Protective Effects of Cervus elaphus and Eucommia ulmoides Mixture (KGC01CE) on Muscle Loss and Function in Aged Rats

Abstract

Sarcopenia is a condition characterized by a progressive loss of muscle mass and function which are influenced by certain factors such as aging, nutritional deficiencies, and chronic diseases. Despite numerous efforts to prevent or treat sarcopenia, effective therapeutic options for this disease remain limited. This study aims to evaluate the effects of KGC01CE treatment, a mixture of Cervus elaphus (Ce) and Eucommia ulmoides (Eu), which are well-known traditional herbal medicines in Asia, on age-related muscle loss and functional decline in aged rats. KGC01CE has been found to be more effective than the individual extracts in inhibiting dexamethasone (DEX)-induced muscle atrophy and improving muscle mass and grip strength in C2C12 cells and aged rats. Moreover, animal studies were conducted to determine the minimum effective dose, and a 12-week oral administration of KGC01CE treatment at doses of 50, 100, and 200 mg/kg to 15-month-old aged rats resulted in a dose-dependent increase in lean mass, muscle mass, grip strength, and muscle cross-sectional area (CSA), which had decreased due to aging. Furthermore, it was shown that KGC01CE activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and inhibited the expression of muscle-degrading proteins MuRF, Atrogin-1, and myostatin. These results suggest that KGC01CE treatment may effectively prevent muscle loss and functional decline, providing a novel therapeutic strategy for sarcopenia.

Keywords: Cervus elaphus; Eucommia ulmoides; KGC01CE; muscle atrophy; sarcopenia.

Figure 1

Representative HPLC chromatograms of sialic acid and pinoresinol diglucoside in KGC01CE (Ce:Eu= 1:3). (A) HPLC chromatograms indicating the sialic acid standard and sialic acid content in KGC01CE. (B) HPLC chromatograms indicating the pinoresinol diglucoside standard and pinoresinol diglucoside content in KGC01CE.

Figure 2

Effect of KGC01CE on DEX-induced muscle atrophy in the C2C12 models. (A) Representative image of muscle atrophy. (B) Myotube diameter. After pre-treatment with 50 μg/mL of individual extracts or a ratio-based mixture in differentiated myotubes, 100 μm DEX treatment was administered for 24 h. Subsequently, the images were captured using a microscope, and the myotube diameter was measured using the ImageJ software. NC: negative control (untreated), C:DEX, Ce: Ce + DEX, Eu: Eu+ DEX, CE(3:1): Ce:Eu (3:1)+ DEX, CE(1:1): Ce:Eu (1:1) + DEX, CE(1:3): Ce:Eu (1:3) + DEX. Data are expressed as mean ± SD. *** p< 0.001, n.s: not significant, scale bar = 50 μm.

Figure 3

Effect of KGC01CE on body weight and food intake in aged rats. (A) Experimental design, (B) body weight, and (C) food intake (g/day). Fifteen-month-old male rats were orally administered with KGC01CE at concentrations of 50, 100, and 200 mg/kg for 12 weeks. NC: young SD rats, C: aged SD rats, KGC01CE − 50:aged SD rats + CE(1:3) 50 mg/kg b.w, KGC01CE − 100:aged SD rats +CE(1:3) 100 mg/kg b.w, KGC01CE − 200: aged SD rats +CE(1:3) 200 mg/kg b.w. Data are expressed as mean ± SD (n = 6). ** p < 0.01, *** p < 0.001, n.s: not significant.

Figure 4

Effect of KGC01CE on lean mass in aged rats. (A) Representative image from DXA, (B) lean mass measured using DXA. After 12 weeks of administration, lean mass was assessed using DXA. Total lean mass was expressed as a ratio to body weight. NC: young SD rats, C: aged SD rats, KGC01CE − 50: aged SD rats + CE(1:3) 50 mg/kg b.w, KGC01CE − 100: aged SD rats + CE(1:3) 100 mg/kg b.w, KGC01CE − 200: aged SD rats + CE(1:3) 200 mg/kg b.w. Data are expressed as mean ± SD (n = 4). * p< 0.05, ** p< 0.01, n.s: not significant.

Figure 5

Effect of KGC01CE on muscle mass in aged rats. (A) Total muscle mass (sum of the GAS, TA, EDL, and SOL muscles), (B) percentage of increased muscle, and (C) weight of the GAS, TA, EDL, and SOL. Muscle weight expressed as a ratio of muscle weight to body weight (g/g). NC: young SD rats, C: aged SD rats, KGC01CE − 50: aged SD rats + CE(1:3) 50 mg/kg b.w, KGC01CE − 100: aged SD rats + CE(1:3) 100 mg/kg b.w, KGC01CE − 200: aged SD rats + CE(1:3) 200 mg/kg b.w. Data are expressed as mean ± SD (n = 6). * p < 0.05, *** p < 0.001, n.s: not significant.

Figure 6

Effect of KGC01CE on grip strength and CSA in aged rats. (A) Grip strength (N/kg), (B) representative H&E image, (C) mean of CSA. Grip strength was assessed after 12 weeks of administration and expressed as a grip strength to body weight ratio. CSA was expressed as a percentage relative to the NC value. NC: young SD rats, C: aged SD rats, KGC01CE − 50: aged SD rats + CE(1:3) 50 mg/kg b.w, KGC01CE − 100: aged SD rats + CE(1:3) 100 mg/kg b.w, KGC01CE − 200: aged SD rats + CE(1:3) 200 mg/kg b.w. Data are expressed as mean ± SD (n = 6). * p < 0.05, *** p < 0.001, n.s: not significant.

Figure 7

Effect of KGC01CE on muscle protein synthesis and degradation pathway in aged rats. (A) Representative Western blot image of phospho-PI3K, phospho-Akt, MuRF-1, atrogin-1, and myostatin. (B) The protein expression levels were normalized to the GAPDH. The proteins extracted from the gastrocnemius muscle were analyzed using Western blotting. The PI3K/Akt signaling pathway for muscle protein synthesis was assessed, and the MuRF-1, atrogin, and myostatin related to muscle breakdown were evaluated. Quantification was performed relative to GAPDH using the ImageJ software and the results were expressed as fold change normalized to the NC.NC: young SD rats, C: aged SD rats, KGC01CE − 50: aged SD rats + CE(1:3) 50 mg/kg b.w, KGC01CE − 100: aged SD rats + CE(1:3) 100 mg/kg b.w, KGC01CE − 200: aged SD rats + CE(1:3) 200 mg/kg b.w.