Abstract
Cortisol is a hormone involved in stress during exercise. The application of natural compounds is a new potential approach for controlling cortisol-induced stress. Tumour suppressor protein p53 is activated during cellular stress. Succinate dehydrogenase complex subunit A (SDHA) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) are considered to be two of the most stable reference genes when measuring stress during exercise in horses. In the present study cells were considered to be in a 'stressed state' if the levels of these stable genes and the highly stress responsive gene p53 were altered. It was hypothesized that a natural organic sulphur-containing compound, methylsulfonylmethane (MSM), could inhibit cortisol-induced stress in racing horse skeletal muscle cells by regulating SDHA, HPRT1 and p53 expression. After assessing cell viability using MTT assays, 20 µg/ml cortisol and 50 mM MSM were applied to horse skeletal muscle cell cultures. Reverse transcription-quantitative PCR and western blot analysis demonstrated increases in SDHA, HPRT1 and p53 expression in cells in response to cortisol treatment, which was inhibited or normalized by MSM treatment. To determine the relationship between p53 and SDHA/HPRT1 expression at a transcriptional level, horse gene sequences of SDHA and HPRT1 were probed to identify novel binding sites for p53 in the gene promoters, which were confirmed using a chromatin immunoprecipitation assay. The relationship between p53 and SDHA/HPRT1 expression was confirmed using western blot analysis following the application of pifithrin-α, a p53 inhibitor. These results suggested that MSM is a potential candidate drug for the inhibition of cortisol-induced stress in racehorse skeletal muscle cells.