Mechanisms of action of chondroitin sulfate and glucosamine in muscle tissue: in vitro and in vivo results. A new potential treatment for muscle injuries?
E. Montell Sr Osteoarthritis and Cartilage VOLUME 26, SUPPLEMENT 1, S404, APRIL 01, 2018
Purpose: In recent years, glycosaminoglycans (GAGs) have been widely evaluated as potential therapies for the treatment of musculoskeletal pathologies. Two of the most studied GAGs, the natural compounds chondroitin sulfate (CS) and glucosamine (GLU), have demonstrated beneficial effects for the treatment of osteoarthritis both in preclinical studies and in clinical trials. Both CS and GLU are considered symptomatic slow-acting drugs for OA and have demonstrated clinical efficacy and safety when administered to OA patients, showing a synergistic effect when combined. Therefore, the mechanisms of action of the combination CS+GLU have been largely studied in articular tissue.
However, its potential therapeutic effects for muscle healing remain still unknown. In our previous work, we demonstrated that CS and GLU administration improves muscle healing and force recovery of the injured skeletal muscle in rats, thus suggesting an important role of these products as potential new therapies for the treatment of muscle injuries in sports medicine. The aim of the present study is to elucidate the mechanisms of action responsible for this interesting benefit.
Methods: Skeletal muscle biopsies were rinsed in a phosphate-buffered saline (PBS) containing 5 μg mL−1 de Amphotericine B and minced into fine pieces. The tissues were further digested in a solution containing 1.25 mg/mL Protease type XIV, for 1 h at 37ºC with intermittent shaking. The resulting tissue suspension were collected by centrifugation at 1500×g for 5 min and the digested muscle pellet was resuspended in warm, sterile PBS, triturated to liberate the human satellite cells. Differential centrifugations were used to enrich the cell fraction. The collected supernatants were filtered through a 100 μm cell strainer and then centrifuged at 1500×g for 5 min. The final cell-pellet was re-suspended in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and pre-plated for 2 h. Cell suspension was then transferred onto cell-culture dishes in Growth media (DMEM/M-199 medium (3:1) with 10% FBS, 10 μg/ml insulin, 2 mM glutamine, 25 ng/ml fibroblast growth factor, and 10 ng/ml epidermal growth factor) and cells were expanded in a growing monolayer. The effect of CS+GLU treatment in primary human skeletal muscle cells was evaluated in a cell proliferation assay. NF-κB intracellular levels were determined by Western Blot. TNF-α production was measured in culture medium supernatants by ELISA.
Results: An enhancement in cell proliferation was found in CS+GLU treatments at a concentration of 100 and 200 μg/ml, increasing 160-fold (p<0,01) and 204-fold (p<0,001), respectively, compared to untreated cells. In addition, myoblasts were then incubated with IL-6 (50 ng/ml) for 72 h in order to induce an inflammatory environment. The results showed an IL-6 induced-reduction on cell proliferation in all groups, although the data did not reach statistical significance. Therefore, an IL-6 inhibitory effect on cell proliferation in human muscle cannot be ensured. We also measured the effect of the combined treatment CS+GLU on NF-κB activation and TNF-α production in human skeletal muscle cells in primary culture. Despite of TNF-α levels were undetectable in cell supernatants, preliminary data showed a slight reduction on NF-κB signaling pathway. Global gene expression profiles, measured by microarrays and GeneChip Human Gene 1.0 ST Arrays (Affymetrix), will also be analyzed.
Conclusions: Musculoskeletal injures are the most common cause for severe, chronic pain and physical disability affecting hundreds of millions of people around the world and represent a major concern also in sports medicine. A preclinical study evaluated the impact of CS and GLU combination on muscle healing. The results showed that daily administration of both oral and intraperitoneal CS and GLU (combined) induced not only an increase in intramuscular CS deposition in the injured area but also improved muscle force and stimulated the growth of regenerating muscle fibers. The mechanisms of action involved in this potential therapeutic effect seem to be related with an increase in muscle cell proliferation, together with blocking NF-κB nuclear translocation and TNFα production. Although further investigation is required, these preclinical data suggests potentially positive effects of CS and GLU administration for the treatment of skeletal muscle injuries in sports medicine.
