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04.22 Circadian clock and fibrosis
  1. Steven O’Reilly
  1. Northumbria University, Ellison Building, Tyne and Wear, NE2 8ST

Abstract

Background and aims Systemic sclerosis (SSc) is an autoimmune inflammatory condition resulting in fibrosis of the skin. The circadian clock is an autonomous clock that is inherent in every cell and perturbations of the clock are associated with a variety of diseases including inflammatory diseases. BMAL1 is a core component of the clock and binds with the protein CLOCK to regulate a variety of processes. We aim to evaluate the role of BMAL1 in fibrosis.

Materials and methods Primary healthy dermal fibroblasts were cultured from healthy donors and then transfected with small interfering RNA to BMAL1 or siRNA scramble after 72 hours the cells were harvested and analysed for BMAL1, collagen1, alpha Sma, Sirtuin-1 and alpha tubulin as a loading control, by immunoblotting. In some experiments the same experimental conditions were followed but some cells were incubated additionally with the Sirtuin-1 activator Resveratrol. In separate experiments TGF-β1 was incubated or not with healthy fibroblasts and after 48 hours the cells were lysed and analysed for Sirtuin-1 levels by immunoblot.

Results The siRNA mediated knockdown of BMAL1 was achieved after 72 hours in healthy dermal fibroblasts. The siRNA-mediated depletion of BMAL1 led to an increase in collagen1 protein levels and also a decreased expression of the HDAC Sirtuin-1. Co incubation of siRNA to BMAL1 and the Sirtuin-1 activator resveratrol led to repression of the increased collagen1 levels. Incubation of the pro-fibrotic TGF-β1 did not alter the levels of Sirtuin-1 convincingly.

Conclusion The circadian clock protein BMAL1 regulates collagen expression through altering the levels of the HDAC Sirtuin-1. Reduced Sirtuin-1 appears important in SSc, however, TGF-β1 does not appear to be reducing this. Circadian clock disruption is important in skin fibrosis.

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