Medium 199 was purchased from Gibco; Thermo Fisher Scientific, Inc. Penicillin-streptomycin solution and FBS were purchased from HyClone; GE Healthcare Life Sciences. Trypsin-EDTA (0.05%) was obtained from Gibco; Thermo Fisher Scientific, Inc. Antibodies specific for β-actin (cat. no. sc-47778) and horseradish peroxidase-conjugated secondary antibodies [goat anti-mouse (cat. no. sc-2005) and anti-rabbit (cat. no. sc-2004)] were obtained from Santa Cruz Biotechnology, Inc. Anti-SDHA antibody (cat. no. ab66484) was purchased from Abcam. The primary antibody against HPRT1 (cat. no. LS-C81245) was purchased from LifeSpan BioSciences, Inc. and the anti-p53 primary antibody (cat. no. ARP37163_T100) was obtained from Aviva Systems Biology Corporation. Pifithrin-α (PFT-α; P4359) was purchased from Sigma-Aldrich; Merck KGaA (32).

A skeletal muscle tissue biopsy was performed on the leg of a male neonatal thoroughbred racehorse to cultivate primary horse skeletal muscle cells. The obtained muscle tissue was first chopped into 1×1 mm sections, washed twice using PBS and subsequently transferred to 15 ml tubes containing 2 ml trypsin/EDTA for 18 h at 4°C. The trypsin was then discarded and the tissue pieces were further incubated with residual trypsin at 37°C for 30 min, followed by the addition of 5 ml media containing 10% FBS. The resulting suspension was centrifuged for 3 min at 670 × g at room temperature to collect the cell pellet. After centrifugation, a filter was used (200 µM; Cell Strainers; cat. no. 08-771-1; Falcon™; Thermo Fisher Scientific, Inc.) to completely disperse any remaining tissues and to collect single cells. The resulting supernatant was centrifuged further (3 min; 670 × g at room temperature) before the cell pellet was collected and cultured in media. This cell isolation protocol was as previously described (33). The Pusan National University-Institutional Animal Care and Use Committee approved the study design (approval no. PNU-2015-0864).

Racehorse skeletal muscle cells were cultured in Medium 199 supplemented with 10% FBS and 1% antibiotic-antimycotic (ABAM; Invitrogen; Thermo Fisher Scientific, Inc.). Horse skeletal muscle cell cultures were incubated in a humidified atmosphere with 5% CO₂ at 37°C. For each experiment, at between 70 and 80% confluence, cells were gently washed twice with PBS and then treated by adding MSM with fresh media. Unless specified otherwise, cells were treated with 50 mM MSM for 24 h at 37°C.

Cell viability was assessed using an MTT assay (Sigma Aldrich; Merck KGaA). Briefly, cells were resuspended in Medium 199 and seeded into 24-well culture plates at a density of 1×10⁴ cells/well, 1 day prior to drug treatment. The next day, culture medium was replaced with fresh Medium 199 (vehicle control) or different concentrations of MSM (5–400 mM), and the cells were incubated for a further 24 h at 37°C. MTT (5 mg/ml) was subsequently added into each well, and the culture dishes were incubated at 37°C for 4 h. Formazan crystals in each well were then dissolved using DMSO (Sigma Aldrich; Merck KGaA), and the absorbance at 550 nm was measured using an Ultra multifunctional microplate reader (Tecan Group, Ltd.). Cell viability was determined from these readings using the calculation % Viability=(fluorescence value of MSM/fluorescence value of non-treated control) ×100. All measurements were performed in triplicate, and experiments were repeated at least three times.

Whole cell lysates were prepared from untreated or MSM-treated racehorse skeletal muscle cells by incubation with radioimmunoprecipitation lysis buffer (EMD Millipore) containing phosphatase and protease inhibitors on ice. Cells were disrupted by aspiration through a 23-gauge needle with the resultant lysate centrifuged at 18,300 × g for 10 min at 4°C to remove cellular debris. Protein concentrations were measured using the Bradford assay (Thermo Fisher Scientific, Inc.). Equal amounts of protein (100 µg/lane) were separated on a 10% SDS-PAGE gel, followed by transferal onto nitrocellulose membranes. The blots were then blocked for 1 h at room temperature with 5% skim milk dissolved in TBS buffer supplemented with 0.1X Tween-20 (TBS-T). The membranes were then probed overnight at 4°C with the relevant primary antibodies [anti-SDHA (1:500), anti-HPRT1 (1:500), anti-p53 (1:500) and anti-β-actin (1:1,000)] diluted in 5% bovine serum albumin (BSA; EMD Millipore) or skim milk (Difco™ skim milk; BD Biosciences). Membranes were then washed with TBS-T and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (1:2,000). Detection was performed using an Enhanced Chemiluminescence Plus detection kit (Amersham; GE Healthcare) and imaged on an ImageQuant™ LAS 4000 imaging device (Fujifilm Corporation). Blots were stripped using Restore™ Western Blot Stripping Buffer (Thermo Fisher Scientific, Inc.). Densitometry values were determined using FUJI FILM Multi Gauge version 3.1 (Fuji Photo Film Co., Ltd.).

Total RNA was extracted with the RNeasy Mini kit (Qiagen GmbH) according to the manufacturer's protocol. RNA was quantified spectrophotometrically at 260 nm. Subsequently, RT-sqPCR analyses were performed to detect SDHA, HPRT1 and GAPDH RNA expression. Briefly, cDNA was synthesized from total RNA at 42°C for 1 h, and at 95°C for 5 min using first-strand cDNA synthesis kit (AccuPower^® RT PreMix; cat. no. K-2041; Bioneer Corporation) and oligo d(T) primers. The RT-PCR Premix kit (AccuPower^® PCR PreMix; cat. no. K-2016; Bioneer Corporation) was used to amplify SDHA, HPRT1 and GAPDH with primers synthesized by Bioneer Corporation. To generate a 200-bp SDHA fragment, the following primers were used: SDHA forward, 5′-CTACAAGGGGCAGGTTCTGA-3′ and reverse, 5′-TCTGCAATACTCAGGGCACA-3′. To generate a 290-bp HPRT1 fragment, the following primers were used: HPRT1 forward, 5′-TCTTTGCTGACCTGCTGGAT-3′ and reverse, 5′-GGGTCCTTTTCACCAGCAAG-3′. To generate a 211-bp p53 fragment, the following primer pair was used: p53 forward, 5′-AGGTTGGCTCTGACTGTACC-3′ and reverse, 5′-TCCTCCTTCTTGCGGAAGTT-3′. Finally, a 320-bp GAPDH mRNA fragment was generated using the following primer pair: GAPDH forward, 5′-AAGGCCATCACCATCTTCCA-3′ and reverse, 5′-ACGATGCCAAAGTGGTCATG-3′ and an 18S mRNA fragment was generated using the following primer pair: 18S forward, 5′-AGCCTTCGGCTGACTGGCTGG-3′ and reverse, 5′-CTGCCCATCATCATGACCTGG-3′. The thermocycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 31 cycles at 95°C for 45 sec, 58°C for 60 sec and 72°C for 60 sec, followed by final extension at 72°C for 10 min. The PCR products were resolved by electrophoresis on a 2% agarose gel, and were visualized using ethidium bromide (cat. no. E7637; Sigma-Aldrich; Merck KGaA) staining. Quantification was performed using FUJI FILM Multi Gauge version 3.1 (Fuji Photo Film Co., Ltd.).

The p53 binding motif was identified using Geneious Prime software (Geneious; version R6.1; http://www.geneious.com). The sequences of SDHA and HPRT1 were screened for the p53 binding motif (AGACAT). The results obtained showed 4 binding motifs for p53 in the SDHA sequence and 6 binding motifs for p53 in the HPRT1 sequence. Primers were designed on the basis of these sequences.

The ChIP assay was performed using the Imprint^® chromatin immunoprecipitation kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, racehorse skeletal muscle cells were fixed using 1% formaldehyde at room temperature for 10 min and quenched using 1.25 M glycine at room temperature. Samples were then mixed and washed with ice-cold PBS by centrifugation at room temperature for 5 min at 180 × g. After washing, the cells were suspended in nuclei preparation buffer and shearing buffer prior to their sonication (30% amplitude for 30 sec followed by 30 Sec rest for 20 cycles) on ice. The sheared DNA was subsequently centrifuged at 4°C for 5 min at 180 × g and the cleared supernatant was used for protein/DNA immunoprecipitation. The clarified supernatant was diluted with buffer at a 1:1 ratio and 5-µl aliquots of the diluted samples were used as internal controls. The diluted supernatant was then incubated in 96 well plates pre-coated with 4 µg/µl anti-p53 antibody for 90 min at room temperature. The negative and positive controls were incubated with 1 µg normal goat IgG and 1 µg anti-RNA polymerase II (Sigma-Aldrich; Merck KGaA), respectively. The unbound DNA was washed using immunoprecipitation wash buffer, and the bound DNA was collected by applying the crosslink reversal method, using DNA release buffer containing proteinase K. The released DNA and DNA from the internal control were subsequently purified using a GenElute™ Binding Column G (Sigma-Aldrich; Merck KGaA), following which they were quantified using conventional PCR. The thermocycling conditions for PCR were as follows: Initial denaturation at 95°C for 10 min, followed by 35 cycles at 95°C for 40 sec, 58°C for 50 sec and 72°C for 50 sec, followed by final extension at 72°C for 10 min. The PCR products were resolved by electrophoresis on a 1.5% agarose gel, and were visualized using ethidium bromide (cat. no. E7637; Sigma-Aldrich; Merck KGaA) staining. Quantification was performed using FUJI FILM Multi Gauge version 3.1 (Fuji Photo Film Co., Ltd.).

All experiments were performed at least three times. Data are presented as the mean ± SEM. Statistical analyses were conducted with one-way ANOVA and Student's t-test. They were performed with Duncan's multiple range test as a post hoc test. Analyses were performed using the SAS 9.3 program (SAS Institute, Inc.). P<0.05 was considered to indicate a statistically significant difference.

A candidate drug for the treatment of stress should be designed in such a way that it causes little or no toxicity in racehorse skeletal muscle cells. Therefore, the effect of increasing concentrations of cortisol and MSM on thoroughbred racehorse skeletal muscle cells was analysed using an MTT assay. It was found that ~70% of the cells remained viable following treatment with 20 µg/ml cortisol (Fig. 1A). Subsequently, the effect of three ascending concentrations of MSM on horse skeletal muscle cell viability was checked in the presence of 20 µg/ml cortisol. Combined with 20 µg/ml cortisol, little difference was observed in cell viability between 50 mM MSM treatment and no MSM treatment (Fig. 1B). Therefore, doses of 20 µg/ml cortisol and 50 mM MSM were selected for subsequent experiments.

It was hypothesized that the expression pattern of SDHA and HPRT1 is a significant factor in cortisol-induced stress. Therefore, in the present study, the expression of SDHA and HPRT1 following treatment with increasing concentrations of cortisol was analysed. The expression levels of SDHA and HPRT1 were elevated in response to 20 µg/ml cortisol for 24 h, at the mRNA and protein levels (Figs. 2A, B and S1). From this, it was hypothesized that cortisol treatment induced stress in horse skeletal muscle cells. To examine whether the impact of cortisol-induced stress, could be minimized using MSM, increasing concentrations of MSM were applied in conjunction with cortisol. Treatment of cells with 50 mM MSM or higher reduced the expression of SDHA and HPRT1 (Figs. 2C, D and S1). MSM at 100 and 200 mM reduced the expression of GAPDH, which may have been due to the increased cytotoxicity caused by the synergistic effect of cortisol and MSM (data not shown). These results suggested that MSM inhibited cortisol-induced stress.

RT-PCR was performed to assess the expression patterns of SDHA, HPRT1 and p53 in cortisol-treated and untreated (control) horse skeletal muscle cells (Fig. 3). MSM treatment appeared to reverse cortisol-induced increases in SDHA, HPRT1 and p53 expression significantly (Fig. 3A and B), an observation that was replicated at the protein level (Fig. 3C and D). These results suggested that p53 may serve an important role in MSM treatment of cortisol-induced stress.

It was hypothesized that p53 serves an important role in cortisol-induced stress through its interactions with SDHA and HPRT1 at a post-translational level. Therefore, the ability of p53 and MSM to transcriptionally regulate SDHA and HPRT1 genes in horse skeletal muscle cells was investigated. p53 binding motifs were discovered at four sites (labelled a-d) in the HPRT1 gene promoter (Fig. 4A) and at six sites (labelled a-f) in the SDHA gene promoter region using Geneious prime software (Geneious; Version R6.1) (Fig. 4A and C). These binding sites were confirmed using a ChIP assay. Positive p53 binding was found at sites a, b and d in the HPRT1 sequence, whereas no binding was observed at site c (Fig. 4B). MSM treatment acted on site b, where it inhibited p53-induced HPRT1 expression (Fig. 4B). Strong positive p53 binding was also found in the SDHA promoter sequence at sites a, b, c, d and e, but a negative result was observed at site f (Fig. 4D). Here, it was also found that MSM reversed cortisol-induced p53 binding to sites a and e in the SDHA promoter. These results suggest that p53 serves a vital role in the cortisol-induced expression of SDHA and HPRT1, which is inhibited by MSM driven regulation of p53 expression.

To confirm the relationship between p53 and SDHA/HPRT1 expression in the presence of cortisol, western blotting analysis was performed using, PFT-α, a p53 inhibitor. Significant increases in SDHA and HPRT1 expression were observed in racehorse skeletal muscle cells in response to 20 µg/ml cortisol, which was significantly reversed with the concomitant addition of PFT-α (Fig. 5A). Significantly elevated p53 expression was also observed in cortisol-treated cells, which was also reversed by PFT-α treatment (Fig. 5B). These results further support the role of p53 in cortisol-induced increases in SDHA and HPRT1 expression.