Pharmacodynamic Mechanisms of Cicadae Periostracum in Parkinson's Disease: A Metabolomics-Based Study

Abstract

Cicadae Periostracum (CP) is a traditional Chinese animal-derived medicine with the potential to treat Parkinson's disease (PD). This study aims to explore the pharmacodynamic mechanisms of CP against PD-based on metabolomics technology and provide a theoretical basis for developing new anti-PD medicine. First, MPP⁺-induced SH-SY5Y cells were used to evaluate the anti-PD activity of CP. In the animal study, an MPTP-induced PD mouse model was employed to assess CP's therapeutic effects. Immunofluorescence (IF) staining and Western blotting (WB) were used to evaluate its neuroprotective activity on neurons. A Serum metabolomics analysis was conducted to examine CP's regulatory effects on metabolites and to identify vital metabolic pathways. Finally, cellular experiments were performed to validate the critical pathways. Cellular activity experiments demonstrated that CP mitigates MPP⁺-induced SH-SY5Y cytotoxicity, inhibits apoptosis, and restores mitochondrial homeostasis. Animal experiments revealed that CP significantly alleviates dyskinesia in PD mice, enhances motor performance, and restores neuronal integrity while reducing α-synuclein (α-syn) aggregation in the striatum (STR), showing its strong anti-PD effect. Metabolomic analysis revealed that CP can significantly improve the metabolic disorders of ten biomarkers that are mainly involved in amino acid metabolism and fatty acid β-oxidation and are closely related to oxidative stress pathways. Finally, pathway verification was performed, and the results show that CP exerted neuroprotective effects against PD through the dual signaling pathways of Bcl-2/Bax/Caspase-3 and Nrf2/HO-1. This study provides a comprehensive strategy for elucidating the mechanisms by which CP exerts its therapeutic effects against PD, highlighting its potential in developing anti-PD drugs.

Keywords: Cicadae Periostracum; Parkinson’s disease; mechanisms of action; metabolomics; oxidative stress.

Figure 1

CP autotoxicity test and protective effect on MPP⁺-induced SH-SY5Y cells; CP reduces apoptosis and protects mitochondrial homeostasis. (A) Cytotoxicity assessment of CP in SH-SY5Y cells. (B) Neuroprotective effect of CP against MPP⁺-induced cytotoxicity in SH-SY5Y cells. (C) Representative images of IF staining; CP reduces MPP⁺-induced cell apoptosis. Scale bar, 50 μm. (D) Representative images of IF staining; CP restores MPP⁺-induced mitochondrial homeostasis in SH-SY5Y cells. Scale bar, 20 μm. ** p < 0.01, and *** p < 0.001 compared with the model group.

Figure 2

MPTP model establishment, behavioral, IF staining, and WB experimental results. (A) MPTP modeling; schematic diagram of drug treatment time. (B) The mice activity trajectory in the open field test (n ≥ 8). (C) Pole test results. (D) Rotarod test result. (E) Open field test results. (F) Representative micrographs of TH IF staining in the SN; scale bar-250 μm. (G) WB analysis of TH and α-syn in the midbrain of different groups. Data are expressed as the mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 compared with the model group.

Figure 3

Multivariate data analysis from UPLC-MS/MS. (A) OPLS-DA score plots of the control and model groups (n = 6). (B) OPLS-DA permutation test of the control and model groups. (C) PCA score plots of all groups. (D) Cluster heat map analysis of differential metabolites in the serum of all groups. The horizontal axis in the figure represents different sample groups, the vertical axis represents all metabolites, and the color blocks at different positions represent the relative expression of metabolites at the corresponding positions. Red indicates a high expression of the substance and blue indicates a low expression.

Figure 4

Statistical analysis of differential metabolites. (A) Heatmap analysis of differential metabolites in the serum of CP mice. (B) Volcano plot of differential metabolites in the serum of mice between the CP and the model group. (C) Correlation analysis of differential metabolites in the serum of mice between the CP and the model group. (D) Z-score analysis of differential metabolites in the serum of mice between the CP and the model group. (E) Histogram of differential metabolites. Comparison of relative contents of the serum differential metabolites in the CP group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to the model group.

Figure 5

Metabolic regulatory pathways analysis. (A) KEGG pathway classification analysis. (B) Rich factor analysis. (C) Bubble plot impact pathway analysis. (D) The regulatory network plot analysis.

Figure 5

Metabolic regulatory pathways analysis. (A) KEGG pathway classification analysis. (B) Rich factor analysis. (C) Bubble plot impact pathway analysis. (D) The regulatory network plot analysis.

Figure 6

CP reduces apoptosis and oxidative stress in MPP⁺-induced SH-SY5Y cells. (A) GSH level in MPP⁺-induced SH-SY5Y cells in different groups. (B) MDA level in MPP⁺-induced SH-SY5Y cells in different groups. (C) WB analysis of Bcl-2, Bax, and cl-caspase-3 in different groups and statistical analysis (CP, μg/mL). (D) WB analysis of Nrf2, HO-1, and iNOS expression in different groups and statistical analysis. Data are expressed as the mean ± standard deviation. * p < 0.05 compared with the model group. ** p < 0.01; *** p < 0.001; **** p < 0.0001.