We recently showed that 5α-reduced metabolites of corticosterone (B), namely 5α-dihydrocorticosterone (5αDHB) and 5α-tetrahydrocorticosterone (5αTHB), possess similar anti-inflammatory properties to B, but have lesser metabolic effects. Here, we explored the anti-inflammatory mechanisms of these 5α-reduced metabolites in vitro. Data are mean % suppression/induction, compared by one way ANOVA, *P<0.05 versus vehicle.
In RAW264.7 murine macrophages stimulated with lipopolysaccharide (LPS, 30 ng/ml, 24 h), TNFα and IL6 release was suppressed by B (1 μM; 61.7%*, 92.2%*, respectively), 5αDHB (1 μM; 20.4%*, 68.8%*) and 5αTHB (1 μM; 40.3%*, 47.5%* respectively), effects that were inhibited by co-incubation with the glucocorticoid receptor (GR) antagonist RU486 (1 μM). Relevance to normal cells was confirmed in LPS-stimulated (100 ng/ml, 24 h) bone-marrow-derived murine macrophages, in which TNFα and IL6 release was suppressed by B (1 μM; 74.2%*, 69.4%*, respectively), 5αDHB (1 μM, 47.1%*, 28.7%*) and 5αTHB (1 μM, 21.9%*, 16.4%*).
To examine underlying mechanisms, RAW264.7 macrophages were incubated with steroids (1 μM, 1 h) prior to LPS stimulation (100 ng/ml, 30 min). Phosphorylation of inflammatory kinases p38 and JNK were suppressed by B (42.5%*, 47.5%*), 5αDHB (35.7%*, 36.6%*) and 5αTHB (34.9%*, 38.4%*, respectively). Protein expression of MKP-1 and IκBα was stimulated by B (160%*, 458%*) and 5αDHB (116%*, 389%*, respectively), whilst 5αTHB induced MKP-1 (104%*) but not IκBα expression.
To examine transcriptional effects, HEK293 cells transfected with NF-κB or AP-1 reporter plasmids, with or without a plasmid encoding GR, were treated with steroids (1 μM) and stimulated with phorbol ester (TPA, 5 ng/ml, 24 h). B and 5αDHB suppressed NF-κB-mediated transcription (42.2%*, 39.5%*) and AP-1-mediated transcription (67.6%*, 82.9%* respectively) in a GR-dependent manner. In contrast, 5αTHB increased NF-κB-mediated transcription (25.0%*) and AP-1-mediated transcription (22.1%*) independently of GR.
In conclusion, 5α-reduced glucocorticoid metabolites exhibit anti-inflammatory properties in macrophages. The mechanisms of actions of the metabolites differ, but both suppress inflammatory kinase pathways. Thus 5α-reduced glucocorticoid metabolites have potential as dissociated anti-inflammatory steroid therapy.