Rapamycin reduces oxidative stress-induced senescence by increasing sirtuin-1 in small airway epithelial cells

Background: Sirtuins (SIRTs) are endogenous anti-ageing molecules which are important in counteracting oxidative stress. In the lungs of COPD patients, high levels of oxidative stress activates the mTOR (mechanistic target of rapamycin) pathway leading to a reduction in SIRT1 and SIRT6 expression and driving accelerated ageing. Therefore, inhibition of mTOR using rapamycin, an inhibitor of mTOR complex 1 (mTORC1), may prevent/reverse the induction of senescence induced by oxidative stress. Aim: To determine the effect of rapamycin in preventing the reduction of SIRT1/6 in H₂O₂-treated healthy human small airway epithelial cells (SAECs). Methods: SAECs isolated from surgically resected lungs from non-smokers (NS) were pre-treated with 0.01-10µM rapamycin (or vehicle control) for 2h prior to stimulation with 300µM H₂O₂ for 48h to induce senescence. Protein expression of SIRT1, SIRT6 and p21^CIP1 (senescence marker) was quantified using Western blot. Results: Exposure of SAEC to H₂O₂ reduced protein expression of SIRT1 by 37±9.4%, n=3 and SIRT6 by 18±1.8%, n=5, and increased p21^CIP1 expression by 36±0.6% n=3. Pre-treatment with rapamycin resulted in a concentration-dependent response, with 10µM rapamycin significantly increasing SIRT1 expression by 71±23.8%, p<0.01, n=3, and reducing p21 expression by 46.1±25.7%, p<0.05, n=3, with no significant effect on SIRT6 expression (n=5). Conclusion: These data suggest that rapamycin treatment concentration-dependently prevents oxidative stress-induced senescence and increases expression of SIRT1 protein but has no effect on SIRT6 protein. This experimental approach may be used to study other potential senotherapies acting on this pathway.