EGR2 and NLRP3 Pathways in Obstructive Sleep Apnea-Related Cognitive and Mood Disorders

"Peripheral blood collection \& PBMC isolation:~Children: 3 mL venous blood drawn from the right antecubital vein preoperatively; Adults: 3 mL fasting venous blood drawn the morning after PSG.~Collected in EDTA tubes; PBMCs separated via Ficoll-Paque density gradient. Flow cytometry: 1×10⁶ PBMCs stained for CD14/CD16, HLA-DR, CD11b, CD80/CD86, CD163/CD206.~RNA extraction: Remaining PBMCs lysed in TRIzol and stored at -80 °C for RT-qPCR of EGR2, NLRP3, and downstream genes Serum/plasma: Within 1 h of collection, centrifuge at 400 × g for 10 min at 4 °C; 1 mL used for ELISA quantification of TNF-α, IL-6, IL-1β, CCL2, IL-17, CRP; remainder stored at -80 °C for future proteomic or metabolomic assays~Saliva sampling \& processing:~2-3 mL unstimulated saliva expectorated into sterile tubes, kept at 4 °C, processed (centrifuged, aliquoted) within 2 h, then stored at -80 °C."

"Subcutaneous fat biopsy (adults undergoing surgery):~100-200 mg obtained intraoperatively, placed in RNAlater at 4 °C for 24 h, then frozen at -80 °C for downstream RNA-seq, RT-qPCR, and Western blot analyses of EGR2, NLRP3, and related inflammatory markers"