Mechanisms of action of 5α- tetrahydrocorticosterone, a novel anti-inflammatory glucocorticoid
Topical glucocorticoids (GCs), such as hydrocortisone (HC), are the main drugs used to treat inflammatory skin conditions including eczema and psoriasis, but their longterm use is limited by the onset of side effects such as skin thinning, impairment of wound healing and systemic metabolic dysfunction. For this reason, there is a substantial need for new compounds with the same anti-inflammatory effects but fewer adverse effects. Previous studies have suggested 5α-tetrahydrocorticosterone (5α-THB) as a new, more selective anti-inflammatory compound; this steroid is a metabolite of the major endogenous GC in rodents, corticosterone (B). Preliminary data indicated that 5α-THB may be as effective as HC in reducing mouse irritant dermatitis, but without the local or systemic side effects of HC. The aim of this thesis is to investigate the mechanisms through which 5α-THB delivers more selective anti-inflammatory effects, with the hypothesis that 5α-THB influences distinct signalling pathways from those of B. A mouse model of irritant dermatitis induced by topical application of croton oil on the ear was developed, and the anti-inflammatory properties of 5α-THB were analysed, in comparison with those of B, after 6 and 24 hours of treatment. In inflamed tissue, B reduced tissue oedema and cell infiltration at both time points; in contrast, 5α-THB did so at 24 but not 6 hours, at a dose five-fold higher than B. Real-time analysis at 24 hours showed that B and 5α-THB similarly reduced the croton oil-induced increase of transcripts of genes encoding vascular and cellular adhesion molecules. Interestingly, while B did not affect the abundance of transcripts of the anti-inflammatory gene Dusp1, 5α-THB increased it in croton oil-treated ears, suggesting a different mechanism of action between 5α-THB and B. The experiment was repeated with the injection of the glucocorticoid receptor (GR) antagonist RU486; RU486 relieved the effect of B on swelling but did not attenuate the anti-inflammatory effects of 5α-THB, indicating a further important difference between the two steroids. Angiogenesis is fundamental for the healing process, and it is known that topical GCs impair wound healing in part by inhibiting angiogenesis; for this reason, the effects of 5α-THB on the formation of new vessels, in comparison with B, were tested in a mouse model of inflammatory angiogenesis induced by sub-cutaneous implantation of polyurethane sponges. 5α-THB, at equipotent doses to B for the reduction of macrophage infiltration, inhibited angiogenesis to a lesser extent than its precursor. In addition, B had systemic effects in that it lowered adrenal gland weights, whereas 5α-THB did not. Histological analysis suggested that while B inhibits formation and maturation of new vessels, 5α-THB may affect only the former process. Molecular analysis showed that B reduced the abundance of transcripts of the majority of the tested genes involved in inflammation, angiogenesis and tissue remodelling, but 5α-THB had more selective effects. Ex vivo studies in mouse bone marrow-derived macrophages stimulated with LPS showed that 5α-THB inhibited release of pro-inflammatory cytokines in a weaker manner compared with B. This inhibition was partially prevented by co-incubation of RU486 with B but not with 5α-THB. In in vitro studies, molecular pathways activated by B and associated with adverse side effects were only weakly activated by 5α-THB. In particular, 5α-THB only weakly induced phosphorylation of GR, and activation of expression of GC-responsive reporter plasmids and endogenous metabolic genes. Interestingly, 5α-THB reduced B-induced trans-activation of some of these genes. In summary, 5α-THB effectively reduces skin inflammation, but, unlike B, has only moderate anti-angiogenic properties, and weakly activates molecular mechanisms associated with adverse metabolic side effects. Most importantly, its action may not be due to activation of GR. This work opens the intriguing possibility that GCs work through mechanisms not yet investigated, and this may be of pivotal importance in the search for new safer anti-inflammatory compounds.