Microbiota-gut-brain axis and neuroendocrine pathways underlie divergent mechanisms of intermittent and continuous theta-burst stimulation in autism spectrum disorder

Theta-burst stimulation, including intermittent (iTBS) and continuous (cTBS) protocols, is a promising neuromodulatory intervention for autism spectrum disorder (ASD). This study aims to elucidate the therapeutic mechanisms of iTBS and cTBS for ASD. Prenatal valproic acid-induced ASD rats were established and were randomized into VPA, VPA + iTBS, and VPA + cTBS groups, with a saline group as control. Core and comorbid ASD behaviors in rats were assessed. Multi-omics analyses included 16 S rRNA sequencing of cecal contents, non-targeted fecal metabolomics, and prefrontal cortex transcriptomics. Key pathways were validated via Western blot, ELISA, and immunofluorescence. Integrative analyses correlated multi-omics data with neuroendocrine findings. Behavioral assessments demonstrated that both iTBS and cTBS significantly ameliorated social deficits and repetitive behaviors in VPA-exposed rats. However, protocol-specific effects on comorbidities were observed: cTBS, but not iTBS, effectively alleviated anxiety-like behaviors, whereas iTBS, but not cTBS, significantly improved learning and memory. The multi-omics approach demonstrated that iTBS primarily modulated inflammatory immune responses and energy metabolism, while cTBS predominantly regulated oxidative stress, lipid metabolism, and nucleotide metabolism. Both interventions suppressed the hyperactivated PI3K/AKT/mTOR signaling pathway, an effect potentially linked to the normalization of hypothalamic-pituitary axis function. Furthermore, we identified a potential interplay between the GH/IGF-1 axis and the gut microbiome in ASD, which was differentially modulated by iTBS and cTBS. iTBS modulated inflammatory-immune responses and energy metabolism, while cTBS regulated oxidative stress, lipid metabolism, and nucleotide metabolism. The inhibition of the central GH/PI3K/AKT/mTOR pathway by both protocols may involve their specific regulation of distinct gut microbiota communities.