Hispidin-enriched Sanghuangporus sanghuang mycelia SS-MN4 ameliorate disuse atrophy while improving muscle endurance

Abstract

Background: Disuse atrophy is a frequent cause of muscle atrophy, which can occur in individuals of any age who have been inactive for a prolonged period or immobilization. Additionally, acute diseases such as COVID-19 can cause frequent sequelae and exacerbate muscle wasting, leading to additional fatigue symptoms. It is necessary to investigate potent functional nutrients for muscle reinforcement in both disuse atrophy and fatigue to ensure better physical performance.

Methods: The effects of Sanghuangporus sanghuang SS-MN4 mycelia were tested on two groups of 6-week-old male mice-one with disuse atrophy and the other with fatigue. The disuse atrophy group was divided into three sub-groups: a control group, a group that underwent hind limb casting for 7 days and then recovered for 7 days and a group that was administered with SS-MN4 orally for 14 days, underwent hind limb casting for 7 days and then recovered for 7 days. The fatigue group was divided into two sub-groups: a control group that received no SS-MN4 intervention and an experimental group that was administered with SS-MN4 orally for 39 days and tested for exhaustive swimming and running on Day 31 and Day 33, respectively. RNA sequencing (RNA-seq) and western blot analysis were conducted on C2C12 cell lines to identify the therapeutic effects of SS-MN4 treatment.

Results: In a disuse atrophy model induced by hind limb casting, supplementing with 250 mg/kg of SS-MN4 for 14 days led to 111.2% gastrocnemius muscle mass recovery and an 89.1% improvement in motor function on a treadmill (P < 0.05). In a fatigue animal model, equivalent SS-MN4 dosage improved swimming (178.7%) and running (162.4%) activities (P < 0.05) and reduced blood urea nitrogen levels by 18% (P < 0.05). SS-MN4 treatment also increased liver and muscle glycogen storage by 34.36% and 55.6%, respectively, suggesting a higher energy reserve for exercise. RNA-seq and western blot studies from the C2C12 myotube showed that SS-MN4 extract upregulates Myh4 and helps sustain myotube integrity against dexamethasone damage.

Conclusions: Supplementation of SS-MN4 (250-mg/kg body weight) with hispidin as active compound revealed a potential usage as a muscle nutritional supplement enhancing muscle recovery, fast-twitch fibre regrowth and fatigue resistance.

Keywords: C2C12; Sanghuangporus sanghuang mycelia; disuse atrophy; fatigue; hispidin.

Scheme 1

Schematic diagram of SS‐MN4 as muscle nutrient for two different mouse models (fatigue and disuse atrophy).

Figure 1

Effects of SS‐MN4 nutrient supplement on disuse atrophy model. (A) Body weight, (B) grip strength, (C) treadmill performance, (D) gastrocnemius muscle mass, (E) soleus muscle mass and (F) serum circulating myostatin (MSTN). Vehicle group (casting only) showed a significant decrease in both body weight and grip strength; 14‐day supplementation of SS‐MN4 can significantly ameliorate the muscle loss effect and is comparable to the control. Treadmill improvement may contribute to the significant difference in gastrocnemius muscle mass rather than soleus muscle mass. Suppression of serum circulating MSTN may aid in improving muscle mass. Data were expressed as mean ± SD (n = 6). *P < 0.05 and ^# P < 0.05 indicate significant differences compared with the selected group.

Figure 2

Effects of SS‐MN4 nutrient supplement on in vivo endurance model. After 39 days of SS‐MN4 supplementation at 250 mg/kg/day, fatigue symptoms were improved. No significant difference was observed in body weight change after long‐term consumption (A). The SS‐MN4 treatment group exhibited a significant improvement in both swimming (~178.7%) and running (~162.4%) compared to the control group (B). The SS‐MN4 supplement significantly reduced lactate production after exercise (C), and post‐exercise blood urea nitrogen (BUN) levels showed a significant reduction in BUN value compared to the control (D). The improvement in fatigue symptoms with long‐term SS‐MN4 treatment might contribute to higher energy reserves in organs (E). Data were expressed as mean ± SD (n = 6). *P < 0.05 indicates significant differences compared with the control group.

Figure 3

Protection of C2C12 myotubes by SS‐MN4 against dexamethasone damage. Pre‐supplementation with SS‐MN4 24 h before dexamethasone damage (A) showed significant myotube protection at a concentration of 25 μg/mL against dexamethasone damage at 50 μM, as demonstrated by H&E staining (B). Quantification of myotube diameter (n = 150) for each group was calculated to determine the overall protective efficiency (C). Data were expressed as mean ± SD (n = 6). *P < 0.05 and ^# P < 0.05 indicate significant differences compared with the selected group.

Figure 4

Pathway analysis of treatment effect of SS‐MN4 using bulk RNA sequencing. Genes involved in the action of SS‐MN4 action on healthy muscle cells (untreated vs. SS‐MN4) (A). Genes that are upregulated are involved in muscle cell differentiation and muscle cell development (B). Pathway analysis on dexamethasone (DEX)‐upregulated gene showed negative regulation of locomotion, cell motility and cellular component movement, which recapitulated the muscle damage process (C). SS‐MN4 supplementation treated the DEX‐damaged group and identified myosin heavy chain‐related genes (Myh9 and Myh6) and myogenesis‐related genes (Myod1 and Myo18b) were upregulated (D). Pathway analysis on upregulated gene revealed involvement in the biological process of muscle tissue development, muscle cell differentiation and response to growth factors (E).

Figure 5

Supplementation with SS‐MN4 can prevent myotube atrophy by upregulating myosin heavy chain‐related genes and proteins. Real‐time analysis showed that 25 μg/mL of SS‐MN4 extract increased Myogenin expression while suppressing the Murf gene expression (A). Fast skeletal Myh4 and Myh1 genes were significantly enhanced compared to the slow fibre gene Myh7 (B). Western blot results demonstrated that SS‐MN4 ethanolic extract and its major active compound, hispidin, significantly improved MYH4 protein expression at a concentration of 25 μg/mL, thus protecting against dexamethasone (DEX) damage (C). At a concentration of 25 μg/mL, SS‐MN4 resulted in significant MYH4 protein protection compared with β‐hydroxy‐β‐methylbutyrate (HMB) at 100 μg/mL (D). Both SS‐MN4 and hispidin increased AMPK and total myosin heavy chain (MYHC) expression when there was no DEX damage (E). *P < 0.05 and ^# P < 0.05 indicate significant differences compared with the selected group.