Nr1d1 mediates microglial inflammatory activation induced by intermittent hypoxia in BV2 cells

Obstructive sleep apnea (OSA) is characterized by recurrent intermittent hypoxia (IH), a major contributor to neuroinflammation and subsequent neurological dysfunction. While microglia are recognized as central regulators of inflammatory responses within the central nervous system, the precise molecular mechanisms driving IH-induced microglial activation remain largely undefined. Current therapeutic strategies for OSA primarily focus on alleviating hypoxia, but often fail to fully address the downstream neuroinflammatory sequelae, highlighting a critical gap in understanding and targeting these specific pathways.

Researchers established an intermittent hypoxia (IH) model in BV2 cells to mimic OSA conditions. Inflammation was quantitatively assessed using western blot for key markers cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS), alongside ELISA to measure pro-inflammatory cytokines Interleukin-6 (IL-6) and Tumour necrosis factor-alpha (TNF-α). To identify transcriptional changes, RNA sequencing was performed on IH-exposed cells. An integrated approach combining differential expression analysis, functional enrichment, protein interaction networks, transcription factor prediction, and machine learning-based screening was employed to pinpoint candidate key genes. Finally, the role of Nr1d1 was evaluated through pharmacological modulation (inhibition) to assess its impact on IH-induced inflammation.

Exposure to intermittent hypoxia (IH) in BV2 cells significantly increased the levels of inflammatory markers, with COX2 and iNOS protein expression, and IL-6 and TNF-α cytokine secretion all showing pronounced elevations. RNA sequencing revealed distinct transcriptional profiles between control and IH-treated groups, indicating a substantial reprogramming of gene expression. Functional enrichment analyses linked these IH-responsive genes to critical biological processes including immune regulation, stress responses, and metabolic pathways. Network analysis further highlighted a prominent circadian gene cluster as a central component of the IH-induced transcriptional network. > Integrated network and machine learning analyses converged on Nr1d1 as a key candidate gene mediating these inflammatory responses. Subsequent pharmacological experiments demonstrated that inhibition of Nr1d1 attenuated the IH-induced inflammatory activation, reducing the observed increases in inflammatory markers.