 1,25(OH) D3 treatment than in
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