A SIRT1-independent mechanism mediates protection against steroid-induced senescence by resveralogues in equine tenocytes

Abstract

Tendinopathy is a common age-related disease which causes significant morbidity for both human athletes and performance horses. In the latter, the superficial digital flexor tendon is an excellent model for human tendinopathies because it is a functional homologue of the human Achilles tendon and a primary site of injuries with strong similarities to the human disease. Corticosteroids have been previously used clinically to treat tendinopathic inflammation, but they upregulate the p53-p21 axis with concomitant reductions in cell proliferation and collagen synthesis in human tenocytes. This phenotype is consistent with the induction of cellular senescence in vitro and in vivo and probably represents an important clinical barrier to their effective use. Because of the many differences in senescence mechanisms between species, this study aimed to evaluate these mechanisms after corticosteroid treatment in equine tenocytes. Exposure to clinically reflective levels of dexamethasone for 48 hours drove equine tenocytes into steroid induced senescence (SIS). This was characterised by permanent growth arrest and upregulation of p53, the cyclin dependent kinase inhibitors p21waf and p16ink4a as well as the matrix degrading enzymes MMP1, MMP2 and MMP13. SIS also induced a distinctive equine senescence associated secretory phenotype (eSASP) characterised by enhanced secretion of IL-8 and MCP-1. Preincubation with resveratrol or the potent SIRT1 activator SRT1720 prevented SIS in equine tenocytes, while treatment with the non-SIRT1 activating resveratrol analogue V29 was equally protective against SIS, consistent with a novel, as yet uncharacterised SIRT1-indendent mechanism which has relevance for the development of future preventative and therapeutic strategies.

Fig 1

A. Viability of tenocytes following dexamethasone treatment by the MTT assay. The results are expressed as the mean ± SD of three independent experiments. One-Way ANOVA with Bonferroni’s Multiple Comparison Test was applied. B. Cell proliferation assay following dexamethasone treatment. The percentage of cell cycling after 48 h dexamethasone and etoposide treatment was measured by EdU and Ki67 labelling. The data represent the average and standard deviation of three independent experiments (n = 3 horses) using one-way ANOVA Bonferroni’s multiple comparison test. (ET: etoposide, DEX: dexamethasone) (*p<0.05, **p<0.01, ***p<0.001). C. Cell proliferation assay in serum free medium containing dexamethasone for 48h. The results are expressed as the Mean ± SD of three experiments using One-Way ANOVA with Turkey’s Multiple Comparison Test. EdU and Ki67 labelling were performed in duplicate and at least 500 cells were inspected to determine the percentage of ki67-positive cells(DEX: dexamethasone) (*p<0.05, **p< 0.01).

Fig 2. Expression of p53, p21 and p16 in dexamethasone-treated tendon derived cells.

Total RNA from TDCs was analysed by qPCR at (2A) 24h and (2B) 72h following dexamethasone (DEX) treatment. P53 was not detected at 72 h (*p<0.05, **p<0.01, ***p<0.001).

Fig 3. Induction of a SASP in tenocytes by dexamethasone.

Cytokines were assayed in culture medium by ELISA at 5 days after dexamethasone treatment. There were no statically significant differences in IL-8 and MCP-1 levels between the 1 and 10μM concentrations of dexamethasone (DEX).

Fig 4. MMPs gene expression.

The gene expression level of MMP-1, MMP-2 and MMP-13 in dexamethasone-treated TDCs 5 days after removing dexamethasone (DEX) was analysed by qPCR. Data were normalized to GAPDH and expressed as fold change over control levels. One-way ANOVA,* p<0.05,** p<0.01.

Fig 5

A. EdU and Ki67 labelling for proliferating cells. Statistically significant differences between 1 or 10μM dexamethasone (DEX) and Resveratrol (RSV)+ DEX are indicated by ** p<0.01. B. p53 and p21 genes expression. The expression level of p53 and p21 in dexamethasone and resveratrol-treated(DEX+RSV) TDCs 24h after removing DEX was analysed by qPCR. *p<0.05, **p<0.01.

Fig 6

A. Quantification of SA-β-gal staining following a 48-hour exposure to 1 and 10μM dexamethasone and 2μM resveratrol (DEX+RSV). Cells were assessed for the percentage of SA-β-gal positive staining seven days post-treatment. SA-β-gal staining was performed in duplicate and at least 100 cells were inspected to determine the percentage of SA-β-gal positive cells. Statistical analysis performed using One-way ANOVA (*p<0.05,**p< 0.01). B. Resveratrol inhibited SASP development in dexamethasone-treated cells. The levels of IL-8 and MCP-1 were measured by cytokine array 5 days after treatment with 1 and 10μM dexamethasone and 2μM resveratrol (DEX+RSV). The values are shown as the mean ± SD. **p<0.01,***p < 0.001. C. The effect of resveratrol on MMPs gene expression in dexamethasone and resveratrol-treated (DEX+RSV) TDCs. The gene expression levels of MMP-1, MMP-2, and MMP-13 were measured by qPCR five days after the removal of treatment. Data were normalized to GAPDH expression and presented as fold change over the control level. Statistical analysis performed using One-way ANOVA (*p<0.05, **p< 0.01, ***p<0.001).

Fig 7. EdU labelling for proliferating cells.

Statistically significant differences between 1 (A) or 10μM dexamethasone (DEX) (B) and Resveratrol (RSV)/ V34 and V29+ DEX are indicated by * p<0.05 ** p<0.01.

Fig 8. The effect of different concentrations of SRT-1720 on TDCs proliferation against dexamethasone treatment.

Data are presented as the Mean ± SD of EDU positive cells observed between 1 and 10μM dexamethasone (DEX) and 0.5 and 1μM SRT-1720 (SIRT). Statistical analysis was performed using One-Way ANOVA with Bonferroni’s Multiple Comparison Test (*p<0.05, **p< 0.01) (n = 3).