Effect of chlorella vulgaris on promotion of muscle regeneration in myoblast cell in culture and animal model

Abstract

Skeletal muscle ageing or sarcopenia is a geriatric syndrome characterized by a dramatic loss of skeletal muscle mass and strength in advancing age. Chlorella vulgaris is microscopic unicellular freshwater green algae which have been known for its high nutritive contents with current reports of its pharmacological effects. This study aimed to determine the effects of C. vulgaris on muscle regeneration in vitro and in an animal model. In in vitro study, human skeletal muscle myoblast (HSMM) cells were continuously subcultured to acquire young and senescent cells. Cell viability assay was performed by real-time monitoring followed by induction of differentiation. Several parameters such as cell cycle profile, myogenic differentiation markers and expression of gene and myomiRs were determined on Day 0, 1, 3, 5 and 7 of differentiation. Acute toxicity test of C. vulgaris was performed on the animal model according to OECD Guidelines 420 on female Sprague Dawley (SD) rats. Twenty-eight male, young (3 months) and aged (21 months) SD rats, respectively were used for the in vivo study which were divided into 3 groups; Group 1, control (given distilled water), Group 2, treated with 150 mg/kg body weight (BW), and Group 3, treated with 300 mg/kg/BW of C. vulgaris for 3 months by oral gavage. On Day 0, 30, 60 and 90, open field test, front and back grip strength and bone density were evaluated. Urine and blood were also collected. Rats were sacrificed on Day 91 and gastrocnemius and quadriceps muscles as well as organs were collected. Our results showed that the highest cell proliferation was achieved at 10 and 100 µg/ml of C. vulgaris. Both dosages were used in the subsequent experiments. Treatment with C. vulgaris resulted in the promotion of myoblasts differentiation as evidenced by increased fusion index, maturation index, size of myotubes and myotube surface area as well as increased population of cells in G0/G1 phase and decreased S phase cells (p<0.05). Gene and myomiRs expression was increased by C. vulgaris treatment throughout the differentiation day (p<0.05). Acute toxicity test demonstrated no toxicity signs or mortality up to 2000 mg/kg/BW of C. vulgaris. Liver function test, renal profile and histology analysis showed no significant difference indicating no toxicity or malignancy in both organs with C. vulgaris treatment. The grip strength of front and hind paws of C. vulgaris-treated young and aged rats was significantly increased on Day 30, 60 and 90 compared to untreated control (p<0.05). Bone mineral content (BMC), bone mineral density (BMD) and lean BMC of C. vulgaris-treated rats was significantly increased on Day 30, 60 and 90 compared to Day 0 in old rats, but no significant difference was observed in young rats (p<0.05). For open field test, the total path was significantly increased in young and old rats treated with 300 mg/kg BW of C. vulgaris at Day 60 and Day 90 compared to Day 0 (p<0.05). Oxidative marker urinary isoprostanes, creatine-kinase MM and malondialdehyde (MDA) with combination of 4-hydroxyalkenals (HAE) decreased significantly with C. vulgaris treatment (p<0.05). In conclusion, C. vulgaris demonstrated its potential in modulating cellular aging of myoblast cell and promotes muscle regeneration in an animal model. Therefore, C. vulgaris supplementation may have beneficial effect in muscle regeneration in aging and prevent or reduce the severity of sarcopenia.