후각 소실에서 비강 내 니코틴아미드 아데닌 디뉴클레오타이드 투여의 치료 효과 연구

Abstract

Olfactory dysfunction is a debilitating condition with no established treatment. This study aimed to assess the efficacy of intranasal (i.n.) administration of nicotinamide adenine dinucleotide (NAD) in restoring olfactory function. Cultured human olfactory stem cells (hOSCs) were used to evaluate the therapeutic efficacy of NAD in vitro. Immunofluorescence (IF) staining, PCR, and Western blot analyses were employed to determine whether NAD treatment facilitated the differentiation of cultured hOSCs into olfactory sensory neurons (OSNs). Four experimental groups were used for the in vivo study: control, NAD i.n. (NAD group), intraperitoneal (i.p.) injection of dexamethasone (DXM group), and anosmia (n = 10 per group). Chronic ZnSO4 i.n. administration induced olfactory dysfunction in the mouse model. Intranasal administration of 20 μL ZnSO4 (170 μM) in each nostril (40 μL in total) was performed three times a week for four weeks. The NAD group received daily NAD i.n, while the DXM group received DXM (1 mg/kg) i.p. injections once a week for four weeks. The control and anosmia groups mice were treated with daily i.n. phosphate-buffered saline (20 μL in each nostril; 40 μL in total). Restoration of olfactory function was evaluated using histological analysis and behavioral tests. Further investigation of the mechanism was performed via comprehensive gene expression analyses using RNA sequencing (RNA-seq). IF staining of OSN markers (OMP, acetylated β-III tubulin) and neuronal stem cell markers (SOX2, nestin) revealed that NAD promoted the differentiation of hOSCs into OSNs in vitro. PCR analyses showed that NAD enhanced the expression of neuronal differentiation-related markers (SOX2, NESTIN, NEUROD1, NEUROG1, and OMP). Additionally, Western blot analysis demonstrated increased levels of SOX2, nestin, and OMP protein levels after NAD treatment. In vivo, the olfactory function of the NAD group significantly improved by day 28 compared with the anosmia group, whereas the DXM group showed no improvement. Histological analyses revealed that the olfactory epithelium (OE) in the NAD group was markedly repaired, with both the thickness of the OE and the number of OSNs significantly higher than those in the DXM and anosmia groups. Bulk RNA-seq of the olfactory turbinate tissue identified two unique gene expression patterns and 201 differentially expressed genes (DEGs). Of these, 113 DEGs (cluster T1) were upregulated in control and NAD group mice, while 88 DEGs (cluster T2) were downregulated exclusively in control mice. Gene ontology terms associated with Cluster T1 included "modulation of chemical synaptic transmission", "regulation of nervous system process", and "response to steroid hormone". Cellular deconvolution using a validated public single-cell RNA sequencing (scRNA-seq) dataset identified candidate genes expressed in olfactory receptor neurons. Twenty-five genes implicated in the modulation of synaptic chemical transmission were upregulated after NAD stimulation. Six candidate genes were identified by intersecting them with neuronal marker genes acquired from scRNA-seq analysis data: ABHD2, DLGAP2, FOXO3, HIPK2, KCNMA1, and PCDH17. These genes were significantly upregulated in the NAD group compared with the anosmia group, with their expression levels restored to those observed in the control group. PCR analysis confirmed significantly higher expression levels of DLGAP2, FOXO3, HIPK2, and PCDH17 in differentiated hOSCs treated with NAD in vitro. This study demonstrated the potential therapeutic efficacy of NAD for regenerating OSNs in vitro and restoring olfactory function in mice with anosmia. Our comprehensive evaluation indicated promising outcomes for the use of NAD as a potential treatment for olfactory dysfunction. Keywords: olfaction, anosmia, intranasal administration, nicotinamide adenine dinucleotide