Modified Anshen Dingzhi Prescription Ameliorates Tourette Syndrome and Comorbid Anxiety by Modulating Synaptic Plasticity and the cAMP/PKA/CREB Signaling Pathway

Technical Summary: Modified Anshen Dingzhi Prescription Ameliorates Tourette Syndrome and Comorbid Anxiety

Problem Statement
Tourette Syndrome (TS) is a complex neurodevelopmental disorder characterized by motor and vocal tics, frequently comorbid with anxiety disorders (affecting 31.4%–72.7% of patients). While tics may attenuate in adulthood, comorbid anxiety often persists, significantly compromising quality of life and increasing suicide risk. Current pharmacological interventions, such as α2-adrenergic agonists and antipsychotics, often suffer from limited efficacy and suboptimal tolerability, struggling to simultaneously address tic suppression and anxiety alleviation. In Traditional Chinese Medicine (TCM), TS tics are attributed to "internal stirring of liver wind," while anxiety stems from an "unsettled heart-spirit." The Modified Anshen Dingzhi Prescription (MADP), a nine-herb formula designed to extinguish liver wind and tranquilize the spirit, has shown clinical promise, yet its multi-component molecular mechanisms remain unelucidated.

Methodology
This study employed a multi-faceted approach combining in vivo animal modeling, chemical profiling, and integrated omics to investigate MADP's efficacy and mechanism.

• Animal Model: A rat model of TS comorbid with anxiety was established using 3,3'-iminodipropionitrile (IDPN) injections (to induce tics) combined with chronic restraint stress (CRS) (to induce anxiety).
• Intervention: Rats were treated with MADP at low (clinical equivalent) and high (2x clinical) doses, or the positive control Tiapride, for three weeks.
• Behavioral Assessment: Stereotypic behaviors were scored using the Diamond method, and anxiety-like behaviors were assessed via the Open Field Test (OFT).
• Histology: Prefrontal cortex (PFC) neuronal integrity was evaluated using Nissl staining, while dendritic spine density and morphology were analyzed via Golgi staining.
• Chemical Profiling: UPLC-MS/MS was used to identify bioactive constituents in the MADP aqueous extract.
• Integrated Pharmacology & Transcriptomics: Network pharmacology predicted hub targets and pathways based on chemical constituents and disease databases. Transcriptomic analysis (RNA-seq) of PFC tissues was performed to identify differentially expressed genes (DEGs).
• Molecular Validation: Western blotting and immunofluorescence were used to validate the expression of proteins in the cAMP/PKA/CREB signaling pathway and synaptic markers (PSD95, Synapsin I+II).

Key Contributions and Results

• Chemical Composition: UPLC-MS/MS identified 135 bioactive constituents in MADP, including saikosaponin A, paeoniflorin, tenuifolin, and α-asarone.
• Behavioral Efficacy: MADP treatment significantly attenuated stereotypic behavior scores and improved abnormal activity patterns in the OFT compared to the untreated model group.
• Structural Restoration: Histological analysis revealed that MADP restored neuronal integrity in the PFC. Specifically, it significantly increased dendritic spine density, total neurite length, and dendritic arbor complexity, reversing the structural deficits induced by IDPN and CRS.
• Mechanistic Pathway: Network pharmacology and transcriptomics converged on the cAMP signaling pathway as a critical intervention route.
• Molecular Validation: Western blot analysis demonstrated that MADP significantly upregulated total protein levels of PKA and CREB, enhanced CREB phosphorylation (p-CREB), and increased the expression of synaptic markers PSD95 and Synapsin I+II. Notably, the study found no significant alteration in p-PKA levels, suggesting the effect is mediated through the activation of the CREB branch of the pathway.

Significance and Claims
The authors claim that this study provides scientific mechanistic evidence supporting the clinical application of MADP for TS comorbid with anxiety. The findings suggest that MADP ameliorates behavioral symptoms by activating the cAMP/PKA/CREB signaling pathway, which subsequently restores synaptic plasticity in the prefrontal cortex. The study posits that stress-induced PFC dysfunction serves as a common pathological bridge between tics and anxiety, and that MADP disrupts this cycle by regulating synaptic protein synthesis and dendritic morphology.

The authors maintain a modest tone regarding future implications, acknowledging that while the study establishes a holistic mechanism, the precise pharmacological material basis (specific blood-brain barrier penetrating components) and direct binding affinities of monomers require further investigation. They also note that the animal model may not fully capture clinical heterogeneity and that the excessive stereotypic behaviors in the model may have masked certain anxiety phenotypes, suggesting the need for diverse behavioral paradigms in future research.