Abstract
<title>Abstract</title> <p> Background Tourette Syndrome (TS) are complex neurodevelopmental conditions frequently comorbid with anxiety. Modified Anshen Dingzhi Prescription (MADP), an empirical Chinese herbal formula, has demonstrated dual therapeutic benefits in suppressing tics and regulating emotions; however, its multi-component mechanisms remain to be fully elucidated. Objective This study investigated the therapeutic efficacy of MADP on TS comorbid with anxiety and explored its molecular mechanisms involving prefrontal cortex (PFC) synaptic plasticity and the cAMP/PKA/CREB signaling pathway. Methods A rat model of TS comorbid with anxiety was induced by 3,3'-iminodipropionitrile (IDPN) and chronic restraint stress (CRS). Therapeutic effects were assessed using stereotypic behavior scores and the open field test (OFT). PFC neuronal morphology and dendritic spine density were observed through Nissl and Golgi staining. Chemical profiling of MADP was performed via UPLC-MS/MS. Integrated network pharmacology and transcriptomics were employed to predict hub targets and pathways, followed by validation using Western blot and immunofluorescence. Results MADP treatment significantly attenuated stereotypic behavior scores and improved abnormal activity patterns. Pathological observations revealed that MADP effectively restored neuronal integrity in the PFC, significantly increasing dendritic spine density, total neurite length, and dendritic arbor complexity. UPLC-MS/MS identified 135 bioactive constituents. Multi-omics integration indicated that the cAMP signaling pathway is a critical intervention route. Molecular validation demonstrated that MADP significantly upregulated protein levels of PKA and CREB, enhanced CREB phosphorylation (p-CREB), and increased the expression of the synaptic markers PSD95 and Synapsin I + II ( <italic>P</italic> < 0.05). Conclusion MADP effectively ameliorates behavioral symptoms in TS comorbid with anxiety by activating the cAMP/PKA/CREB signaling pathway and restore PFC synaptic plasticity. These findings provide scientific mechanistic evidence supporting the clinical application of MADP. </p>