Dingyue Chai,1 Yuzi Sun,1 Jiamin Lu,1 Yuhui Yao,2 Chunyu Jiang,1 Lihui Wu,1 Qianqian Cai3 

1Departments of Basic Medicine and Forensic Medicine, Hangzhou Medical College, Hangzhou, People’s Republic of China; 2Department of Paediatrics, The First Hospital of Jiaxing, Zhejiang, People’s Republic of China; 3Shanghai Key Laboratory of Molecular Imaging, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences, Shanghai, 201318, People’s Republic of China

Correspondence: Lihui Wu, Email jaemny@163.com Qianqian Cai, Email caiqq@sumhs.edu.cn

Purpose: Attention-Deficit/Hyperactivity Disorder (ADHD) is one of the most prevalent neurodevelopmental disorders in childhood, with a globally increasing incidence. This study aims to investigate alterations in prefrontal cortical lipid metabolism in ADHD model rats and following transcutaneous auricular vagus nerve stimulation (taVNS) intervention, and to elucidate the regulatory effects of the sphingosine kinase inhibitor SKI II on the SPHK-S1P signaling pathway.
Methods: Lipidomic analysis was performed to profile the lipid spectrum in prefrontal cortex tissues from Wistar-Kyoto (WKY) control rats, spontaneously hypertensive rat (SHR) ADHD models, sham-operated rats, and taVNS intervention rats. Key sphingolipid metabolic enzymes were assessed by RT-qPCR and Western blot, while sphingosine-1-phosphate (S1P) levels were quantified via ELISA. ADHD model rats received intraperitoneal administration of SPHK inhibitor SKI II (15 mg/kg, 16 days). Behavioral tests evaluated hyperactivity, impulsivity, and anxiety-like phenotypes. Western blot analyzed expression of dopamine synthesis rate-limiting enzyme.
Results: The ADHD model group exhibited significantly elevated ganglioside and lysophospholipid levels. taVNS intervention specifically reduced sphingomyelin content. SPHK1 and SPHK2 mRNA and protein expression were markedly upregulated in ADHD models, concomitant with increased S1P levels. taVNS selectively decreased Sphk2 mRNA expression (without altering protein levels). SKI II administration significantly ameliorated hyperactivity, impulsivity, and anxiety-like behaviors in ADHD models, concurrently restoring dopamine β-hydroxylase expression.
Conclusion: The SPHK-S1P signaling axis is a core pathway driving sphingolipid metabolic dysregulation in the prefrontal cortex in ADHD. Targeted inhibition of this pathway synergistically modulates the expression levels of proteins associated with dopaminergic neurotransmission. The limited regulatory effect of taVNS on sphingolipid metabolism suggests its clinical benefits may stem from multi-pathway synergistic mechanisms.

Keywords: attention-deficit/hyperactivity disorder, sphingolipid metabolism, SPHK-S1P, taVNS