S66 Targeting anti-ageing molecule AMPK restores corticosteroid sensitivity in COPD

Rationale

Chronic obstructive pulmonary disease (COPD) is an irreversible inflammatory lung disease and is currently the fourth greatest burden of disease of worldwide. However a key issue is that patients show a lack of response to corticosteroid treatment. Corticosteroid insensitivity is mainly caused by oxidative stress which directly stimulates inflammatory transcription factors and reduces the activity of co-adaptor proteins essential for the inhibitory actions of corticosteroids. AMP-activated kinase (AMPK) is a serine/threonine protein kinase that regulates cellular energy homeostasis and anti-oxidant defences, and has recently been labelled as an anti-ageing molecule. We hypothesised that activation of AMPK using Quercetin reverses corticosteroid resistance caused by cigarette smoke extract (CSE) in a monocytic cell line.

Methods

Human monocytic cell line, U937s, were initially incubated with Quercetin (20µM) for 24 hours and then exposed to CSE for 2 hours. Cells were then treated with dexamethasone (1x10^-11 to 1x10^-6M) for 45 minutes and stimulated with TNF-a (10ng/ml) for 24 hours. Supernatants were collected and CXCL-8 was measured using ELISA. Corticosteroid resistance was calculated as the ability of dexamethasone to inhibit 25% of TNF-a-induced CXCL-8 (IC₂₅). Activation of AMPK by Quercetin was measured using the levels of the phosphorylated AMPK by Western Blot. Nuclear factor erythroid related factor 2 (Nrf-2) levels and glucocorticoid receptor (GR) nuclear translocation were also assessed using Western Blot.

Results

CSE induced corticosteroid resistance in U937s (IC₂₅ = 30nM vs IC₂₅ = 5nM). Interestingly Quercetin restored corticosteroid sensitivity by approximately 3 fold (IC₂₅ = 11nM) compared to CSE. Quercetin increased levels of activated AMPK and also up-regulated the expression of Nrf-2. However, Quercetin was unable to restore GR nuclear translocation.

Conclusions

Quercetin was found to be a potential novel therapy for restoration of corticosteroid sensitivity in COPD. Although the mechanism of action remains to be elucidated, Nrf-2 and AMPK activations which increase anti-oxidant levels and prevents oxidative damage could be a key the mechanism of action Activation of AMPK could therefore be a potential novel mechanism for the restoration of corticosteroid sensitivity and Quercetin could be used as an add-on treatment to corticosteroids in COPD.