Abstract

Objective: To investigate the neuroprotective effect of glycosides of Cistanches (GCs) in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) mouse model and to explore the potential mechanism.

Methods: Forty mice were randomly divided into a control group, PD group, GCs group, and levodopa (L-DOPA) group (n = 10 per group). The control group received intraperitoneal injections of normal saline, whereas the other groups were intraperitoneally injected with MPTP (30 mg/kg) once daily for 5 consecutive days. After model establishment, the control and PD groups were administered normal saline intragastrically once daily for 28 consecutive days. The GCs group and L-DOPA group received intragastric administration once daily for 28 consecutive days at doses of 100 mg/kg and 50 mg/kg, respectively. The open field test and rotarod test were used to evaluate motor function. Immunohistochemistry was performed to detect the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra of the midbrain. Transmission electron microscopy (TEM) was used to observe mitochondrial ultrastructure in the substantia nigra. Biochemical assays were used to determine glutathione (GSH), malondialdehyde (MDA), total superoxide dismutase (T-SOD), and reactive oxygen species (ROS) levels in substantia nigra tissues. Western blotting was used to assess TH, nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap-1), and heme oxygenase-1 (HO-1) protein expression in substantia nigra tissues.

 

 

Results: Compared with the control group, mice in the PD group exhibited markedly impaired motor function, significantly decreased T-SOD activity, obvious reductions in TH-positive neurons and GSH content, and elevated MDA and ROS levels in the substantia nigra (P < 0.01). Compared with the PD group, mice in the GCs group showed improved motor function, increased T-SOD activity, a higher number of TH-positive neurons, and elevated GSH content, accompanied by decreased MDA and ROS levels (P < 0.05). In the control group, substantia nigra mitochondria displayed normal morphology without blurred membrane structures or vacuolar degeneration. The PD group showed mitochondrial swelling, disordered cristae, and vacuolar changes; these pathological alterations were alleviated in the GCs group. Compared with the control group, TH, Nrf2, and HO-1 protein levels were significantly downregulated, while Keap-1 protein expression was markedly upregulated in the PD group (P < 0.01). Compared with the PD group, the GCs group showed significant upregulation of TH, Nrf2, and HO-1 protein levels and a notable reduction in Keap-1 protein level (P < 0.05).

Conclusion: GCs ameliorate motor dysfunction and attenuate dopaminergic neuronal and mitochondrial damage in MPTP-induced PD model mice. These effects may be associated with modulation of Nrf2/HO-1 pathway-related protein expression, enhancement of antioxidant capacity, and alleviation of oxidative stress.

Key words: Parkinson's disease; glycosides of Cistanches; oxidative stress; Nrf2/HO-1 pathway; dopaminergic neuron; ROS

 

Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. Patients typically present with motor symptoms such as resting tremor, muscular rigidity, bradykinesia, and postural balance impairment. With accelerating population aging, the number of individuals with PD continues to increase, severely affecting quality of life [1]. Current clinical treatments mainly relieve symptoms and still have difficulty halting disease progression. Therefore, screening low-toxicity and effective neuroprotective agents from natural products has become a research hotspot.

Glycosides of Cistanches (glycosides of cistanches, GCs) are major active constituents of Cistanche and exhibit antioxidant, anti-apoptotic, and neuroprotective effects [2]. Oxidative stress refers to an imbalance between the production of reactive oxygen species (ROS) and the body's antioxidant defense mechanisms, resulting in cellular injury, inflammation, and other pathological changes [3]. Previous studies [4] have shown that nuclear factor erythroid 2-related factor 2 (Nrf2), as a key transcription factor regulating redox homeostasis, is a crucial signaling target that mediates neuronal protection. Thus, targeting the Nrf2 anti-oxidative stress pathway may represent a potentially effective intervention strategy for PD prevention and treatment.

Our preliminary research indicated that GCs can reduce the loss of dopaminergic neurons in the substantia nigra of PD model mice and improve motor function [5]. However, whether GCs exert anti-Parkinsonian effects via the Nrf2/HO-1 signaling pathway remains unclear. In this study, we established an MPTP-induced PD mouse model to evaluate the neuroprotective effects of GCs and further explore the underlying molecular mechanism.

 

1 Materials and Methods

1.1 Materials

1.1.1 Experimental animals

Forty 8-week-old SPF-grade healthy male C57BL/6 mice (body weight 21–23 g) were purchased from Beijing SBF Biotechnology Co., Ltd., production license number: SCXK (Beijing) 2024-0001. Mice were acclimated for 1 week before experiments. Housing conditions were temperature (23±1)∘C(23 \pm 1)^\circ\text{C}(23±1)∘C, relative humidity 50%–60%, ad libitum food and water, and a 12 h/12 h light/dark cycle. All procedures strictly complied with the "Regulations on the Administration of Laboratory Animals" and were approved by the Animal Ethics Committee of Baotou Medical College (Approval No.: 包医伦审〔2022〕第60号).

1.1.2 Reagents and instruments

GCs (content ≥ 85%) were provided by Baoji Kosmai Plant Polysaccharide Development Co., Ltd. (Lot No.: KM20220609011). MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Cat. No.: S31504). Primary antibodies: tyrosine hydroxylase (TH; Cat. No.: 25859-1-AP), Nrf2 (Cat. No.: 16396-1-AP), Keap-1 (Cat. No.: 10503-2-AP), and HO-1 (Cat. No.: 10701-1-AP) were from Proteintech (USA). GAPDH (Cat. No.: 48620), β-actin (Cat. No.: 21338-1), and secondary antibody (Cat. No.: L3012) were from Signalway Antibody (USA). Universal two-step detection kit (Cat. No.: PV-9000) and DAB kit (Cat. No.: ZLI-9017) were from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. PAGE gel accelerator (Cat. No.: T8090) was from Beijing Solarbio Science & Technology Co., Ltd. 30% acrylamide solution (Cat. No.: G2004), lysis buffer (Cat. No.: R0010), T-SOD (Cat. No.: G4306), GSH (Cat. No.: G4305), MDA (Cat. No.: G4302), and ROS kit (Cat. No.: G1746) were from Wuhan Servicebio Technology Co., Ltd. Rotarod and open field apparatus were from Shanghai XinRuan Information Technology Co., Ltd. Electrophoresis instrument was from Beijing Liuyi Biotechnology Co., Ltd. Microplate reader was from Thermo Fisher (USA). Paraffin microtome and light microscope were from Leica (Germany). Gel imaging system was from Shanghai Tanon Technology Co., Ltd.

1.2 Methods

1.2.1 Animal grouping and treatments

Mice were randomly allocated to four groups: Control group, PD group, GCs group, and L-DOPA group (n = 10 per group). According to previous literature [6], an MPTP intraperitoneal injection protocol was used to establish a PD mouse model. Except for the Control group, all other groups received MPTP at 30 mg/kg intraperitoneally once daily for 5 consecutive days; the Control group received intraperitoneal injections of an equal volume of normal saline.

After modeling, drug interventions were initiated: the GCs group received GCs 100 mg/kg by gavage; the L-DOPA group received L-DOPA 50 mg/kg by gavage; the Control and PD groups received equal volumes of normal saline by gavage. All groups were treated once daily for 28 consecutive days.

1.2.2 Assessment of motor function

1.2.2.1 Open field test

A standardized open field apparatus was used. Mice were placed in the central area of a 50 cm × 50 cm × 50 cm chamber and allowed to acclimate for 5 min. Then, a video tracking system recorded locomotor trajectories and total travel distance over 5 min.

1.2.2.2 Rotarod test

Mice were placed on a rotarod device. Speed increased from 0 to 30 r/min within 30 s, with a total test duration of 300 s. The latency to first fall was recorded. Each mouse was tested three times, and the mean value was used.

1.2.3 Tissue collection

One hour after the final administration, mice were anesthetized via intraperitoneal injection of 0.3% sodium pentobarbital (0.15 mL/10 g). In each group, three mice were randomly selected for perfusion fixation: the heart was exposed quickly, and mice were perfused with normal saline until the liver turned pale, followed by perfusion with 4% paraformaldehyde. Whole brains were removed and post-fixed in 4% paraformaldehyde for 24 h, followed by dehydration, embedding, and sectioning. The remaining mice were perfused only with normal saline; whole brains were collected, the substantia nigra region of the midbrain was dissected on ice, and samples were stored at −80°C for assays.

1.2.4 Immunohistochemistry (IHC)

Midbrain substantia nigra tissues were paraffin-embedded and cut into 5 μm sections. Sections were placed in sodium citrate buffer, heated to boiling, then cooled naturally and washed with PBS three times. Endogenous peroxidase blocking reagent was applied at room temperature for 10 min, followed by PBS washing. Sections were incubated with diluted TH primary antibody at 4°C overnight, washed with PBS, incubated with enhancer reagent at room temperature for 20 min, washed, incubated with enzyme-labeled secondary reagent at room temperature for 20 min, and washed again. DAB working solution (50 μL) was applied at room temperature for 2 min. Sections were rinsed, differentiated, blued, dehydrated, cleared in xylene, and mounted with neutral resin. TH-positive cell numbers were quantified using ImageJ.

1.2.5 Determination of oxidative stress indicators

Substantia nigra tissue was weighed, homogenized, and centrifuged for 10 min (12,000 r/min, 4°C). Supernatant was collected. Assays were conducted strictly according to kit instructions. Standard curves were generated and used to calculate T-SOD activity, MDA, GSH, and ROS levels in samples.

1.2.6 Western blotting

Substantia nigra tissues were lysed with protein lysis buffer and protease inhibitors, thoroughly homogenized, and lysed on ice for 30 min. Lysates were centrifuged at low temperature for 10 min (12,000 r/min). Protein concentration was determined by BCA assay. Samples were prepared, denatured at 100°C for 10 min, and stored for use. SDS-PAGE was performed followed by transfer to membranes and blocking. After TBST washes, membranes were incubated with primary antibodies at 4°C overnight, washed, incubated with secondary antibody at room temperature for 2 h, washed again, and developed with chemiluminescent substrate. ImageJ was used for densitometric semi-quantification.

1.2.7 Transmission electron microscopy (TEM)

Substantia nigra tissues from three groups were sampled. Each sample was rapidly fixed with 2.5% glutaraldehyde electron microscopy fixative and kept at 4°C overnight, followed by fixation with 1% osmium tetroxide at low temperature, ethanol gradient dehydration, and epoxy resin infiltration/embedding. Ultrathin sections (60 nm) were stained with 1% uranyl acetate and lead citrate. TEM was used to observe mitochondrial ultrastructural changes and to capture representative images.

1.3 Statistical analysis

GraphPad Prism 10.0 was used for data analysis. Normality and homogeneity of variance were tested. Measurement data are expressed as mean ± SD. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post hoc test was used for pairwise comparisons. P < 0.05 was considered statistically significant.

 

2 Results

2.1 Effects of GCs on motor function in PD mice

Open field test results showed that compared with the Control group, the PD group exhibited a reduced total travel distance (P < 0.01). Compared with the PD group, the GCs group and L-DOPA group showed increased total travel distance (P < 0.05) (see Fig. 1A, 1B). Rotarod test results showed that compared with the Control group, the PD group exhibited a shortened latency to fall (P < 0.01). Compared with the PD group, the GCs and L-DOPA groups showed prolonged latency to fall (P < 0.05) (see Fig. 1C).

 

Fig.1 Behavioral performance of mice in each group ( x  s, n=10) A, B: Open field test; C: Rotarod test; a: Control group; b: PD group; c: GCs group; d: L-DOPA group; **P<0.01 vs Control group; #P<0.05 vs PD group.

Fig.2 Expression of TH-positive cells in mice in each group and corresponding bar charts ( x  s, n=3) a: Control group; b: PD group; c: GCs group; d: L-DOPA group; **P<0.01 vs Control group; #P<0.05 vs PD group.

Fig.3 TH protein expression bands of mice in each group and corresponding bar charts ( x  s, n=3) a: Control group; b: PD group; c: GCs group; d: L-DOPA group; **P<0.01 vs Control group; #P<0.05 vs PD group.

 

2.2 Effects of GCs on TH in the substantia nigra of PD mice

IHC results showed that compared with the Control group, the number of TH-positive cells in the substantia nigra pars compacta was reduced in the PD group (P < 0.01). Compared with the PD group, the GCs group and the L-DOPA group showed increased TH-positive cell numbers (P < 0.05) (see Fig. 2). Western blot results showed that compared with the Control group, TH protein expression in the substantia nigra was reduced in the PD group (P < 0.01). Compared with the PD group, TH protein expression in the substantia nigra was increased in the GCs and L-DOPA groups (P < 0.05) (see Fig. 3).

 

Fig.4 Comparison of ROS content in PD model mice ( x  s, n=3) a: Control group; b: PD group; c: GCs group; **P<0.01 vs Control group; #P<0.05 vs PD group.

 

2.3 Effects of GCs on ROS in the substantia nigra of PD mice

Compared with the Control group, substantia nigra ROS levels were increased in the PD group (P < 0.01). Compared with the PD group, substantia nigra ROS levels were decreased in the GCs group, and the difference was statistically significant (P < 0.05) (see Fig. 4).

2.4 Effects of GCs on MDA, T-SOD, and GSH in the substantia nigra of PD mice

Compared with the Control group, substantia nigra T-SOD activity and GSH content were decreased in the PD group (P < 0.01), while MDA content was increased (P < 0.01). Compared with the PD group, the GCs group exhibited increased T-SOD activity and GSH content (P < 0.05) and decreased MDA content (P < 0.05) (see Fig. 5).

 

Fig.5 The effects of GCs on MDA, T-SOD and GSH in the midbrain substantia nigra of mice in each group ( x  s, n=3) a: Control group; b: PD group; c: GCs group; **P<0.01 vs Control group; #P<0.05 vs PD group.

 

2.5 Effects of GCs on neuronal mitochondrial ultrastructure in the substantia nigra of PD mice

In the Control group, substantia nigra neuronal mitochondria were mostly oval, with intact inner and outer membranes and clear boundaries; mitochondrial cristae were orderly. Compared with the Control group, the PD group displayed obvious mitochondrial swelling, unclear inner/outer membrane boundaries, disorganized and partially broken cristae, and vacuole-like changes. Compared with the PD group, mitochondrial ultrastructural damage was significantly alleviated in the GCs group: mitochondrial morphology was more regular, swelling and vacuole-like changes were reduced, and membrane boundary and cristae clarity improved (see Fig. 6).

 

Fig.6 The effects of GCs on the ultrastructure of substantia nigra neurons in PD mice ×20 000 A: Control group; B: PD group; C: GCs group; Red arrows indicate mitochondria.

 

2.6 Effects of GCs on Nrf2/HO-1 pathway-related proteins in the substantia nigra of PD mice

Western blot results showed that compared with the Control group, Nrf2 and HO-1 protein expression levels were decreased in the PD group, while Keap-1 protein expression was increased; differences were statistically significant (all P < 0.01). Compared with the PD group, the GCs group exhibited increased Nrf2 and HO-1 protein expression and decreased Keap-1 protein expression; differences were statistically significant (all P < 0.05) (see Fig. 7).

Fig.7 Representative Western blot bands and quantitative bar graphs of Nrf2, Keap-1, and HO-1 protein expression in mice in each group ( x  s, n=3) a: Control group; b: PD group; c: GCs group; **P<0.01 vs Control group; #P<0.05 vs PD group.

 

3 Discussion

The core motor symptoms of PD include bradykinesia, rigidity, resting tremor, and postural instability or gait disturbance [7]. In PD animal models, reduced spontaneous activity and impaired motor coordination can be assessed by the open field and rotarod tests, respectively [8]. Previous studies [9] reported that MPTP-induced PD mice show abnormal motor function, such as reduced travel distance and shortened rotarod latency to fall. Consistent with these reports, our study found that after MPTP administration, mice exhibited significantly reduced travel distance and shortened latency to fall, indicating impaired spontaneous activity and motor coordination. L-DOPA, a commonly used clinical drug for PD, significantly improved MPTP-induced motor dysfunction in mice, further supporting the reliability of the model and behavioral evaluation. After GCs intervention, both travel distance and latency to fall were significantly improved, suggesting that GCs can alleviate MPTP-induced PD-like motor dysfunction and may provide protection against related neuronal injury. These findings provide an experimental basis for further pharmacodynamic evaluation and mechanistic studies.

Progressive degeneration and loss of dopaminergic neurons in the substantia nigra is a core pathological feature of PD, leading to dysfunction of the nigrostriatal dopaminergic system [10]. Previous studies [11] assessed dopaminergic neuron damage by TH immunohistochemistry and observed a significant decrease in TH-positive cells in PD model mice, accompanied by motor abnormalities. Yildirim et al. [12] similarly reported that the number of TH-positive cells in the substantia nigra was significantly reduced in MPTP-induced PD mice. Consistent with these studies, our results showed that MPTP treatment decreased TH-positive cell numbers and induced motor dysfunction, indicating damage to the substantia nigra dopaminergic system. L-DOPA intervention partially inhibited the decline in TH-positive cells. GCs intervention also significantly attenuated the MPTP-induced reduction in TH-positive cells, although not fully restoring them to normal levels. This suggests that GCs can partially improve MPTP-induced substantia nigra dopaminergic system injury and may exert protective effects on dopaminergic neurons.

Studies [13–14] have reported that MPTP induces oxidative stress injury in mice, manifested as mitochondrial structural disruption, decreased T-SOD activity, reduced GSH content, and increased ROS and lipid peroxidation product MDA levels. In our study, MPTP-treated mice exhibited mitochondrial structural damage in the substantia nigra, elevated ROS and MDA levels, and decreased T-SOD activity and GSH content, confirming that the MPTP-induced PD model used here is reliable. After GCs treatment, mitochondrial structural damage was significantly inhibited, ROS and MDA levels decreased, and T-SOD activity and GSH content increased, indicating an antioxidant effect.

Nrf2 is a core transcriptional regulator maintaining cellular redox homeostasis. Its activation promotes the expression of antioxidant proteins such as HO-1, thereby enhancing cellular antioxidant defense capacity [15]. Lu et al. [16] found that Nrf2 protein expression was significantly reduced in MPTP-induced PD mice. In line with this, we observed that MPTP reduced Nrf2 and HO-1 protein expression, accompanied by decreased antioxidant enzyme activity and increased lipid peroxidation, suggesting suppression of antioxidant defense. GCs treatment significantly reversed the MPTP-induced downregulation of Nrf2 and HO-1, implying that GCs may enhance antioxidant defense by regulating the Nrf2/HO-1 signaling pathway.

In addition, our study showed that GCs reduced Keap-1 protein expression in PD model mice. Previous research [17] demonstrated that Keap-1 is a key negative regulator of Nrf2. Under homeostatic conditions, Keap-1 binds Nrf2 and promotes its ubiquitination and proteasomal degradation, maintaining low Nrf2 expression. When Keap-1 function is inhibited, Nrf2 is stabilized and translocates to the nucleus to induce antioxidant genes such as HO-1. Therefore, GCs may weaken Keap-1-mediated inhibition of Nrf2, promote Nrf2 stabilization and nuclear translocation, and enhance downstream HO-1 expression, thereby exerting anti-oxidative stress effects.

GCs are an effective fraction mixture composed of multiple components such as phenylethanoid glycosides and iridoid glycosides. Previous studies [18] confirmed that GCs can exert neuroprotective effects through antioxidant, anti-apoptotic, anti-inflammatory actions, and maintenance of mitochondrial homeostasis, improving functional deficits in models of cerebral ischemia, Alzheimer's disease, PD, and other neurological injuries. Current PD-related research on GCs mostly focuses on overall pharmacodynamic evaluation, while the integrated mechanism involving mitochondrial structure and the Nrf2/HO-1 pathway has not been systematically clarified. In this study, GCs activated the Nrf2/HO-1 pathway, inhibited mitochondrial structural damage, and reduced ROS-mediated oxidative stress, thereby exerting neuroprotective effects against PD.

Echinacoside is the most abundant and most active marker component in GCs and has been reported to have significant neuroprotective activity [7]. However, whether echinacoside mediates anti-PD neuroprotection via activation of the Nrf2/HO-1 pathway remains to be further verified. Future studies should focus on major monomers such as echinacoside to identify the key active constituents responsible for Nrf2/HO-1 pathway activation and the core neuroprotective effect.

In summary, GCs improved motor dysfunction in PD model mice. The mechanism may involve regulation of the Nrf2/HO-1 signaling pathway, enhancement of antioxidant enzyme activity, and attenuation of ROS-driven oxidative stress, thereby producing neuroprotective effects.

 

References

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Sample Purchase / Factory Information (Cistanche Extract Supplier)

Chengdu Wecistanche Bio-Tech Co., Ltd. (WECISTANCHE) is described as a major manufacturer/supplier focused on Cistanche herb industrialization and Cistanche extract products, established in 2003 and headquartered in Luopu County, Xinjiang, China. The profile states it has an integrated Cistanche industrial chain including seed breeding, cultivation bases, fresh material collection and storage capacity, and a GMP production facility. The "About Us" content lists certifications such as China SC, HACCP, organic-related certifications (including NOP), and indicates multiple Cistanche-related patents (e.g., membrane separation technology).

 

Contact (from the provided About Us content):

Email: wallence.suen@wecistanche.com

Address: No.1, Floor 26, Unit 1, Building 6, No.133 Sheng'an Street, High-tech District, Chengdu, China (Sichuan) Pilot Free Trade Zone.

Source: "About Us - WECISTANCHE" (https://www.xjcistanche.com/about-us)

 

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