CNS neuroscience & therapeutics · 2026
Olfml3 Regulates Microglial Inflammation and Neuronal Injury in Obstructive Sleep Apnea via Cybb-Mediated TLR4/NF-κB Pathway.
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Kong Deqiu, Wang Yaowen, Chen Xianjun, Hu Cihao, Zhang Bin, Chen Xing
Аннотация
BACKGROUND: Neurocognitive impairment in obstructive sleep apnea (OSA) is primarily driven by intermittent hypoxia (IH)-triggered neuroinflammation, where microglia play a pivotal role. The involvement of Olfml3 in IH-induced neuroinflammation remains unclear.
METHODS: Single-cell RNA sequencing (scRNA-seq) data from the hippocampi of IH-induced OSA mice were analyzed to identify cell subpopulations, with further focus on Olfml3's differential expression, enriched pathways, and differentiation trajectories in microglia. An in vitro OSA model was established using IH-treated microglia. qRT-PCR and western blot (WB) were utilized to assess Olfml3 and cytochrome b (Cybb) expression. Microglial polarization was evaluated via flow cytometry, while Enzyme-Linked Immunosorbent Assay (ELISA) was applied to quantify inflammatory cytokines. Reactive oxygen species (ROS) were detected using fluorescent probes, and TLR4/NF-κB pathway activation was verified by WB assessment of Toll-like receptor 4 (TLR4), phosphorylated-p65 (p-p65), and p65 expression. Neuronal injury was assessed by treating neurons with microglial-conditioned medium, followed by CCK-8 for viability assessment and flow cytometry for apoptosis analysis. An in vivo OSA model was constructed by exposing mice to IH treatment. Cognitive deficits of mice were evaluated using the Morris water maze and blood oxygen saturation measurement, while pathological changes in brain tissue and cell apoptosis were examined via HE and TUNEL staining. Immunohistochemistry staining was employed to detect Olfml3 and Cybb protein levels. An immunofluorescence assay was conducted to measure Iba1 for evaluating microglial activation. ROS levels were detected by using fluorescent probes. The expression of TLR4/NF-κB pathway proteins was assessed by WB. CD86/CD206 ratios were analyzed by flow cytometry, and the expression of inflammatory cytokines was analyzed by ELISA.
RESULTS: scRNA-seq revealed reduced microglial proportions under hypoxia, with further analysis revealing that Olfml3 in microglia had a negative correlation with Cybb. An IH-induced OSA model confirmed that Olfml3 overexpression alleviated microglial inflammation and neuronal injury by suppressing the TLR4/NF-κB pathway via Cybb. In vivo experiments further validated Olfml3's protective role against IH-induced neuroinflammation in OSA.
CONCLUSION: Olfml3 in microglia mitigates IH-induced proinflammatory activation and neuronal injury via the Cybb/TLR4/NF-κB axis, thereby conferring neuroprotection against OSA-associated neuroinflammation.
METHODS: Single-cell RNA sequencing (scRNA-seq) data from the hippocampi of IH-induced OSA mice were analyzed to identify cell subpopulations, with further focus on Olfml3's differential expression, enriched pathways, and differentiation trajectories in microglia. An in vitro OSA model was established using IH-treated microglia. qRT-PCR and western blot (WB) were utilized to assess Olfml3 and cytochrome b (Cybb) expression. Microglial polarization was evaluated via flow cytometry, while Enzyme-Linked Immunosorbent Assay (ELISA) was applied to quantify inflammatory cytokines. Reactive oxygen species (ROS) were detected using fluorescent probes, and TLR4/NF-κB pathway activation was verified by WB assessment of Toll-like receptor 4 (TLR4), phosphorylated-p65 (p-p65), and p65 expression. Neuronal injury was assessed by treating neurons with microglial-conditioned medium, followed by CCK-8 for viability assessment and flow cytometry for apoptosis analysis. An in vivo OSA model was constructed by exposing mice to IH treatment. Cognitive deficits of mice were evaluated using the Morris water maze and blood oxygen saturation measurement, while pathological changes in brain tissue and cell apoptosis were examined via HE and TUNEL staining. Immunohistochemistry staining was employed to detect Olfml3 and Cybb protein levels. An immunofluorescence assay was conducted to measure Iba1 for evaluating microglial activation. ROS levels were detected by using fluorescent probes. The expression of TLR4/NF-κB pathway proteins was assessed by WB. CD86/CD206 ratios were analyzed by flow cytometry, and the expression of inflammatory cytokines was analyzed by ELISA.
RESULTS: scRNA-seq revealed reduced microglial proportions under hypoxia, with further analysis revealing that Olfml3 in microglia had a negative correlation with Cybb. An IH-induced OSA model confirmed that Olfml3 overexpression alleviated microglial inflammation and neuronal injury by suppressing the TLR4/NF-κB pathway via Cybb. In vivo experiments further validated Olfml3's protective role against IH-induced neuroinflammation in OSA.
CONCLUSION: Olfml3 in microglia mitigates IH-induced proinflammatory activation and neuronal injury via the Cybb/TLR4/NF-κB axis, thereby conferring neuroprotection against OSA-associated neuroinflammation.