Rice Germ Attenuates Chronic Unpredictable Mild Stress-Induced Muscle Atrophy

Abstract

Chronic stress leads to hypothalamic-pituitary-adrenal axis dysfunction, increasing cortisol levels. Glucocorticoids (GCs) promote muscle degradation and inhibit muscle synthesis, eventually causing muscle atrophy. In this study, we aimed to evaluate whether rice germ supplemented with 30% γ-aminobutyric acid (RG) attenuates muscle atrophy in an animal model of chronic unpredictable mild stress (CUMS). We observed that CUMS raised the adrenal gland weight and serum adrenocorticotropic hormone (ACTH) and cortisol levels, and these effects were reversed by RG. CUMS also enhanced the expression of the GC receptor (GR) and GC-GR binding in the gastrocnemius muscle, which were attenuated by RG. The expression levels of muscle degradation-related signaling pathways, such as the Klf15, Redd-1, FoxO3a, Atrogin-1, and MuRF1 pathways, were enhanced by CUMS and attenuated by RG. Muscle synthesis-related signaling pathways, such as the IGF-1/AKT/mTOR/s6k/4E-BP1 pathway, were reduced by CUMS and enhanced by RG. Moreover, CUMS raised oxidative stress by enhancing the levels of iNOS and acetylated p53, which are involved in cell cycle arrest, whereas RG attenuated both iNOS and acetylated p53 levels. Cell proliferation in the gastrocnemius muscle was reduced by CUMS and enhanced by RG. The muscle weight, muscle fiber cross-sectional area, and grip strength were reduced by CUMS and enhanced by RG. Therefore, RG attenuated ACTH levels and cortisol-related muscle atrophy in CUMS animals.

Keywords: chronic unpredictable mild stress; cortisol; gamma-aminobutyric acid; muscle atrophy; rice germ.

Figure 1

Rice germ (RG) decreases the adrenocorticotropic hormone (ACTH) and cortisol levels, which are increased by stress. (A) Schematic diagram of chronic unpredictable mild stress (CUMS)-induced animal experimental design. The CUMS procedure was conducted for 5 weeks. After the CUMS procedure for 5 weeks, RG (90 mg/kg/day) and GABA (30 mg/kg/day) were administered orally at the same time as the CUMS procedure for 4 weeks. After 4 weeks of oral administration, grip strength was performed, and samples (Serum, spleen, adrenal gland, and gastrocnemius muscle) were collected. (B) These are representative images of the spleen (upper) and adrenal gland (lower). Scale bar = 500 μm. (C,D) The quantitative graphs of the spleen (C) and adrenal gland (D) weight were normalized to the body weight. (E,F) Protein levels of ACTH (E) and cortisol (F) in serum were measured using an enzyme-linked immunosorbent assay (ELISA). (G) Protein levels of glucocorticoid receptor (GR) in gastrocnemius muscles were measured via Western blotting. In order to account for variations in protein loading, the Western blotting quantification was adjusted by normalizing it with β-actin, which served as a control protein for loading. Data are represented as the mean ± standard error (Sample size, n = 5). ***, p < 0.001, non-CUMS/Saline vs. CUMS/Saline; $$$, p < 0.001, CUMS/Saline vs. CUMS/RG or GABA; #, p < 0.05, ##, p < 0.01 or ###, p < 0.001, CUMS/RG vs. CUMS/GABA (Tukey’s test).

Figure 2

RG decreases the levels of the KLF transcription factor 15 (Klf15) and DNA damage inducible transcript 4 (Ddit4, also known as Redd-1) and increases the levels of the mechanistic target of rapamycin kinase (mTOR), s6K, and 4E-BP1. (A) Binding of cortisol to GR in the gastrocnemius muscle was measured via ELISA. (B,C) mRNA levels of Klf15 (B) and Redd-1 (C) in the gastrocnemius muscle were measured via quantitative reverse transcription–polymerase chain reaction (qRT–PCR). (D) Protein levels of mTOR, phosphorylated mTOR (pmTOR), s6k, ps6k, 4E-BP1, and p4E-BP1 in the gastrocnemius muscle were measured via Western blotting. In order to account for variations in protein loading, the Western blotting quantification was adjusted by normalizing it with β-actin, which served as a control protein for loading. To ensure consistency, the values for each blot were expressed relative to the average of the non-chronic unpredictable mild stress (CUMS) group. Data are represented as the mean ± standard error (Sample size, n = 5). ***, p < 0.001, non-CUMS/Saline vs. CUMS/Saline; $, p < 0.05 or $$$, p < 0.001, CUMS/Saline vs. CUMS/RG or GABA; #, p < 0.05 or ###, p < 0.001, CUMS/RG vs. CUMS/GABA (Tukey’s test).

Figure 3

RG increases insulin-like growth factor 1 (IGF-1) and protein kinase B (AKT) levels and decreases forkhead box O3A (FoxO3a), Atrogin-1, and Murf1 levels. (A) Protein levels of IGF-1, AKT, phosphorylated AKT (pAKT), FoxO3a, and phosphorylated FoxO3a (pFoxO3a) in the gastrocnemius muscle measured via Western blotting. (B–D) Quantitative graph for Western blotting of IGF-1 (B), pAKT/AKT (C), and pFoxO3a/FoxO3a (D). In order to account for variations in protein loading, the Western blotting quantification was adjusted by normalizing it with β-actin, which served as a control protein for loading. To ensure consistency, the values for each blot were expressed relative to the average of the non-chronic unpredictable mild stress (CUMS) group. (E,F) mRNA levels of Atrogin-1 (E) and Murf1 (F) in the gastrocnemius muscle were measured via qRT–PCR. Data are represented as the mean ± standard error (Sample size, n = 5). ***, p < 0.001, non-CUMS/Saline vs. CUMS/Saline; $, p < 0.05, $$, p < 0.01 or $$$, p < 0.001, CUMS/Saline vs. CUMS/RG or GABA; #, p < 0.05 or ##, p < 0.01, CUMS/RG vs. CUMS/GABA (Tukey’s test).

Figure 4

RG decreases inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), and p53 levels and increases cyclin-dependent kinase 2 (CDK2) and cyclin D1 levels. (A) iNOS expression levels in the gastrocnemius muscle measured via Western blotting. (B) Quantitative graph for Western blotting of (A). (C) SOD activity in the gastrocnemius muscle measured via ELISA. (D) Protein levels of p53, ace-p53, CDK2, and Cyclin D1 in the gastrocnemius muscle measured via Western blotting. (E,F,H) Quantitative graph for Western blots of ace-p53/p53 (E), CDK2 (F), and cyclin D1 (H) levels. To account for variations in protein loading, the Western blotting quantification was adjusted by normalizing it with β-actin, which served as a control protein for loading. To ensure consistency, the values for each blot were expressed relative to the average of the non-CUMS group. (G) Proliferating cell nuclear antigen (PCNA) expression levels in the gastrocnemius muscle measured via immunohistochemistry (The arrow points to the PCNA positive signal). Scale bar = 60 μm. (I) Quantitative graph for immunohistochemistry of (G). Data are represented as the mean ± standard error (Sample size, n = 5). ***, p < 0.001, non-CUMS/Saline vs. CUMS/Saline; $, p < 0.05, or $$$, p < 0.001, CUMS/Saline vs. CUMS/RG or GABA; #, p < 0.05, ##, p < 0.01 or ###, p < 0.001 CUMS/RG vs. CUMS/GABA (Tukey’s test).

Figure 5

RG attenuates muscle atrophy caused by stress. (A) These are representative images of the gastrocnemius muscle. Scale bar = 5 mm. (B) A quantitative graph of gastrocnemius muscle weight was normalized to body weight. (C) This is a graph of and the longest transverse length of the gastrocnemius muscle. (D,E) The gastrocnemius muscle fiber cross-sectional area was measured via hematoxylin and eosin (H&E) staining. Scale bar = 30 μm. (F) Grip strength was measured using a meter. (G) Summary of this study. RG decreased (↓) stress hormone (ACTH and cortisol) secretion and downregulated (↓) the expression of GR, Klf15, and Redd-1, leading to the increased (↑) activation of the mTOR/s6k/4E-BP1 pathway. Additionally, RG elevated (↑) the IGF-1/AKT levels, decreased (↓) the FoxO3a/Atrogin-1/MuRF1 levels, and reduced (↓) the iNOS and acetylated p53 levels, resulting in enhanced (↑) expression levels of CDK2 and cyclin D1. Furthermore, RG improved (↑) the gastrocnemius muscle fiber CSA and grip strength in chronic unpredictable mild stress (CUMS) animals. Data are represented as the mean ± standard error (Sample size, n = 5). ***, p < 0.001, non-CUMS/Saline vs. CUMS/Saline; $, p < 0.05, $$, p < 0.01 or $$$, p < 0.001, CUMS/Saline vs. CUMS/RG or GABA; #, p < 0.05 or ###, p < 0.001, CUMS/RG vs. CUMS/GABA (Tukey’s test).