Exploring the Therapeutic Mechanism of Jianpi Zhidong Decoction on Tourette Syndrome Based on Proteomics and Network Pharmacology

Abstract

Purpose: To explore the pharmacological effects of Jianpi Zhidong Decoction (JPZDD) on Tourette Syndrome (TS) using proteomics and network pharmacology.

Materials and methods: Chemical components of JPZDD were identified via UPLC-MS/MS. Chronic restraint stress TS model was established by intraperitoneal injection of iminodipropionitrile (IDPN) for 1 week with restraint stress for 3 weeks. Sixty male SD rats were divided into control, model, Tiapride (Tia), and JPZDD groups. After the intervention of 28 days, behavioral tests, Nissl staining, Western blot, immunofluorescence, colorimetry, and ELISA were performed to evaluate the pharmacological effects of JPZDD. Proteomics and network pharmacology predicted targets, validated by Western blot.

Results: JPZDD alleviated stereotypic behaviors, hippocampal pathology, and modulated glucose metabolites (GLU, pyruvate, lactate, ATP). It downregulated GLUT1, GLUT3, HK2, and LDHA levels while upregulating PDHA level. Besides, JPZDD balanced M1/M2 microglial phenotypes, reducing IL-1β and IL-6 and increasing IL-4 and IL-10. UPLC-MS/MS identified 44 active ingredients and 123 targets; proteomics revealed 28 differentially expressed proteins. GO/KEGG analysis implicated that the PI3K/AKT/mTOR pathway may be the molecular target. JPZDD inhibited PI3K, AKT, and mTOR phosphorylation.

Conclusion: JPZDD (16 g·kg⁻¹·d⁻¹) alleviates motor tics, modulates microglial activation and glucose metabolism, and downregulates the PI3K/AKT/mTOR pathway, providing a mechanistic basis for its therapeutic role in TS.

Keywords: Jianpi Zhidong decoction; Tourette syndrome; glucose metabolism; microglia; network pharmacology.

Graphical abstract

Figure 1

Flow chart of animal studies.

Figure 2

UPLC-MS/MS analysis of the chemical components of JPZDD. (A) Chromatogram of the positive ion mode. (B) Chromatogram of the negative ion mode. The abscissa shows the retention time (Rt) of the metabolites, and the ordinate shows the ion current intensity, intensity unit: count per second (cps). (C) Quantity and proportion of Class I compounds.

Figure 3

JPZDD had certain efficacy in the treatment of the symptoms of TS. (A) Body weight changes of rats in each group (n = 15 per group). (B) Evaluations of stereotypic behavior scores (n = 15 per group). (C–F) Evaluations of in the open field tests in each group (n = 15 per group). Data are expressed as mean ± SD (post-hoc LSD test). #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group. ***P < 0.001 vs Model group.

Figure 4

Observed the damage degree of neurons in hippocampus of chronic restraint stress TS rats by Nissl staining (× 200, Scale bars = 200 μm, n = 6 per group).

Figure 5

Hippocampus levels of glucose metabolites and glucose metabolizing enzymes in different groups. (A–D) The levels of GLU, Pyruvate and LD in hippocampus (n = 6 per group). (E) GLUT1,GLUT3, HK2, LDHA and PDHA proteins expressions in hippocampal tissue after treatment for 28 days were determined by Western blotting assay. Tubulin was used as an internal control (n = 6 per group). (F–J) Representative the relative protein expression of GLUT1,GLUT3, HK2, LDHA and PDHA. All data were quantified by Image J software (n = 6 per group). Data are expressed as mean ± SD (post-hoc LSD test). #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group. *P < 0.05, **P < 0.01, ***P < 0.001 vs Model group.

Figure 6

JPZDD regulates microglia polarization and inflammation. (A) Representative IHC Images of Iba-1, CD86 and CD206 proteins in the hippocampal tissues of different groups (arrows point to positive cells). (B) M1 and M2 cells were labeled with triple immunofluorescence staining. Cell nuclei were stained with DAPI (blue), microglia cells were stained with Iba-1 (red), M1 cells were stained with iNOS (yellow), M2 cells were stained with CD163 (green). (× 200, Scale bars = 200 μm, n = 4). (C–F) The levels of IL-1β, IL-6, IL-4, IL-10 in hippocampal tissues of different groups (n = 6 per group). Data are expressed as mean ± SD (post-hoc LSD test). #P < 0.05, ##P < 0.01 vs Control group. *P < 0.05 vs Model group.

Figure 7

Network pharmacology analysis. (A) Venn diagram of 123 intersecting genes (JPZDD vs Tourette Syndrome). (B) PPI network of intersecting genes. (C) Composite target network of potential therapeutic targets for JPZDD. Yellow rhombus nodes indicate JPZDD; green rectangle nodes indicate active ingredients in JPZDD; blue and Orange circle nodes represent the potential targets. (D) The GO enrichment analysis of potential therapeutic targets. (E) The KEGG enrichment analysis of potential therapeutic targets.

Figure 8

Proteomics analysis of the DEPs. (A) The volcano plot of the differential proteins (JPZDD group vs Model group). (B) The GO enrichment analysis of DEGs (JPZDD group vs Model group). (C) The KEGG enrichment analysis of DEGs (JPZDD group vs Model group).

Figure 9

JPZDD could improve tic symptom by inhibiting PI3K/AKT/mTOR signaling pathway. (A) Protein expression levels of PI3K, AKT, mTOR, p-PI3K, p-AKT and p-mTOR. (B–D) Statistical analysis of phosphorylation and total protein levels of PI3K, AKT and mTOR. Data are expressed as mean ± SD (post-hoc LSD test). #P < 0.05, ##P < 0.01, ###P < 0.001 vs Control group. *P < 0.05, **P < 0.01, ***P < 0.001 vs Model group. and P < 0.05, vs Tia group.

Figure 10

JPZDD could improve tic symptom by inhibiting PI3K/AKT/mTOR signaling pathway.