Chronic diazepam administration increases the expression of Lcn2 in the CNS

Benzodiazepines (BZDs), which bind with high affinity to gamma-aminobutyric acid type A receptors (GABAA-Rs) and potentiate the effects of GABA, are widely prescribed for anxiety, insomnia, epileptic discharge, and as anticonvulsants. The long-term use of BZDs is limited due to adverse effects such as tolerance, dependence, withdrawal effects, and impairments in cognition and learning. Additionally, clinical reports have shown that chronic BZD treatment increases the risk of Alzheimer's disease. Unusual GABAA-R subunit expression and GABAA-R phosphorylation are induced by chronic BZD use. However, the gene expression and signaling pathways related to these effects are not completely understood. In this study, we performed a microarray analysis to investigate the mechanisms underlying the effect of chronic BZD administration on gene expression. Diazepam (DZP, a BZD) was chronically administered, and whole transcripts in the brain were analyzed. We found that the mRNA expression levels were significantly affected by chronic DZP administration and that lipocalin 2 (Lcn2) mRNA was the most upregulated gene in the cerebral cortex, hippocampus, and amygdala. Lcn2 is known as an iron homeostasis-associated protein. Immunostained signals of Lcn2 were detected in neuron, astrocyte, microglia, and Lcn2 protein expression levels were consistently upregulated. This upregulation was observed without proinflammatory genes upregulation, and was attenuated by chronic treatment of deferoxamine mesylate (DFO), iron chelator. Our results suggest that chronic DZP administration regulates transcription and upregulates Lcn2 expression levels without an inflammatory response in the mouse brain. Furthermore, the DZP-induced upregulation of Lcn2 expression was influenced by ambient iron.