Effect of Wenshen-Yanggan Decoction on Movement Disorder and Substantia Nigra Dopaminergic Neurons in Mice with Chronic Parkinson's Disease

Abstract

This study aimed to explore the protective effects of Wenshen-Yanggan decoction on dopaminergic (DA) neuron injury in a rotenone-induced mouse model with chronic Parkinson's disease (PD) and explore its mechanism of action. Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to measure the content of six main components in the Wenshen-Yanggan decoction. The chronic PD mouse model was established by treating 10-month-old healthy wild C57BL/6 male mice with rotenone 30 mg/kg/day for 28 days in succession. The pole test and rotarod test were applied to detect the rescue effect of Wenshen-Yanggan decoction in high, medium, and low dosages, respectively, on PD-like behaviors in mice with chronic PD. The protective effect of Wenshen-Yanggan decoction on the mesencephalic nigrostriatal DA neuron injury was determined employing tyrosine hydroxylase (TH) immunofluorescence staining. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the inflammatory cytokines in serum, including TNF-α (tumor necrosis factor-alpha), IFN-γ (interferon gamma), NF-κB (nuclear factor kappa-B), and IL-1β (interleukin-1 beta). Western blotting was performed to quantify the expression of phosphorylated c-Jun N-terminal kinase (p-JNK), cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), and NF-κB in the brain. Our results showed that the Wenshen-Yanggan decoction in high, medium, and low dosages reduced the turning time of mice (P < 0.01, P < 0.01, and P < 0.05). The high and medium dosages shortened the total climbing time of PD mice in the pole test (P < 0.01 and P < 0.05). Meanwhile, the high, medium, and low dosages increased the rod-standing time of PD mice in the rotarod test (P < 0.01, P < 0.05, and P < 0.05). Besides, the decoction reversed the decrease in TH-positive neurons induced by rotenone, upregulated TH protein expression, and downregulated the α-syn expression in the PD model. Moreover, the decoction in high dosage significantly inhibited the expression of p-JNK, cleaved caspase-3, and NF-κB in the midbrain of PD mice (P < 0.05, P < 0.05, and P < 0.01), upregulated the expression of Bcl-2 (P < 0.05), and decreased the content of TNF-α, IFN-γ, NF-κB, and IL-1β in the serum (P < 0.001, P < 0.001, P < 0.001, and P < 0.001). Taken together, the Wenshen-Yanggan decoction could protect mice from rotenone-induced chronic PD, which might be related to the reduction of the DA neuron apoptosis via suppressing the inflammatory reaction and the neuronal apoptosis pathway.

Figure 1

The total ion chromatogram of the Wenshen-Yanggan decoction extract. TIC and MRM chromatograms of the sample (A, TIC of the negative sample solution; B, TIC of the mixed reference solution; C, TIC of the sample solution; (1) diosgenin; (2) rhynchophylline; (3) linderane; (4) echinacoside; (5) paeoniflorin; (6) protocatechuic acid).

Figure 2

Effects of the Wenshen-Yanggan decoction on behavioral tests of the PD chronic model of mice. In the pole test, (a) refers to the turning time and (b) refers to the total time; (c) refers to the retention time in the rotarod test. Saline, blank group; Model, rotenone model group; Wsyg-H, high-dosage group; Wsyg-M, medium-dosage group; Wsyg-L, low-dosage group; Sinemet, positive control. ^∗∗P < 0.01 and ^∗∗∗P < 0.001 vs. Saline; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. Model.

Figure 3

Immunofluorescence staining of TH and α-syn protein in the substantia nigra of mice. Saline, blank group; Model, rotenone model group; Wsyg-H, high-dosage group; Wsyg-M, medium-dosage group; Wsyg-L, low-dosage group; Sinemet, positive control. ^∗∗∗P < 0.001 vs. Saline; ^##P < 0.01 and ^###P < 0.001 vs. Model.

Figure 4

Changes of serum inflammatory factors including (a) TNF-α, (b) IFN-γ, (c) NF-κB, and (d) IL-1β in mice. Saline, blank group; Model, rotenone model group; Wsyg-H, high-dosage group; Wsyg-M, medium-dosage group; Wsyg-L, Low-dosage group; Sinemet, positive control. ^∗∗∗P < 0.001 vs. Saline; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. Model.

Figure 5

Protein expression of p-JNK, cleaved caspase-3, Bcl-2, and NF-κB. Saline, blank group; Model, rotenone model group; Wsyg-H, high-dosage group; Wsyg-M, medium-dosage group; Wsyg-L, low-dosage group; Sinemet, positive control. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. Saline; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. Model.