Neurosteroid allopregnanolone (3α5α-THP) inhibits inflammatory signals induced by activated MyD88-dependent toll-like receptors

摘要

RAW264.7 cells (n = 5–10/grp) were treated with Pam3Cys (10 µg/ml; 30 min) (A), imiquimod (IMQ; 3 µg/ml; 24 h) (B), or Poly(I:C) (25 µg/ml; 24 h) (C) with or without 3α,5α-THP (1 µM). Cells were harvested at 24 h after treatment initiation and examined for the expression of MyD88-dependent (A, B) and TRIF-dependent (C) signal activation. A Pam3Cys caused a significant increase in the levels of TRAF6 (n = 8/grp), pERK1/2 (n = 10/grp), pCREB (n = 10/grp), pATF2 (n = 10/grp), and TNF-α (n = 5/grp) relative to vehicle control (CTL), and these increases were completely blocked by 3α,5α-THP (One-way ANOVA, Tukey’s post hoc test: *p < 0.05; **p < 0.01). B IMQ caused a significant increase in the levels of pIRF7 (n = 9/grp) and TNF-α (n = 5/grp) relative to CTL, and these increases were completely blocked by 3α,5α-THP (One-way ANOVA, Tukey’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001). C Poly(I:C) significantly increased the levels of pIRF3 (n = 5/grp), IP-10 (n = 6/grp), and TNF-α (n = 5/grp) relative to CTL (One-way ANOVA, Tukey’s post hoc test: *p < 0.05). The increases of pIRF3, IP-10, and TNF-α were not inhibited by 3α,5α-THP (One-way ANOVA, Tukey’s post hoc test: p > 0.05). D 3α,5α-THP does not target the non-activated TLR signals. RAW264.7 cells untreated with TLR agonist but exposed to vehicle or treated with 3α,5α-THP (1 µM) were harvested after 24 h. The levels of TRAF6 (n = 6/grp), pERK1/2 (n = 6/grp), pCREB (n = 6/grp), pATF2 (n = 6/grp), TNF-α (n = 6/grp), pIRF7 (n = 6/grp), IP-10 (n = 6/grp), and pIRF3 (n = 6/grp) were similar in the 3α,5α-THP-treated and untreated cells (t-test, p > 0.05). The data indicate that 3α,5α-THP specifically targets only the activated TLR signal.