Anti-Atrophic Effects of Dichotomine B from Stellaria dichotoma During Starvation-Induced Skeletal Muscle Atrophy

Abstract

Muscle atrophy is defined as reductions in muscle size and function and represents a critical concern affecting elderly populations, immobilized patients, and individuals following specific dietary regimens, such as fasting and low-protein diets. This study investigated the protective effects of Stellaria dichotoma root extract and its isolated bioactive compounds during muscle atrophy using both in vitro and in vivo experimental models. First, S. dichotoma root extract prevented dexamethasone (DEX)-induced atrophy in C2C12 myotubes. Through systematic solvent partitioning and resin chromatography, five compounds (1-5) were successfully isolated from the n-butanol fraction. Dichotomine B (2) was identified as the most abundant and bioactive constituent. Treatment with dichotomine B significantly preserved the myotube diameter, enhanced the fusion index, and maintained the myosin heavy chain protein level while suppressing key atrophic biomarkers, including FoxO3a, MuRF-1, and Atrogin-1, in DEX-treated myotubes. Furthermore, dichotomine B (2) reduced proteolysis in serum-free cultured C2C12 myotubes and in mice subjected to 48 h of fasting, preserving muscle mass and strength. These findings suggest that S. dichotoma root extract and its principal compound, dichotomine B (2), have promising therapeutic potential and provide an opportunity to develop novel pharmacological interventions against muscle wasting through suppression of proteolysis pathways.

Keywords: Stellaria dichotoma; dexamethasone; dichotomine B; skeletal muscle atrophy; starvation.

Figure 1

The effects of S. dichotoma extract and its sub-fractions on the diameter of DEX-induced C2C12 myotubes. (A) Cell morphology and (B) relative changes in the diameter of DEX-induced C2C12 myotubes cotreated with each S. dichotoma extract (30 μg/mL) observed under a microscope (200×) and analyzed by ImageJ2 software. The data are presented as means ± SDs (n = 3). ^## p < 0.01 vs. control. ** p < 0.01 vs. dexamethasone treatment. *** p < 0.001 vs. dexamethasone treatment.

Figure 2

The effects of isolated compounds 1–5 from S. dichotoma on the diameter of DEX-induced C2C12 myotubes. (A) The chemical structures of compounds 1–5 isolated from the n-butanol fraction. (B) The cell morphology and (C) relative changes in myotube diameter of DEX-induced C2C12 myotubes cotreated with each isolated compound (1–5) under a microscope (scale bar = 250 μm) and analyzed by ImageJ software. These results are presented as means ± SDs (n = 3). ^## p < 0.01 vs. control. * p < 0.05 vs. dexamethasone treatment. ** p < 0.01 vs. dexamethasone treatment.

Figure 3

The effects of dichotomine B during DEX-induced C2C12 myotube atrophy. (A) Immunofluorescence staining of MHC after cotreatment with DEX and dichotomine B (DB) (10 or 30 μM) in C2C12 myotubes for 24 h. (B) The MHC-positive area and (C) fusion index were analyzed and determined by ImageJ2 software. The effects of DB on muscle atrophy biomarkers in DEX-stimulated C2C12 myotubes. (D) Western blot analysis of MHC, Atrogin-1, and MuRF-1 from lysates of DEX-induced C2C12 myotubes cotreated with DB (10 or 30 μM) for 24 h. The experiment was performed in triplicate independently (from the left panel to the right panel). (E) The relative changes in the protein levels of MHC, Atrogin-1, and MuRF-1 in DB-treated myotubes. The experiments were performed in triplicate. The data are presented as means ± SDs (n = 3). # p < 0.05 vs. control. ## p < 0.01 vs. control. ### p < 0.001 vs. control. * p < 0.05 vs. dexamethasone treatment. ** p < 0.01 vs. dexamethasone treatment. *** p < 0.001 vs. dexamethasone treatment.

Figure 4

The effects of dichotomine B on the myotube diameter and MHC expression in C2C12 myotubes under serum-free conditions. (A) Immunofluorescence staining of MHC after treatment with dichotomine B (DB) (1 or 10 μM) in C2C12 myotubes under serum-free conditions for 24 h. (B) The myotube diameter and (C) the fusion index were analyzed and determined by ImageJ2 software. (D) Western blot analysis of MHC, Atrogin-1, and MuRF-1 from lysates of C2C12 myotubes incubated with DB (1 or 10 μM) under starvation conditions for 24 h. The relative changes in the protein levels of (E) MHC, (F) FoxO3a, (G) Atrogin-1, and (H) MuRF-1 in DB-treated myotubes. The data are presented as means ± SDs (n = 3). # p < 0.05 vs. control. ### p < 0.001 vs. control. * p < 0.05 vs. starved myotubes. ** p < 0.01 vs. starved myotubes. *** p < 0.001 vs. starved myotubes.

Figure 5

The effects of dichotomine B on grip strength, muscle weight, and atrophy biomarkers in fasting C57BL/6J mice. (A) The body weights of mice fed controlled standard chow or fasting with or without administration of dichotomine B (DB) at 10 mg/kg and followed for up to 2 days. (B) The grip strength test was performed on day 3 of the experiment. (C,D) The tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (GA), and soleus (SOL) were collected on day 3 of the experiment to measure their diameters and weights. (E) Western blot analysis of MHC and Atrogin-1 from mouse TA muscles. The relative changes in the protein levels of (F) MHC and (G) Atrogin-1 in each group. The data are presented as means ± SDs (n = 3). # p < 0.05 vs. the control group. ## p < 0.01 vs. the control group. ### p < 0.001 vs. the control group. * p < 0.05 vs. the starved group. *** p < 0.001 vs. the starved group.