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

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.


Introduction
Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. It is characterized by progressive loss of dopamine (DA) neurons mainly in the substantia nigra pars compacta (SNpc) and the appearance of cytoplasmic inclusion, which is called Lewy bodies, and its main component is α-synuclein. e autopsy analysis of PD patients and experimental animals indicates that proinflammatory factors and nigral dopaminergic neuron apoptosis were significantly increased, the common characteristics of the PD brain [1][2][3]. Previous pathology studies in PD patients and animal models and the neuroinflammation target treatment studies all indicated that the neuroinflammation and apoptosis response can mediate PD-related progressive death of dopaminergic neurons, suggesting inflammation and apoptosis could be considered as a promising target for PD treatment [4,5].
Rotenone (RT) is a mitochondrial complex I inhibitor that can selectively impair dopaminergic neurons and induce PD-like behaviors [6].
is toxin features high lipophilicity and can easily access dopaminergic neurons beyond the blood-brain barrier without any transporter [7]. It is one of the neurotoxic drugs used to establish the neurotoxin model of PD in recent studies [8]. e rotenone model summarizes the majority of pathological features observed in PD patients, including the absence of dopaminergic neurons in the substantia nigra pars compacta and the enhanced oxidative stress and neuroinflammation in the nigrostriatal dopaminergic pathway [9].
Levodopa is the most effective drug for PD treatment at present; however, it cannot postpone the disease progression. Moreover, after levodopa intervention for 3-5 years, nearly all patients with PD develop motion complications such as reduced efficacy, on-off phenomenon, and dyskinesia. In traditional Chinese medicine, PD is called "tremor palsy," which is characterized by tremors, numbness, and weakness of limbs. Accumulating literatures have reported the application of herbal prescriptions in alleviating PD symptoms, and the efficacy of Chinese herbal medicines has attracted increasing attentions. e Wenshen-Yanggan decoction is a formula used for PD treatment at Nanjing TCM Hospital, which is composed of Cistanches Herba, Paeoniae Radix Alba, Dioscoreae Rhizoma, Linderae Radix, Fructus Alpiniae Oxyphyllae, and Uncariae Ramulus Cum Uncis. It has various functions, for instance, warming the kidney yang, nourishing the liver blood, and quenching the liver wind. In the preliminary study, we found that echinacoside, one of the main components of the decoction, exerts remarkable neuroprotective effects on neurodegenerative diseases including PD [10][11][12][13]. However, the experimental study on the effect of Wenshen-Yanggan decoction on PD has not yet been reported. erefore, in this study, we aimed to investigate the neuroprotective effects of Wenshen-Yanggan decoction on PD model mice and explore its underlying mechanism. e decoction contents were detected, and stable quality control was carried out. Rotenone was used to elicit chronic PD in C57BL/6 mice.

Animals and Groups.
10-month-old C57BL/6J male mice weighing 30-35 g were used and bred in the animal facility with a 12 h circadian rhythm (room temperature 22 ± 2°C and humidity 55 ± 5%). Mice were given free access to eat and drink. e animals were acclimated to the environment for 2 weeks before the experiment. en, they were randomly divided into the blank group (Saline), the model group (RT), the Wenshen-Yanggan decoction in high dosage (Wsyg-H), the decoction in medium dosage (Wsyg-M), the decoction in low dosage (Wsyg-L), and the positive control group (Sinemet, carbidopa/levodopa, 25/100 mg. 50 mg/kg), with 10 mice in each group.

Preparation of the Wenshen-Yanggan Decoction.
Linderae Radix (20 g), Fructus Alpiniae Oxyphyllae (30 g), Paeoniae Radix Alba (30 g), and Uncariae Ramulus Cum Uncis (20 g) were mixed together, immersed in 60% alcohol (12 times weight), and decocted for 1.5 h. e extract was filtered through six-layer gauzes. e abovementioned procedures were repeated twice. Residues were collected, and then Dioscoreae Rhizoma (20 g) and Cistanches Herba (30 g) were added, immersed in pure water, and decocted for 1.5 h. is procedure was repeated twice and half volume of the liquid was evaporated. Alcohol was added to the mixture until 50% alcohol content, precipitated it for 48 h, and then recovered and eliminated alcohol until nonalcoholic taste, and the remaining extract is then combined with precipitation solution for concentration until every 1 ml mixture contains 4 g crude drugs.
e high-, medium-, and lowdosage solutions of decoction is configured corresponding to 4 g, 2 g, and 1 g crude drugs. e total ion chromatogram and main components of the Wenshen-Yanggan decoction extract are displayed in Figure 1 and Table 1.

Modeling and Medication of the Chronic PD Model.
e mice in the model group, Wsyg groups, and Sinemet group were intragastrically administrated with rotenone (30 mg/kg) daily for 4 weeks at 10 : 00 am, while the mice in the saline group were treated with equal volume of normal saline. From the 5 th week, the mice in the Wsyg groups were treated with low, medium, and high dosage (0.1 ml/10 g) once daily for 4 weeks at 10 : 00 am and the mice in the Sinemet group were treated with Sinemet solution (0.1 ml/ 10 g). e saline group was treated with equal volume of normal saline. 24 h after the last medication, behavioral tests were carried out. All animals were sacrificed 24 h after the behavioral tests.

Pole Test.
According to Chen's method [10], the mouse was placed on the spherical protrusion with its head upward. e time the mouse spent to turn downward (T-turn) and to the bottom of the pole with its posterior limbs touching the ground (T-total) was recorded. Each mouse was tested 3 times at an interval of 2 min, and then the average value was calculated.

Rotarod Test.
e rotarod test was performed using a mouse rotarod fatigue meter (ZH-600 Anhui Zhenghua). e mice were placed on a shifting drum (rotation speed: 5 rpm-40 rpm) for 5 minutes 3 times. e time of mice standing on the drum was recorded and averaged.

Immunofluorescence Staining.
After the left ventricle perfusion, the brains of mice were rapidly taken and placed in 4% paraformaldehyde overnight. Sucrose solution was used for gradient dehydration, and OCT gel was used for embedding. We prepared the brain tissue sections (30 μm) using a frozen slicer and chose nigral brain slices in line with the brain atlas [14]. After being washed with PBS, goat anti-mouse IgG H&L (1 :1000) was added and incubated at room temperature in dark for 1 h. After rinsing with PBS, the antifluorescence quenching tablet was added to seal the tablet. An Olympus BX63 fluorescent microscope was used to observe the slices.

Western Blotting.
e midbrains of mice were placed in RIPA buffer, the concentration was adjusted evenly, and then they were stored at −70°C. en, polyacrylamide gel electrophoresis was used. 50 μg of total protein from each sample was separated by SDS-PAGE (10%, 12%, or 15%) and transferred to the polyvinylidene fluoride (PVDF) membrane using the wet transfer approach. Afterward, the PVDF membrane was blocked in 5% BSA for 1h at room temperature and incubated overnight at 4°C with the following primary antibodies: α-syn On the next day, the membrane was rinsed with Tris-HCL buffered salt-Tween (TBST) solution and incubated with the secondary antibody (1 : 2000) for 1h at room temperature; then, the electrochemiluminescent substrate was added. Blots were visualized and analyzed using the Tanon-5200 automatic chemiluminescence image analysis system. e experiment was repeated 3 times.
2.10. Enzyme-Linked Immunosorbent Assay (ELISA). 1.5 ml blood of each mouse was collected from the orbit. e blood samples were centrifuged for 10 min at 4°C, 3000 r/min, and 10 cm centrifugal radius. en, the upper serum was stored at −20°C. e contents of inflammatory factors in the serum of mice including TNF-α, IFN-c, IL-1β, and NF-κB were detected by using the enzyme-linked immunosorbent assay kit, strictly according to the manufacturer's instructions.

Statistical
Processing. All data were expressed as mean ± SEM and analyzed by one-way ANOVA followed by post hoc analysis of the Student-Newman-Keuls test and LSD multiple comparison test. P < 0.05 was considered statistically significant. and low-dosage group (Wsyg-L) (P < 0.01, P < 0.01, and P < 0.05). In addition, the mice in Model featured significantly longer total time of climbing than those in Saline (P < 0.01), whereas the total time of climbing in Wsyg-H and Wsyg-M groups was significantly decreased (P < 0.01 and P < 0.05). In the rotarod test, the rod-standing time of Model was significantly shorter than that of Saline (P < 0.001); meanwhile, the rod-standing time in Wsyg-H, Wsyg-M, and Wsyg-L groups was significantly longer than that of Model (P < 0.01, P < 0.05, and P < 0.05) (Figure 2).  e results of western blotting showed that the expression of TH after rotenone modeling was significantly downregulated in comparison with that of Saline (P < 0.001), whereas TH expression levels in Wsyg-H, Wsyg-M, and Wsyg-L were profoundly increased relative to Model (P < 0.001, P < 0.01, and P < 0.01) (Figure 3(b)). Similar results were obtained in the immunofluorescence experiment (Figure 3(a)). Meanwhile, the expression of α-syn in the midbrain of mice in Model was significantly elevated compared with Saline (P < 0.001). Nonetheless, Wsyg-H, Wsyg-M, and Wsyg-L significantly decreased the expression of α-syn (P < 0.001, P < 0.001, and P < 0.001) (Figure 3(b)).

Effects of the Wenshen-Yanggan Decoction on the Protein Expression of p-JNK, Cleaved Caspase-3, Bcl-2, and NF-κB in the Midbrain of Chronic Rotenone Mice.
Western blotting analysis showed that rotenone induced a marked increase in p-JNK, cleaved caspase-3, and NF-κB protein levels in the midbrain (P < 0.01, P < 0.01, and P < 0.001), while the expression of Bcl-2 was significantly decreased (P < 0.05). After treating with high dosage of Wsyg, the expression of p-JNK, cleaved caspase-3, and NF-κB was significantly reduced (P < 0.05, P < 0.05, and P < 0.01), and the level of Bcl-2 was noticeably increased (P < 0.05). In addition, p-JNK and cleaved caspase-3 and NF-κB protein levels in the midbrain were significantly downregulated (P < 0.01, P < 0.05, and P < 0.05) in Wsyg-M, while the expression of Bcl-2 was significantly upregulated (P < 0.01). e low dosage of the decoction reduced the expression of p-JNK and cleaved caspase-3 (P < 0.001 and P < 0.05) and enhanced the level of Bcl-2 (P < 0.001), but did not affect NF-κB Figure 5.

Discussion
PD is a slowly progressive neurodegenerative disorder. e pathological changes of PD are characterized by the presence of SNpc degeneration and the occurrence of Lewy body, which are related to autophagy, apoptosis, oxidative stress, and other complex mechanisms including immune response, inflammatory response, and excitotoxicity [15]. e focus of PD research studies is to study its etiology and develop therapies that possess disease-modifying effect and are able to slow down the disease process.
Although the pathogenesis of PD is still unclear, it is believed that neuroinflammation is one of the main pathological mechanisms of PD and a potential target for PD treatment [16]. Upon autopsy of animal models and PD patients, the expression of proinflammatory cytokines in cerebrospinal fluid (TNF-α, IL-1β, IFN-c, and so on) was increased in PD [17]. In this study, the same phenomenon occurred in the rotenone model group, while the Wenshen-Yanggan decoction could reduce these inflammatory factors in PD mice. NF-κB, an important nuclear transcription factor, plays a significant role in cell growth and proliferation and is involved in the etiology and anti-inflammatory effects of PD. e inflammatory response can activate NF-κB, leading to increased synthesis of the proinflammatory factor TNF-α protein [18]. e Wenshen-Yanggan decoction reduced the level of NF-κB in both the serum and midbrain, which might partially account for Wenshen-Yanggan decoction-generated inhibition of inflammatory response in PD mice.
Neuronal apoptosis, specifically the apoptosis of dopaminergic neurons in the dense substantia nigra, has been reported to be the final outcome of PD. [19]. Similarly, we observed the upregulation of cleaved caspase-3 in the brain of the PD model mice in this experiment. e activation of caspase-3 is related to the apoptosis of neurons. e JNK signaling pathway is involved in the regulation of cell growth, differentiation, apoptosis, and so on, and its abnormality is closely related to PD-related apoptosis in the substantia nigra [5,20,21].
Caspase-3 is a main apoptosis-related enzyme, which is capable of activating the caspase cascade. e activation of     JNK signaling pathway can upregulate the expression of COX-2 and caspase-3. Once JNK is activated, p-JNK stimulates the Bcl-2/Bax family and its downstream substrates, promoting apoptosis. [22] Bcl-2 is a membranebound antiapoptotic protein regulated by the upstream JNK, [23] which can attenuate the antiapoptotic effects after activation [24].
In conclusion, this study showed that (1) the Wenshen-Yanggan decoction could improve the PD-like behaviors in the rotenone-induced PD model mice, reduce the expression level of α-syn, and prevent the loss of substantia nigra DA neurons; (2) the rotenone-induced PD model mice displayed neuroinflammatory symptoms including increased expression of inflammatory factors in the serum and midbrain, which were improved by the Wenshen-Yanggan decoction; (3) the Wenshen-Yanggan decoction restored the elevated expression of the key apoptotic protein, cleaved caspase-3, antiapoptotic protein, Bcl-2 protein, and the apoptotic pathway-related protein, p-JNK, in rotenone-induced PD model mice, suggesting that the Wenshen-Yanggan decoction might suppress apoptosis in PD mice through JNK controlled inhibition of inflammatory response.
is experiment has proven that the Wenshen-Yanggan decoction could exert protective effects in PD nerve injury and illustrated its potential mechanism. e present study has shed light on the development of traditional Chinese medicine decoction for PD treatment.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.