Xifeng Zhichou decoction mitigates tic disorder on juvenile rats by regulating neuroinflammation and neurotransmitter homeostasis: dual modulation of Nr4a2 and gut microbiota

Abstract Background Xifeng Zhichou decoction (XFZCD), a modified formulation of Chinese medicine, demonstrates significant clinical efficacy in treatment of tic disorder (TD). However, its efficacy and therapeutic mechanisms remain insufficiently characterized. Objective This study aims to comprehensively evaluate the anti-TD effect of XFZCD in a rat model, and to elucidate the underlying mechanism. Methods Ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) was used to quality analyze of XFZCD, while network pharmacology was employed to predict core compounds and potential mechanisms. A juvenile TD rat model was established via intraperitoneal injection of 3,3-iminodipropionitrile, and the therapeutic effects of XFZCD were evaluated using behavioral tests, cytokine assays, and immunohistochemistry. Transcriptomics and 16S rRNA sequencing were employed to elucidate the mechanisms by investigating regulatory gene and gut-brain axis. Western blot (WB) and enzyme-linked immunosorbent assay were utilized to focus and verify the potential mechanisms. Untargeted metabolomics was conducted to confirm the crucial pathway from the perspective of terminal metabolites. Furthermore, Nr4a-targeted siRNA knockdown in Lipopolysaccharides (LPS)-challenged SH-SY5Y cells was performed to verify the core regulatory role of Nr4a2 in XFZCD-mediated neuroprotection. Results The UPLC-MS analysis identified 66 compounds in XFZCD. Network pharmacology highlighted serotonergic and dopaminergic synapses, and JAK-STAT signaling in enrichment analysis, suggesting XFZCD treats TD by regulating neuroinflammation and neurotransmitter homeostasis. XFZCD significantly mitigated tics, abnormal motor behavior, and anorexia in rat models, while also attenuating striatal neuroinflammation and oxidative stress, thereby exhibiting a substantial therapeutic effect on TD. Transcriptomic analysis identified Nr4a2 as a pivotal regulatory gene, which exhibited strong binding affinity with the core components identified via network pharmacology. 16S rRNA sequencing revealed that XFZCD modulated intestinal function by restoring the Firmicutes / Bacteroidetes ratio. WB and ELISA experiments confirmed that XFZCD could suppress inflammatory responses and restore central neurotransmitter homeostasis. Untargeted metabolomics indicated XFZCD reversed TD-caused abnormal bile acid metabolism, which was closely correlated with gut microbial remodeling. Cellular experiments confirmed that Nr4a2 silencing largely abrogated XFZCD’s inhibitory effects on neuroinflammation and its regulatory function on neurotransmitter balance. Conclusion XFZCD demonstrates significant efficacy in the treatment of TD by inhibiting inflammation and balancing neurotransmitters through dual modulation of Nr4a2 and intestinal flora. Notably, the regulation of Nr4a2 and bile acid metabolism emerges as a promising therapeutic avenue for the treatment of TD.