<b>Decoding the peripheral transcriptomic and meta-genomic response to music in Autism Spectrum Disorder </b><b><i>via</i></b><b> saliva-based RNA sequencing</b>

Saliva-based RNA sequencing (RNA-seq) poses technical challenges due to high bacterial content, RNA degradation, and sample heterogeneity. This study investigates the transcriptional effects of music exposure in individuals with autism spectrum disorder (ASD) using this non-invasive approach. To address saliva-specific limitations, we employed two complementary library preparation methods, Poly-A selection and Human-Enriched protocols, allowing us to maximize human transcript detection and ensure reproducibility. By merging them, we ensured reproducibility and captured both host and microbial signals. While each dataset individually revealed a limited number of differentially expressed genes (DEGs), their integration enhanced biological resolution. Among the consistently modulated genes were HERC6, TSPAN5, and REM2, pointing to music-induced transcriptional changes relevant to neurodevelopmental and immune processes. Functional enrichment analyses highlighted pathways involved in immune regulation, oxidative phosphorylation, and epithelial differentiation. These findings align with evidence of immune dysregulation, mitochondrial dysfunction, and altered cellular communication in ASD. Importantly, co-expression network analysis identified modules significantly correlated with music exposure. Notably, the AKNA module, previously associated with ASD risk, was downregulated and enriched for Ras-related GTPase signaling and immune pathways, suggesting that music may modulate intracellular signaling and inflammation. Conversely, upregulation of the UBE2D3 module pointed to activation of endoplasmic reticulum stress response mechanisms, a known contributor to ASD neurodevelopmental deficits. These results suggest that music engages specific stress-adaptive and immunomodulatory networks in buccal cells, potentially reflecting systemic effects. Our exploratory metagenomic analysis highlights 15 microbial species with consistent abundance shifts across both methods. Notably, Acidipropionibacterium acidipropionici and Propionibacterium freudenreichii, associated with propionic acid production, emerged as music-responsive taxa. Elevated propionic acid has been implicated in ASD-like behaviors and neuroinflammation, suggesting a microbiota-mediated pathway. Music may influence both host gene expression and oral microbiota, potentially affecting neuroimmune processes via the microbiota–brain axis. Although exploratory, the results support the feasibility of using saliva for integrated molecular profiling in ASD.基于唾液的RNA测序（RNA-seq）因样本细菌含量高、RNA易降解及样本异质性等问题，面临诸多技术挑战。本研究借助这一无创检测手段，探究音乐暴露对孤独症谱系障碍（ASD）患者转录组的调控效应。为克服唾液样本特有的技术局限，我们采用了两种互补的建库方案：Poly-A富集（Poly-A selection）与人类转录本富集建库方法（Human-Enriched protocols），以此最大化人类转录本的检出效率并保障实验可重复性。通过整合两种方法，我们既确保了结果的可重复性，同时捕获到宿主与微生物的双重信号。尽管单一数据集仅能检出少量差异表达基因（DEGs），但二者的整合分析显著提升了生物学解析度。在持续受音乐调控的基因中，HERC6、TSPAN5与REM2的表达变化提示，音乐可诱导与神经发育及免疫过程相关的转录组改变。功能富集分析显示，免疫调控、氧化磷酸化及上皮细胞分化相关通路显著富集。上述发现与孤独症谱系障碍中存在免疫失调、线粒体功能障碍及细胞通讯异常的既有研究证据相符。值得注意的是，共表达网络分析鉴定出与音乐暴露显著相关的基因模块。其中，此前被证实与孤独症谱系障碍风险相关的AKNA模块出现表达下调，且该模块富集于Ras相关GTP酶信号通路与免疫通路，提示音乐可能调控细胞内信号转导与炎症反应。与之相反，UBE2D3模块的表达上调则表明内质网应激应答机制被激活，而这一过程已被证实与孤独症谱系障碍的神经发育缺陷密切相关。上述结果表明，音乐可激活口腔颊黏膜细胞中特定的应激适应与免疫调控网络，这一现象或可反映机体的系统性效应。本研究的探索性宏基因组分析共鉴定出15种丰度呈现一致性变化的微生物物种。其中，与丙酸生成相关的嗜酸丙酸杆菌（Acidipropionibacterium acidipropionici）和费氏丙酸杆菌（Propionibacterium freudenreichii）被鉴定为音乐响应性微生物类群。高浓度丙酸已被证实与类孤独症行为及神经炎症相关，这提示存在一条由微生物群介导的作用通路。音乐或可同时调控宿主基因表达与口腔微生物群，进而通过微生物群-脑轴影响神经免疫过程。尽管本研究属于探索性工作，但其结果证实了采用唾液样本开展孤独症谱系障碍整合分子谱分析的可行性。