Daidzein Inhibits Muscle Atrophy by Suppressing Inflammatory Cytokine- and Muscle Atrophy-Related Gene Expression

Abstract

Background: Sarcopenic obesity, which is associated with a poorer prognosis than that of sarcopenia alone, may be positively affected by soy isoflavones, known inhibitors of muscle atrophy. Herein, we hypothesize that these compounds may prevent sarcopenic obesity by upregulating the gut metabolites with anti-inflammatory effects.

Methods: To explore the effects of soy isoflavones on sarcopenic obesity and its mechanisms, we employed both in vivo and in vitro experiments. Mice were fed a high-fat, high-sucrose diet with or without soy isoflavone supplementation. Additionally, the mouse C2C12 myotube cells were treated with palmitic acid and daidzein in vitro.

Results: The isoflavone considerably reduced muscle atrophy and the expression of the muscle atrophy genes in the treated group compared to the control group (Fbxo32, p = 0.0012; Trim63, p < 0.0001; Foxo1, p < 0.0001; Tnfa, p = 0.1343). Elevated levels of daidzein were found in the muscles and feces of the experimental group compared to the control group (feces, p = 0.0122; muscle, p = 0.0020). The real-time PCR results demonstrated that the daidzein decreased the expression of the palmitate-induced inflammation and muscle atrophy genes in the C2C12 myotube cells (Tnfa, p = 0.0201; Il6, p = 0.0008; Fbxo32, p < 0.0001; Hdac4, p = 0.0002; Trim63, p = 0.0114; Foxo1, p < 0.0001). Additionally, it reduced the palmitate-induced protein expression related to the muscle atrophy in the C2C12 myotube cells (Foxo1, p = 0.0078; MuRF1, p = 0.0119).

Conclusions: The daidzein suppressed inflammatory cytokine- and muscle atrophy-related gene expression in the C2C12 myotubes, thereby inhibiting muscle atrophy.

Keywords: C2C12 myotubes; daidzein; high-fat diet; high-sucrose diet; mice; muscle atrophy; sarcopenia; sarcopenic obesity; soy isoflavones.

Figure 1

The C57BL6/J mice fed a high-fat high-sucrose diet (HFHSD) and administered water containing 0.1% isoflavone (Iso group) showed significant improvements in glucose tolerance, obesity, metabolic disorders, and muscle loss compared to the C57BL6/J mice without the isoflavone (Ctrl group). (A) Mice were fed HFHSD ± water containing 0.1% isoflavone for 12 weeks, starting at 8 weeks of age. (B) Changes in body weight (n = 6 in each case). (C) Oral intake of 0.1% isoflavone water. (D,E) The results of the intraperitoneal glucose tolerance test (2 g/kg body weight) for 20-week-old mice and the area-under-the-curve (AUC) analysis (n = 6 in each case). (F,G) The results of the insulin tolerance test (0.75 U/kg body weight) for the 20-week-old mice and the AUC analysis (n = 6 in each case). (H) Relative grip strength (n = 6 in each case). (I–K) Serum levels of alanine aminotransferase (ALT), total cholesterol (T-Chol), and triglycerides (TG) (n = 6 in each case). (L–N) The relative weight of the epididymal fat, soleus muscle, and plantaris muscle (n = 6 in each case). The data are expressed as mean ± standard deviation (SD). The data were analyzed using an unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Effects of Isoflavone on muscle morphology and gene expression. (A,B) The cross-sectional areas of the soleus and plantaris muscles in the 20-week-old mice (n = 6 in each case). (C–F) The relative mRNA expressions of (C) Fbxo32, (D) Trim63, (E) Foxo1, and (F) Tnfa mRNA expression in the soleus muscle normalized to the expression of Gapdh (n = 6 in each case). The data are expressed as mean ± standard deviation (SD). The data were analyzed using an unpaired t-test. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns not significant.

Figure 3

The administration of the isoflavone increased the concentration of the daidzein in the serum, feces, and muscles. The concentrations of the daidzein in (A) serum, (B) feces, and (C) soleus muscle (n = 6 in each group). The concentrations of the genistein in (D) serum, (E) feces, and (F) soleus muscle (n = 6 in each group). The concentrations of the equol in (G) serum, (H) feces, and (I) soleus muscle (n = 6 in each group). The data are expressed as mean ± standard deviation (SD). The data were analyzed using an unpaired t-test. * p < 0.05, ** p < 0.01, ns not significant.

Figure 4

Impact of daidzein on gene expression in C2C12 myotube cells. The relative mRNA expression of (A) Tnfa, (B) Il-6, (C) Fbxo32, (D) Hdac4, (E) Trim63, and (F) Foxo1 all normalized to the expression of Gapdh in the C2C12 cells (n = 6 in each case). The data were analyzed using a one-way ANOVA followed by Holm–Šídák’s multiple-comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant.

Figure 5

Effect of daidzein on muscle atrophy proteins in C2C12 myotube cells. (A) Western blot analysis depicting the levels of Foxo1, MuRF1, and Gapdh in the C2C12 myotube cells. (B,C) The relative optical density of Foxo1 (B) and MuRF1 (C) normalized to Gapdh. The values are expressed as mean ± s.e.m. * p  <  0.05, ** p  <  0.01, as determined using a one-way ANOVA.