How does echinacoside from cistanche treat Parkinson’s disease?

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Part Ⅰ：Neuroprotective Effects And Related Mechanisms Of Echinacoside in MPTP-Induced PD Parkinson’s disease (PD) Mice

Zhen-Nian Zhang'*Zhen Huil Chang Chen Yan Liang Li-Li Tang'Su-Lei Wang Cheng-Cheng Xul Hui Yang'Jing-SiZhang²Yang Zhao

Objective: To explore the neuroprotective effect and the related mechanisms of echinacoside (ECH) from cistanche in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease (PD) mice. Methods: Parkinson’s disease is induced in mice by MPTP and the neurobehaviors of mice in different groups are observed. Then, immunohistochemistry and Western blot analysis are adopted to measure the expression of tyrosine hydroxylase (TH) and α-synuclein in the substantia nigra (SN). The content of dopamine (DA) and other neurotransmitters in the brain is detected by high-performance liquid chromatography. The expression of nerve growth factors and inflammatory factors in SN in mice in each group is measured by quantitative polymerase chain reaction. Finally, the expression of oxidative stress-related parameters in each group is measured. Results: Compared with the model group, the pole-climbing time among mice in the moderate and high-dose echinacoside groups is significantly reduced (P < 0.01). The rotarod staying time, as well as fore and hind-limb strides, shows a significant increase (P < 0.01), as does spontaneous activity (P < 0.01). Moreover, the expression levels of TH, DA, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor in SN in mice show significant increases in these two groups (P < 0.01). The content of superoxide dismutase, catalase, and glutathione peroxidase indicates significant increases in the low, moderate, and high-dose echinacoside groups (P < 0.01), and the content of MDA was reduced (P < 0.01). In the high-dose echinacoside group, the expression of interleukin (IL) 6 and tumor necrosis factor-α is significantly reduced (P < 0.01), while the expression of IL-10 shows a marked increase (P < 0.01) alongside a decrease in the expression of α-synuclein (P < 0.01). Conclusion: Echinacoside from cistanche improves neurobehavioral symptoms in Parkinson’s disease mice and significantly increases the expression of TH and DA. The neuroprotective effect potentially correlates with anti-inflammation and anti-oxidation actions, promotes the expression of nerve growth factors, and reduces the accumulation of α-synuclein.

Cistanche for treating Parkinson's Disease

Keywords: Parkinson’s disease, MPTP, echinacoside, α-synuclein, oxidative stress, neuroprotection, BDNF, GDNF, IL-6, TNF-α, IL-10

Introduction

Parkinson's disease(PD) is a chronic and progressive neurodegenerative disease. A typical pathological feature of Parkinson’s disease is the formation of Lewy bodies, in which α-synuclein and ubiquitin are the primary components. Although the pathogenesis of Parkinson’s disease has not been fully clarified, ever more evidence indicates that environmental factors, oxidative stress, mitochondrial dysfunction, down-regulation of neurotrophic factors, immune inflammation, excitatory amino-acid toxicity, calcium overload, and other pathology mechanisms interact and participate in the occurrence or development of Parkinson’s disease. Studies suggested that risk factors, such as increased oxidative stress and mitochondrial dysfunction, can lead to misfolding and abnormal aggregation of α- synuclein. This in turn may aggravate oxidative stress and mitochondrial dysfunction. These factors may interact with one another and continuously amplify the effect of injury, eventually leading to progressive degeneration and death of dopaminergic neurons.3 Considering that no drug can prevent the progression of Parkinson’s disease at present, combined with the pathogenesis and core pathological characteristics of Parkinson’s disease, targeting α-synuclein may indicate an important direction for finding drugs that can delay the disease’s progression and probe the neuroprotective effects.

Recent studies showed that echinacoside (ECH) from cistanche has a wide range of pharmacological effects, including anti-inflammatory, antioxidative, and neuroprotective functions; additionally, it may contribute to improving learning and memory, provide hepatic protection, and immune regulation, and provide anti-tumor effects.5 Existing studies conducted by the present study group also confirmed that echinacoside from cistanche may be able to improve abnormal gait in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced Parkinson’s disease mice model. It may also significantly reduce the expression of Bax/Bcl-2 and inhibit the reduction of dopaminergic neurons in the striatum, as well as inhibit the activation of microglia and new types of glial cells.6–10 In the pathogenesis of Parkinson’s disease, the activation of microglia can secrete many neurotoxic substances such as chemokines, inflammatory factors, and reactive oxygen-free radicals, and aggravate damage to the neurons through oxidative stress, inflammation, and the induction of apoptosis. Researchers have found that in the MPTP-induced Parkinson’s disease mice model, inhibiting the activation of microglia can prevent the death of dopaminergic neurons.8 Cell models showed that cistanche echinacoside may be able to reduce the expression of inflammatory factors induced by 6-hydroxydopamine. However, there are few reports on the effects of echinacoside from cistanche and its related therapeutic mechanisms for the clearance of α-synuclein, the core pathological product in Parkinson’s disease. In the present study, the elimination of α-synuclein was further studied by combining existing studies, considering the neuroprotective mechanisms of ECH (anti-inflammatory, anti-oxidative stress), and the enhancement of glial cell-derived nerve growth factor (GDNF) and brain-derived nerve growth factor (BDNF).

Materials and Apparatus

Experimental Animals: The experimental group comprised 108 eight-week-old male C57BL/6J mice (specific-pathogen-free (SPF) grade) weighing 20–22 g. Four mice were kept per cage at 22°C–25°C. The mice were provided by the Shanghai Center for Laboratory Animals, Chinese Academy of Sciences, housed at the Nanjing University of Chinese Medicine (Nanjing, China). Animals were treated humanely according to the National Institutes of Health guidelines in an SPF-grade room with a temperature of 22°C– 25°C, 55% relative humidity, and under a 12-h circadian rhythm. The mice had free access to food and water. Primary Reagents: The primary reagents used in the current study were as follows: MPTP (Sigma-Aldrich, Product number: M0896), mouse anti-tyrosine hydroxylase (TH) monoclonal anti-body (Sigma-Aldrich Company), Alexa Fluor 555 anti-rabbit antibody (Biyuntian Institute of Biotechnology), Alexa Fluor 488 anti-rabbit antibody (Biyuntian Institute of Biotechnology), selegiline (Sigma-Aldrich), anti- horseradish peroxidase (HRP)-goat anti-rabbit secondary antibody (Biyuntian Institute of Biotechnology), rabbit polyclonal α-synuclein antibody (Cell Signaling Technology), HRP-goat anti-mice secondary antibody (Biyuntian Institute of Biotechnology), and cistanche echinacoside (Chengdu Linghangzhe Biotechnology Co., Ltd.). Preparation of MPTP: 100 mg MPTP powder was added to 10 mL 0.9% normal saline, thoroughly shaken and mixed to avoid light, and then divided into 10 1.5 mL Eppendorf tubes and stored at –20°C. Prior to use, the solution was diluted once to achieve a final concentration of 5 mg/mL.

Experimental Methods

Grouping of Experimental Animals: The 108 C57BL/6J mice were randomly divided into six groups with 18 mice in each. The details of grouping were as follows: the normal control group (the normal saline group, MPTP group, low-dose echinacoside group [the EL group], moderate-dose echinacoside group [the EM group], high-dose echinacoside group [the EH group], and the selegiline group [the SL group]). Modeling of the Experimental Animals and Medication: The MPTP dose for intraperitoneal injection was 30 mg/ kg/d for seven consecutive days. The selegiline gavage dose was 1 mg/kg/d. The dose of echinacoside in EL, EM, and EH groups was 10 mg/kg/d, 20 mg/kg/d, and 30 mg/kg/d, respectively. Among these groups, the mice in the normal saline group were intragastrically administered with0.1 mL of saline and injected intraperitoneally with 0.1 mL of saline during the modeling period. The gavage was started in the three-dose echinacoside groups and the SL group, 3 days before the MPTP injection, and the mice continued to receive gavage for 7 days after the termination of the MPTP injection.

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