Tourette syndrome (TS), also known as tourette disorder, is a neurodevelopmental condition characterized by persistent vocal and motor tics that cause distress and impair daily functioning. (First et al., 2012). Estimates suggest that TS affects between 0.1% and 1% of children. (Nilles et al., 2023; Efron and Dale, 2018). The presence of psychiatric comorbidities, such as attention deficit-hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD), can have a more significant impact than the tics themselves. (Eapen et al., 2016a; Ogundele and Ayyash, 2018; Cavanna et al., 2020). Research indicates that a large proportion, up to 85%, of children with TS also experience other neurodevelopmental or mental health conditions. (Kattner, 2022). Additionally, individuals with TS commonly exhibit symptoms of anxiety, behavior problems, learning difficulties, sleep disturbances, impaired social interaction, and sensory processing challenges. (Nilles et al., 2023; Lin et al., 2022; Set and Warner, 2021). Numerous studies consistently show that both children and adults with TS have a reduced quality of life. (Eapen et al., 2016b; Gilbert et al., 2024).

The pathophysiology is of TS remains unclear, including various defects in neurobiology caused by inherited genetics, surrounding environment, infections, and psychosocial elements. (Kattner, 2022; Lin et al., 2022; Johnson et al., 2023; Billnitzer and Jankovic, 2020). Abnormalities in the pathways that connect the cerebral cortex and basal ganglia have been identified as causing neuronal disinhibition in the motor and limbic systems. (Cavanna et al., 2020; Ramkiran et al., 2019). Evidence also suggests that the sensory limbic and executive corticostriatal loops play a role in this process. (Felling and Singer, 2011). In terms of biochemistry, multiple neurochemical pathways are implicated, involving imbalances in amine neurotransmitters and other signaling molecules such as endogenous opioid peptides. (Szejko et al., 2022). Besides, TS appears to have a strong genetic foundation, with an inheritance pattern most likely controlled by multiple genes. (Lin et al., 2022; Domènech et al., 2021). The diagnosis and clinical management of the disease are significantly influenced by the immune microenvironment which consists of immune cells, inflammatory and growth factors, extracellular matrix and etc. (Tsetsos et al., 2024) In some cases, abnormalities in immune activation can lead to inflammation, which appears to play a pathogenic role. (Lin et al., 2022; Martino et al., 2020).

The treatment approach for Tourette Syndrome (TS) varies depending on the type and severity of the injury sustained by the patient. (Hsu et al., 2021a; Leckman et al., 2005). Acceptance, understanding, education and are typically enough for most patients. However, psychological or pharmacological treatments might become crucial in more serious situations. (Pringsheim et al., 2019a; Pringsheim et al., 2019b). It is important to note that medication is still the main treatment of TS. (Seideman Mf Fau - Seideman and Seideman, 2020). However, current drugs for TS are primarily psychiatric medications, which often have significant side effects. (Chen et al., 2020). The US Food and Drug Administration (FDA) has approved some medications to manage tics in TS, all of which are antipsychotics that act on the D2 receptor. Despite their effectiveness, both first- and second-generation D2 receptor antagonists are associated with side effects such as somnolence, dysphoria, and the risk of drug-induced movement disorders like dystonia, akathisia, withdrawal dyskinesias, and rarely, tardive dyskinesia. (Pringsheim et al., 2019b; Buse et al., 2013). Moreover, the use of D2 receptor antagonists in TS is limited due to the risk of increased weight gain, elevated prolactin, dyslipidemia, and hyperglycemia, including diabetes. However, one of the main challenges in developing drugs for TS is the lack of clear therapeutic targets for its pathogenesis.

Traditional Chinese Medicine (TCM) offers potential alternative treatments for TS. Yangxue Xifeng Decoction (YXD), a herbal formula composed of 10 herbs, has been shown to significantly improve clinical symptoms in TS patients in China. Nevertheless, the therapeutic targets and mechanisms of Chinese medicines (CM), including YXD, are not yet clear due to the complexity of their components. Network pharmacology presents a novel method for examining the correlation between medications and illnesses. By combining multidirectional pharmaceutical biology, systems biology, computer science, and bioinformatics, network pharmacology allows for the systematic investigation of molecular regulatory mechanisms. (Zhou et al., 2020). The multi-target and multi-drug model enables the construction of more efficient networks, which helps in understanding the complex composition and multiple therapeutic targets of CM formulas. (Luo et al., 2020; Shang et al., 2023). Network pharmacology offers an evidence-based approach for researches of clinical efficacy and quality control on CM formulas. (Luo et al., 2020; Zhou et al., 2020; Niu et al., 2022).

In present study, the material basis and action mechanism of YXD for TS, and the potential biomarkers for TS were investigated. Various network pharmacology methods were performed to predict the Targets and Pathways of YXD for TS. SVM algorithm analyses and LASSO regression were performed for identification of TS core genes. Additionally, the relationship between immune cells and central genes, signaling pathways, key genes transcriptional regulation, and genes that synergize with key genes were analyzed.

Although TS is usually considered non-life threatening, it can significantly impact their social interactions and physical and mental health, due to the presence of tics and associated comorbidities. (Eapen et al., 2016a). However, the insufficient comprehension of the pathological mechanisms of TS led to less than ideal therapy outcomes. (Hartmann and Worbe, 2018). The management strategy entails addressing the symptoms that result in impairment, which may necessitate psychological and/or pharmacological intervention. (Billnitzer and Jankovic, 2020; Seideman Mf Fau - Seideman and Seideman, 2020). Antipsychotics, the solitary approved category of pharmacotherapy for tic suppression, possess an undesirable risk profile associated with weight gain, metabolic irregularities, electrocardiogram (ECG) modifications, and movement disorders. (Kim et al., 2018). Developing knowledge of the biological basis of TS will promote TS treatments instead of just symptom management. The precise pathophysiology of TS still remains elusive. Thus, treatment approaches often rely on empirical methods. (Chou et al., 2023).

There are various strategies in Traditional Chinese medicine (TCM) about tics treatment, but almost all the traditional Chinese strategies agree that the root cause lies in liver wind. YXD have specific effects in TCM theory, effectively alleviating symptoms such as muscle twitching, vocal tics, and obscene speech throughout the body. Besides definite efficacy, TCM has few side effects and drug dependence. (Xiao and Tao, 2017). However, lack of a clear material basis and action mechanism for YXD hinders the wider clinical application. In this study, we utilized drug target and disease target databases to identify that all active ingredients of YXD have 96 targets related to TS, confirming its multi-component and multi-target action. Searching for key genes associated with TS aims to identify biomarkers for accurate diagnosis. Currently, diagnosing the tic disorder relies on clinical history and observation, as there are no definitive laboratory tests or medical markers available. (Liu et al., 2020). This poses a challenge for clinicians in making precise diagnoses. (Johnson et al., 2023). In this study, two core genes, OPRM1 and VIM, were identified among 96 target genes using LASSO regression and SVM algorithm analyses. Furthermore, these two core genes demonstrated superior predictive efficacy in the ROC curves for diagnostic validation, suggesting that OPRM1 and VIM have the potential to predict the development of TS disease. The reliable biomarkers may contribute to accurate diagnose for TS in future, helping for timely and effective treatment. (Xi et al., 2022).

The involvement of immunological pathways is observed in various neurodevelopmental processes such as the neural circuits refinement and formation. However, there is still a lack of information on the efficacy of interventions targeting immune system modulation. (e.g., through environmental modifications) for treating disorders such as TS. (Martino et al., 2020). It is believed that immune mechanisms might function in the developmental changes that underlie the behavioral anomalies seen in TS. (Hsu et al., 2021a; Martino et al., 2015). The post mortem studies and animal models have suggested that microglia played vital role in the crosstalk between the neural and immune systems. Clinical research investigating immune cell subpopulations, cytokine and immunoglobulin levels and etc., have indicated the systemic immune response overactivity. The evidence indicated the disease mechanisms in TS, akin to other neurodevelopmental disorders such as autism, could involve impaired communication between the neural and immune systems. Consequently, this may result in changes in the development of brain pathways that control different behavioral aspects and could potentially affect the coordination of stress and immune responses (Martino et al., 2015). A better understanding of the key molecular pathways influencing neuroimmune interactions in TS can facilitate the discovery of effective biomarkers and more personalized therapeutic approaches. Our findings reveal elevated neutrophil levels in the TS disease cohort compared to controls, consistent with existing literature. Previous studies have observed increased neutrophil-lymphocyte ratios (NLR) across multiple psychiatric conditions. This suggests that abnormal NLR may indicate a general pathological brain process rather than being disorder-specific. Such results support the neuroinflammation hypothesis in psychiatric etiology. Future investigations should explore the links between NLR and specific diagnostic and behavioral constructs to further elucidate these relationships (Bhikram and Sandor, 2022). Our immune infiltration analysis confirmed that the core genes are involved in regulating the immune microenvironment. This results is consistent with the previous section in which we found that TNF and IL-17 signaling pathways mainly involved in YXT. Investigations into TS reveal heightened inflammation indicators. Notably, tumor necrosis factor-alpha and various interleukins (e.g., IL-1β through IL-17) show increased presence. Abnormal cytokine levels can mimic TS symptoms, suggesting a potential link between this condition and chronic inflammatory processes (Leckman et al., 2005; Hsu et al., 2021b).

Immune disorders have been implicated in the progression of TS, but no specific targets was reported for treating TS by modulating the immune system. This may explain why immune system modulators are not included in the drugs used for the clinical treatment of TS. As a complementary finding, our results suggest that OPRM1 and VIM could serve as potential drug targets to regulate the expression levels of immune inhibitors and immune simulators, thereby improving the immune microenvironment. Immune simulators related to OPRM1 include TNFRSF13B, while immune inhibitors include KIR2DL3 and KIR2DL1. Immune simulators related to VIM include CXCR4 and CD48, while immune inhibitors include TNFSF9 and CD276. The specific roles of these immune-related genes in TS warrant further exploration. In conclusion, this study provides evidence that immune regulation participated in the mechanism of action of YXT in treating TS and suggests that OPRM1 and VIM could be potential targets for regulating the immune microenvironment.

OPRM1 and VIM, as core genes, can not only serve as biomarkers and drug targets for TS but also provide a research entrance for further discovery of TS-related molecular mechanisms. Existing studies have shown a greater inclination towards finding evidence that OPRM1 affects TS compared to VIM. (Depienne et al., 2019; Widomska et al., 2023). In fact, a TS cohort was screened for additional variants in OPRM1 using Sanger sequencing, which identified eight rare variants. (Tchalova et al., 2021). Interestingly, all OPRM1 variations were inherited from a parent without symptoms, suggesting that variants in opioid receptors may be susceptibility factors for TS. (Tchalova et al., 2021). Furthermore, frequent variants of OPRM1 have been correlated with impulse control disorders in Parkinson patients recently. (Cormier-Dequaire et al., 2018). The discovery supported that mutations in opioid receptors play a role in impulsive disorders, which are often comorbid with TS (Paschou et al., 2013; Elamin et al., 2013). A recent study aimed to construct a molecular landscape of TD, which revealed signs of increased expression of candidate genes involved in TD in four regions of the brain and the pituitary gland.

The landscape of TS offered opportunity to underly molecular mechanism of TS, and provided potential targets for drug intervention in Tic Disorders (TD). OPRM1, CX3CL1-CX3CR1, NAALAD2, FLT3 are examples of these potential targets. (Widomska et al., 2023). In line with previous research, we have identified OPRM1 and VIM as core genes in TS and established a network of target pathways. Previous studies have shown that OPRM1 exhibits regional specificity in TD, being highly expressed in the TD patient brains but specifically decreased in the striatum postmortem. (Lennington et al., 2016). We also investigated the possible signaling pathways related with OPRM1 and VIM, and conducted enrichment analyses using GO and KEGG. Additionally, we explored potential mechanisms for the transcriptional regulation. As a result, 59 pairs of mRNA-miRNA interactions related to OPRM1 and 34 pairs related to VIM were predicted. Furthermore, we found remarkable association between the OPRM1 and VIM expression, and other TS-related genes, including MAPT and MAPT. These findings provide evidence supporting the use of OPRM1 and VIM as biomarkers and drug targets in the context of TS.

Gene expression regulation heavily relies on transcription factors (Dec. Erga et al., 2018). We utilized RcisTarget to identify significant binding motifs and their associated transcription factors for OPRM1 and VIM genes. This approach allows prediction of potential binding sites through motif sequence extraction, enabling further investigation of underlying molecular mechanisms. (Kuret et al., 2022). MicroRNAs (miRNAs) are short non-coding RNA sequences, spanning 21-25 nucleotides, that can bind to the 3′ UTR of target mRNAs, resulting in their degradation or translation suppression. (He and Hannon, 2004). Our mRNA-miRNA regulatory network analysis revealed miR-155, miR-204, miR-124, and miR-129-5p as the most highly connected among the four hub genes. Research has shown that miRNAs can influence inflammatory signaling, potentially leading to uncontrolled neuroinflammation and associated pathologies like Tourette Syndrome (TS) (Rizzo et al., 2015). For instance, miR-155 acts as a pro-inflammatory mediator in the central nervous system and plays a crucial role in neuroinflammatory disorders such as multiple sclerosis and Alzheimer’s disease (Ghafouri-Fard et al., 2021). miR-124, while predominantly expressed in the brain, is also found in various human and animal tissues. It contributes to the pathogenesis of several disorders, and its abnormal expression has been linked to various neurological conditions due to its vital role in nervous system development (Zingale et al., 2021). Targeting miRNA expression offers a promising therapeutic avenue for addressing pathological neuroinflammation. In this research, we constructed an mRNA-miRNA regulatory network of hub genes to identify various miRNAs that may influence TS development and progression.

Network pharmacology is a promising technique that offers advantages such as low cost and high efficiency (Luo et al., 2020; Zhou et al., 2020). However, it still has some limitations. This study identifies three main limitations. Firstly, the study’s objectives were sourced from diverse databases, each with distinct areas of emphasis. It is crucial to acknowledge that conducting an integrated analysis of multiple databases carries inherent risks owing to their inherent dissimilarities. Secondly, additional foundational experimental and clinical investigations can be performed to corroborate the precision of the results. Network pharmacology theoretically analyzes the material basis of TCM for treating a certain disease, but ignores the feasibility from prediction to realization. For example, there is a wide variety of ingredients and their contents vary greatly in herbs. Network pharmacology cannot take into account the reduction in likelihood by lower ingredient content. In fact, some ingredients, although known to be effective, cannot be purified because of their extremely low content or cannot be industrially synthesized because of their complex structure. Thirdly, the qualitative phase of network pharmacology research on TCM formula investigation still focuses primarily on exploring novel targets and understanding the drug’s mechanism. However, it is crucial to establish a relationship between the dosage of the medication and the specific ailment.

TS, a disorder with multiple pathogenic mechanisms and undefined pathological targets, is often associated with obsessive-compulsive disorder, sleep disorders, mood disorders, and other psychobehavioral problems. This may explain why existing drugs are not effective in treating TS. YXD, a compound prescription of Traditional Chinese Medicines, has multiple targets and mechanisms of action, providing more satisfactory clinical efficacy. Network pharmacology was utilized to map the component-target-pathway network of YXD for the treatment of TS. Through this approach, we identified two core genes, OPRM1 and VIM, and predicted their molecular mechanisms in affecting TS through immune infiltration and transcriptional regulation. Based on the reported evidence, OPRM1 appears to be a plausible biomarker for TS. The active ingredients, targets and molecular mechanisms were elucidated to some extent, which will promote the clinical application of YXD. In future applications of YXD in clinical therapy or drug development, it is important to consider the challenges posed by the content and structure of the individual active ingredients.