The characterization of neuroimmune actions of [3α,5α]3-hydroxypregnan-20-one in alcohol-preferring rat brain.

The endogenous neurosteroid (3α,5α)3-hydroxypregnan-20-one (3α,5α-THP) has anti-depressive, anti-convulsive, sedative, anesthetic, anxiolytic, and anti-inflammatory properties. 3α,5α-THP inhibits the myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptors (TLRs) in murine and human macrophages and the brain of alcohol-preferring (P) rats. The activation of TLRs is associated with high alcohol intake in alcohol use disorder (AUD) in human and rodent brains. Previous activation of the immune system increases the risk for AUD development. In the ethanol naïve P rats, TLR4 is innately activated, monocyte chemoattractant protein-1 (MCP-1) is overexpressed, and fractalkine (CX3CL1) is depleted, predisposing the rats to binge drinking. 3α,5α-THP inhibits MCP-1 and increases CX3CL1 in the P rats’ nucleus accumbens (NAc) and central amygdala (CeA). A prolonged pro-inflammatory state can lead to weakened blood-brain barrier, invasion of peripheral immune cells, and neurodegeneration. This work investigates the impact of 3α,5α-THP on the cellular expression of MCP-1 and CX3CL1 in neurons, microglia, and astroglia using P rats as a model of neuroinflammation. We confirmed that innate overexpression of the pro-inflammatory signaling is observed in the NAc and CeA of the P rat by examining the expression of the TLR ligand danger-associated molecular pattern, high mobility group box (HMGB1) compared to their parental strain, the Wistar rat. 3α,5α-THP inhibited HMGB1 expression in P rats, not Wistar rats. We observed that 3α,5α-THP inhibited MCP-1 in neurons, microglia, and astroglia, while concurrently decreasing the microglia marker ionized calcium-binding adaptor protein-1 (IBA1) without affecting the neuronal marker neuronal nuclear antigen (NeuN) or the astroglia marker glial fibrillary acidic protein (GFAP). Subsequently, we observed that 3α,5α-THP influences CX3CL1 with sex and brain region specification. 3α,5α-THP decreased neuronal CX3CL1 in the NAc in females, amplified neuronal CX3CL1 in the CeA in females and males, and had no effect in the NAc of males. Lastly, 3α,5α-THP did not alter the residential microglia marker transmembrane protein 119 (TMEM119). Our results report that 3α,5α-THP modulates pro-inflammatory signaling and macroglia in the naïve P rats. This adds to evidence of the neuroprotective properties of 3α,5α-THP as a beneficial treatment for neuronal-driven inflammatory disorders.