Cigarette Smoke and Heated Tobacco Product Exhibit Distinct Biochemical Pathways of Microglial Activation Under Hypoxia-Reoxygenation

Tobacco smoking remains the leading cause of preventable death globally. This study examines the effects of cigarette smoke (1R6F) and heated tobacco product (HTP) aerosols on microglial activation, cell proliferation, and proteomic changes under hypox-ia-reoxygenation (H/R) conditions, focusing on nicotine's role in oxidative stress, in-flammation, and Nrf2 pathway activation. H/R conditions significantly activated micro-glia, consistent with prior evidence linking hypoxic stress to neuroinflammatory re-sponses. Cigarette smoke exposure reduced microglial activation, while HTP aerosol and nicotine maintained cellular function, suggesting potentially lower cytotoxicity of HTPs. This supports findings that HTPs may produce fewer toxicants than traditional cigarettes, though long-term brain health impacts remain uncertain. Proteomic analysis indicated that H/R altered microglial protein expression, with 97 proteins related to RNA metab-olism, oxidative phosphorylation, and cellular stress responses. The increased expression of RNA-binding proteins suggests an adaptive response to oxidative damage. Both cigarette smoke and HTP aerosols influence oxidative stress-related proteins differently. Confocal microscopy showed that HTP and nicotine maintained Nrf2 nuclear transloca-tion, an antioxidant response, while cigarette smoke impaired Nrf2 activation, indicating higher oxidative stress and potential cellular damage. The differential activation of the Nrf2 antioxidant pathway suggests that HTPs may be less harmful than traditional cig-arettes, though their long-term effects on cerebrovascular health warrants further as-sessment.