关键词: 香精香料, 高通量测序, 神经递质, 卷烟, RNA-seq分析, snRNA-seq分析

Abstract: BACKGROUND: Fragrances and flavorts are closely associated with the brain’s nervous system; however, their long-term effects on brain tissue under the context of cigarette smoke delivery systems remain poorly understood.
OBJECTIVE: To focus on the molecular mechanisms by which fragrances and flavors influence the neural system of brain tissue and reveal their potential neuroregulatory value in cigarette products by systematically evaluating the effects of different flavoring interventions on mouse neurobehaviors and cellular molecular networks.
METHODS: Adult male C57BL/6J mice were randomly assigned to two experimental systems. In Experiment I, mice were divided into a solvent aerosol group, a control flavoring aerosol group, and a modified flavoring aerosol group. In Experiment II, mice were divided into a control group, a blank flavored cigarette group, a control flavored cigarette group, and a modified flavored cigarette group. Mice were concentrated exposed to flavors and fragrances either via aerosol inhalation or through combustion in cigarette smoke for 45 consecutive days, twice daily, with each exposure lasting 45 minutes. Open field tests were conducted at weeks 2 and 4 to evaluate spontaneous locomotor activity within a 10-minute observation period. RNA sequencing was performed to identify differentially expressed genes and enriched Kyoto Encyclopedia of Genes and Genomes pathways in brain tissue, while single-nucleus RNA sequencing was employed to characterize major cellular subpopulations and associated biological processes of Gene Ontology. 
RESULTS AND CONCLUSION: (1) In Experiment I, open field test results demonstrated that mice exposed to both control and modified flavoring aerosols exhibited significantly enhanced locomotor activity and increased central zone exploration compared with the solvent group (P < 0.05). (2) RNA sequencing results revealed a markedly higher number of differentially expressed genes in the modified flavoring aerosol group compared with the solvent group. Kyoto Encyclopedia of Genes and Genomes pathway enrichment indicated significant involvement of glutamatergic synapse, amphetamine addiction, and neuroactive ligand-receptor interaction pathways, with notable upregulation of the G protein-coupled receptor ligand binding pathway in the modified group. (3) snRNA sequencing results identified neurons and microglia as the primary responsive cell populations following flavoring aerosol exposure. Differentially expressed genes in neuronal subpopulations were predominantly enriched in processes such as trans-synaptic signaling, mitochondrial electron transport chain, and oxidative phosphorylation, while oligodendrocyte lineage cells were predominantly enriched in electron transport, myelination formation, and their regulatory pathways. (4) In Experiment II, mice exposed to modified flavored cigarettes also demonstrated significantly increased locomotor activity and exploratory behavior in the central zone compared with the blank flavored cigarette group (P < 0.05). (5) RNA sequencing analysis showed comparable numbers of differentially expressed genes between the groups. Among them, the control flavored cigarette group exhibited enrichment in neuroactive ligand-receptor interaction, cholinergic and glutamatergic synapses, whereas the modified group was predominantly associated with neurotransmitter homeostasis, excitability regulation, and metabolic pathways. (6) Single-nucleus RNA sequencing analysis revealed significant alterations in neuronal and microglial subpopulations. In the modified flavored cigarette group, neuronal differentially expressed genes were enriched in GABAergic synapse, membrane potential regulation, transmembrane transport, and chemical synaptic transmission; oligodendrocyte-related differentially expressed genes were enriched in dendrite development, postsynaptic neurotransmitter receptor regulation, and synaptic organization. These findings suggest that modified flavors and fragrances, through targeted effects on neurons and oligodendrocytes, modulate synaptic plasticity, neurotransmitter metabolism, myelination, and mitochondrial energy pathways, thereby altering neuronal excitability and functional states in the brain. This study offers a theoretical foundation and research paradigm for investigating the regulatory functions of flavors, fragrances, and their applied products in the nervous system of brain tissue. 

Key words: flavors and fragrances, high-throughput sequencing, neurotransmitters, cigarettes, RNA-seq analysis, snRNA-seq analysis