Cistanche Tubulosa Protects Dopaminergic Neurons Through Regulation Of Apoptosis And Glial Cell-Derived Neurotrophic Factor: in Vivo And in Vitro-Ⅱ

Behavioral Tests 

Swimming Test (Zhu et al., 2014) 

The coordination of body movement in mice was measured by Swimming test. The mice were individually placed in a water tank (25 cm in height and 10 cm in diameter) containing 10 cm of water and tested in a quiet environment to record their stationary duration over 5 min. 

Open Field Test (Kawai et al., 1998) 

Locomotor activity was measured by using the Open field test. The mice were tested in a quiet and dimly lit environment and individually placed in a 30 cm × 30 cm × 15 cm transparent acrylic container with a 6 cm × 6 cm separation grid at the bottom. The mice were given 10 min to adapt to the environment and then the ambulation of the grid number and rearing frequency of individual mice were measured five consecutive times to obtain mean values. 

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Brain Tissue Sampling 

Before tissue sampling, the mice were fed ad libitum with free access to water and received drug intervention for 14 consecutive days. Four mice in each group were selected and quickly decapitated. The SN (Bregma: −2.75 mm −2.92 mm) from each animal was isolated and placed on ice. Brain tissues were rinsed with 0.9% ice-cold sodium chloride solution to remove any blood and dried on filter paper before storing at −80◦C. Four mice from each group were anesthetized intraperitoneally and their chests opened. An infusion needle was then inserted into the left ventricle of each animal. To remove blood in the circulatory system, the right atrial appendage was cut and the animal was infused with 4◦C normal saline until the liver turned pale to ensure successive perfusion. Once the effluent of the right atrium became clear, each animal was perfused with 4% paraformaldehyde fixative. After the perfusion, the brain tissue of each animal was then dissected carefully and post-fixed in 4% paraformaldehyde for 24 h. Fixed brain tissues were then rinsed under running water, dehydrated in a graded series of ethanol solutions, and cleared in xylene solution. This was followed by paraffin immersion and embedding. 

Changes of DA Quantity Measured by HPLC 

The nanopowdered SN from each group was placed in an ice bath containing 0.9% sodium chloride solution (1:9 ratio). The brain tissue was homogenized using an ultrasonic cell disruptor and centrifuged at 1200 rpm for 20 min at 4◦C to obtain the supernatant. For HPLC, a Hypersil AA-ODS column (2.1 mm × 200 mm, 5 µm) at 30◦C column temperature was used. Fluorescence detection was performed at 280 nm λex and 340 nm λem. The injection volume was 10 µL. 

Expression of TH, GDNF, GFRα1 and Ret Detected by Immunohistochemistry 

Paraffin sections (5 µm thick) of individual brain tissue were isolated from each animal and placed in a 40◦C warm water bath for flattening and adhering to glass slides. All of the tissue slides were incubated in a 60◦C oven for 3–6 h, followed by xylene dewaxing, gradient ethanol dehydration, and antigen retrieval by incubating in a citric acid buffer and heating in a microwave for 20 min. The tissue slides were then incubated in a 3% H2O2 solution at room temperature for 10 min. After being washed three times in PBS, the tissue slides were incubated with normal serum in a closed chamber at room temperature for 20 min. Immunohistochemical staining was conducted according to the manufacturer's instructions. A Motic Med 6.0 image analyzer was used to calculate the value of integrated optical density in the positively stained cells. 

Western Blot Analysis in Brain Tissues of Mice 

This study assessed the protein expression of tyrosine hydroxylase (TH), GDNF, GFRα1, Ret, Bcl2, and Bax. The brain lysate from each group was homogenized for 30 min on ice, followed by low-temperature centrifugation, 20,000 rpm, at 4 ◦C for 5 min to collect the supernatant. The protein samples were separated under constant pressure using a 10% SDS-PAGE gel as described above. The primary antibody concentration: TH 0.15 mg/ml, GDNF 0.5 mg/ml, GFRα1 0.8 mg/ml, Ret 0.63 mg/ml, Bcl-2 0.34 mg/ml, and Bax 0.11 mg/ml. The procedure was the same as above. 

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Statistical Analysis 

This study used SPSS 20.0 statistical software for data processing and analysis. Parameter values were expressed as mean ± standard deviation (¯x ± S). ANOVA was used for single-factor data analysis. LSD or the Games-Howell test was used to compare the groups. P < 0.05 (or P < 0.01) was considered as a statistically significant difference.

RESULTS 

The Active Components of C. tubulosa Nanopowder 

In the range of 200–400 nm scanning, ECH in C. tubulosa and VER in 330 nm had the maximum absorption peak, which appeared within 20 min. ICA had maximum absorption peaks at 270 nm and appeared after 20 min (Figure 1A). The results showed that the negative samples did not interfere with the detection (Figure 1B). The samples and control had the same chromatographic peaks and the negative sample had none. This showed that the other ingredients in the sample did not interfere with the component being measured. Moreover, the three components and the adjacent peaks can reach the separation baseline and the separation degree was greater than 1.5. 

C. tubulosa Nanopowder Reduced MPP+ -Induced Cytotoxicity in MES23.5 Cells 

The viability of MES23.5 cells was significantly reduced with increasing concentrations of MPP+. Figure 2F shows the significant cytotoxicity of different concentrations of MPP+. 

C. tubulosa nanopowder reduced MPP+-induced cytotoxicity and enhanced the viability of MES23.5 cells. Figure 2G shows that C. tubulosa nanopowder dosages of 10–250 µg/mL exerted dose-dependent protective effects on the MPP+-treated MES23.5 cells. 

Cytomorphological Effect of C. tubulosa Nanopowder 

Normal MES23.5 cells had good cell adhesion and were spindle-shaped with clear cell boundaries and synapses. MPP+-damaged MES23.5 cells displayed poor cell adhesion and shrinkage, and many were suspended in the media with contracted synapses. These cells were aggregated, shrunken and round with vacuoles inside, and the nuclei were disintegrated or collapsed. C. tubulosa nanopowder in different dosages improved the cytomorphology of MES23.5 cells in different degrees by improving cell adhesion and synaptic clearance of the vehicle group. MES23.5 cells in the high-dose C. tubulosa treatment group showed morphology that was similar to the normal control group (Figures 2A–E). 

Effect of C. tubulosa Nanopowder on TH Expression and Apoptosis in the Cells 

Figure 3 shows a significant reduction in the TH protein expression in the vehicle group. The TH protein expression increased differently in groups treated with different dosages of C. tubulosa. However, the LSD test showed there was no significant difference between the three treated groups. 

Figure 4 shows the results of the apoptosis assessment using flow cytometry. The rate of apoptosis in the vehicle group was significantly higher than in the other groups. Cells treated with different dosages of C. tubulosa nanopowder showed different degrees of decline in the apoptotic rate compared with the vehicle group. Cells in the middleand high-dose C. tubulosa treatment group had the most significant improvement in apoptotic rate compared with the other C. tubulosa treatment groups. LSD test showed there was no significant difference between the two treated groups but a significant difference between the low-dose group too. 

Effect of C. tubulosa Nanopowder on Bcl2/Bax Protein Expression in the Cells 

Figure 5 shows that the expression of Bcl2 protein in the cells of the vehicle group was significantly lower compared with the normal control group. In contrast, the expression of Bax protein in cells of the vehicle group was significantly higher than in the normal control group. C. tubulosa treatment groups showed increased Bcl2 protein expression and decreased Bax protein expression in MPP+-treated MES23.5 cells. Between the three treated groups, there were significant differences in the LSD test. These effects were dose-dependent. 

Behavioral Tests 

The results of the Swimming test suggested that the mice in the vehicle group had relatively long stationary durations, which increased over time. At day 14, the mice in the vehicle group had a significantly longer stationary duration than the mice in the normal control group. The stationary duration of the mice in 

the low-dose C. tubulosa treatment group was not significantly different from that of the mice in the vehicle group. However, the stationary duration of the mice in the high-dose C. tubulosa treatment group was significantly less than that of the mice in the vehicle group. 

The results of the Open field test suggest that after MPTP-induced damage in the mice, the mice in the vehicle group demonstrated a significant decline in their ability for spontaneous activity as shown by the rearing frequency. After a 14-day administration of C. tubulosa nanopowder, the mice in the moderate- and high-dose treatment groups had significantly higher rearing frequencies compared with the mice in the vehicle group (Figures 6A, B). 

Effect of C. tubulosa Nanopowder on DA Content in Mice 

Changes in the DA content of the SN were determined by HPLC. It was found that the DA content in the brain of the vehicle group was significantly reduced. The DA content in the brains of PD mice in the low-dose C. tubulosa treatment group did not differ significantly from the mice in the vehicle group. However, C. tubulosa treatment increased the DA levels in the brains of PD mice in a dose-dependent manner. The brains of PD mice treated with high-dose C. tubulosa had a significantly higher DA content than the brains of mice in the vehicle group (Figure 6C). 

Effect of C. tubulosa Nanopowder on TH Expression in Mice 

The number of TH-positive cells and the level of TH protein expression in the SN of MPTP-induced PD mice were lower compared with mice in the control group. After the C. tubulosa treatment, the number of TH-positive cells and the level of TH protein expression in the SN of MPTP-induced PD mice increased, with a significant difference between the high-dose C. tubulosa treatment group and the vehicle group by LSD test; and there were significant differences between the three treated groups (Figure 7). 

Effect of C. tubulosa Nanopowder on Protein Expression of GDNF and its Receptors, GFRα1 and Ret in Mice 

The protein expression of GDNF and its receptors, GFRα1 and Ret, in the positively stained cells, was evaluated using immunohistochemistry. Western blot analysis was used to evaluate the protein expression levels in the SN of the different groups of mice. The findings for the different groups were similar using the two detection methods. The expression of GDNF and its receptor proteins, GFRα1 and Ret, in positively stained cells in the SN of the mice in the vehicle group, was significantly lower than in the mice in the normal control group. Different dosages of C. tubulosa treatment increased the number of GDNF-, GFRα1-, and Ret-positive cells (Figures 8A–S). 

The protein expression of GDNF, GFRα1, and Ret in the SN of the mice in the vehicle group was significantly lower than in the mice in the control group. Increasing treatment concentrations of C. tubulosa nanopower significantly enhanced the expression of these proteins. The protein expression of GDNF, GFRα1, and Ret in the SN of the mice in the high-dose C. tubulosa treatment group was significantly higher than in the mice in the vehicle group (P < 0.01; Figures 8T, U). 

Effect of C. tubulosa Nanopowder on Protein Expression of Bcl2/Bax in Mice 

Bcl2 protein expression was significantly reduced and Bax protein expression was significantly enhanced in the SN of mice in the vehicle group (P < 0.01) compared with the mice in the normal control group. High-dose C. tubulosa treatment significantly increased Bcl2 protein expression and significantly reduced Bax protein expression in the brains of the vehicle mice (P < 0.01; Figure 9). LSD test showed there was no significant difference between the middle-dose and high-dose groups but a significant difference between the low-dose group. 

DISCUSSION 

PD and Apoptosis

PD is a neurodegenerative disorder. According to Zhang et al. (2005), the prevalence is 10.7% in the Chinese population aged over 55 and 1.67% in those aged over 65. The number of PD patients has increased annually with the acceleration of global aging, placing a heavy financial burden on the families of patients and society at large. In the brain, dopaminergic neurons are mainly involved in the synthesis and secretion of DA. They are widely distributed in the central nervous system and located primarily in the SN (80%). TH is the key rate-limiting enzyme for DA synthesis. Thus, the inhibition of TH activity reduces DA synthesis (Huot and Parent, 2007). The main pathological and biochemical changes of PD are apoptosis of dopaminergic neurons in the SN, a significant reduction of nigrostriatal DA, and the formation of Lewy bodies in dopaminergic neurons (Dexter and Jenner, 2013). The etiology of PD involves associated genetic factors, environmental factors, and aging of the nervous system (Allam et al., 2005). The pathogenesis of PD remains unclear in modern medicine. Since the late 1960s, levodopa replacement therapy has been used successfully to treat PD and has been recognized as a major turning point in PD treatment. However, the long-term application of this therapy causes side effects and the therapy does not treat the underlying causes of PD (Del Sorbo and Albanese, 2008). Therefore, active research for new drugs or treatment methods targeting the protection of dopaminergic neurons is crucial for treating PD.

In earlier studies, the application of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling indicated that 0.6%–4.8% of dopaminergic neurons in the SN of PD patients showed apoptosis (Mochizuki et al., 1996). Electron microscopy showed apoptotic features, including chromatic condensation and apoptotic bodies in dopaminergic cells (Anglade et al., 1997). Tompkins et al. (1997) performed an ultrastructural analysis of brain tissue autopsies from patients with PD, AD, and diffuse Lewy body disease (DLBD). They found apoptotic bodies in the dense layer of the SN in PD and DLBD patients, providing conclusive evidence of neuronal apoptosis in PD and related diseases. Therefore, the reduction or suppression of apoptosis in dopaminergic neurons is fundamental for PD treatment.

Previous studies showed that MPTP induced PD-like symptoms. MPTP crosses the blood-brain barrier and is metabolized by type B monoamine oxidases in astrocytes. It is subsequently converted to toxic MPP+, which accumulates in the mitochondria of dopaminergic neurons through the protein intake of the DA transporter. It thus generates excess oxygen free radicals that inhibit complex I activity of the mitochondrial respiratory chain and ATP synthesis. These events further promote free radical formation and oxidative stress reactions, and eventually lead to the degeneration and death of dopaminergic neurons. Hence, this study used MPP+ to establish an in vitro vehicle in MES23.5 dopaminergic neurons and MPTP to induce a vehicle mouse for mutual verification. According to the results of the MTT assay, MPP+ significantly reduced the viability of MES23.5 cells, suggesting that MPP+ was cytotoxic to dopaminergic neurons. The results also demonstrated that C. tubulosa effectively enhanced the expression of anti-apoptotic proteins and inhibited the increase of MPP+-induced apoptosis.

PD and GDNF 

GDNF is a neurotrophic factor, which was first isolated by Lin et al. (1993). Lin et al. (1993) also showed that GDNF had specific nutritional effects on dopaminergic neurons in the midbrain of rats. GDNF, neurturin (NTN), persephin (PSP), and artemin (ART) constitute the GDNF family. They are structurally similar and functionally related secretory proteins (Kotzbauer et al., 1996; Baloh et al., 1998; Milbrandt et al., 1998; Woodbury et al., 1998). 

The GDNF receptor consists of two components. The first component, GFRα, is fixed to the outer membrane of glycosylphosphatidylinositol (GPI) and anchored to the surface of connexin. The second component is the Ret protein. Research has shown that there are four different types of GFRα: GFRα1, GFRα2, GFRα3 and GFRα4. GFRα1 is a high-affinity receptor of GDNF (Onochie et al., 2000; Chen et al., 2001; Lindahl et al., 2001). Ret protein is a functional receptor of GDNF. The homodimer molecule of GDNF directly binds to GFRα1 to form complexes and interacts with Ret, resulting in the dimerization and activation of Ret. Due to the autophosphorylation of Ret, Ret activates several common TH signaling pathways. In the absence of Ret protein, GDNF causes protein phosphorylation of MAPK, PI-3, and PLC-γ, in addition to mRNA expression and functional activity of C-fos through its receptor protein, GFRα1 (He et al., 2008). 

Studies have demonstrated that GDNF had the strongest protective effect on dopaminergic neurons (Rangasamy et al., 2010; Campos et al., 2012). In vehicles using MPTP and 6-hydroxydopamine (6-OHDA) to induce damage in dopaminergic neurons, GDNF protects dopaminergic neurons by reducing apoptosis and promoting axonal growth to induce stem cell differentiation (Lucas et al., 2012; Littrell et al., 2013). Lin et al. (1993) showed that GDNF specifically promoted viability, differentiation, and axonal growth of dopaminergic neurons to promote the uptake of DA in neurons. The study also showed that GDNF not only prevented acute toxicity but also alleviated long-term toxicity of MPP+ or 6-OHDA in dopaminergic neurons, furthermore preventing cell death in stressed or damaged cells (Yu et al., 2010). In addition, GDNF promoted neural stem cell proliferation and differentiation toward dopaminergic neurons in the midbrain (Lindsay, 1995) to rescue dopaminergic neurons from retrograde degeneration (Hong-Juan et al., 2011). 

Studies have shown that GDNF expression in the SN was significantly reduced in animal vehicles (Yang et al., 2010). This suggests that it may be one of the mechanisms of pathogenesis in PD rats. The injection of 5–15 µg/d GDNF into the lateral ventricle or striatum of an MPTP-induced vehicle animal for three consecutive months promoted nigrostriatal repair of the dopaminergic system in the vehicle animal (Grondin et al., 2002). Studies of GDNF treatment for PD in animal vehicles have shown that intracerebral injection of GDNF into different brain regions, such as the SN, caudate nucleus, and lateral ventricle, improved movement disorders associated with PD animal models, including decreased motor activity, muscle rigidity and tremor (Grondin et al., 2002). However, GDNF cannot directly pass through the blood-brain barrier. Therefore, local cerebral injection of GDNF involves substantial risk and difficulties in clinical application. Applications to introduce exogenous GDNF via controlled-release microspheres, sustained-release capsules, and viral genes are still being studied (Liang et al., 2010; Yang et al., 2010; Qiao et al., 2012). Given the limitations of various techniques to introduce exogenous GDNF into the brain, neuroprotective agents that promote the release of endogenous GDNF are significant for clinical application.

PD and C. tubulosa Nanopowder 

PD is more common in middle-aged adults and the elderly. The theory of TCM considers that PD is primarily located in the brain and is mainly due to liver and kidney deficiency, in addition to vital energy and blood insufficiency. According to this theory, PD treatment should thus focus on invigorating kidney and bone marrow. Yang et al. (2010) used randomized, double-blind, and placebo-controlled clinical trials and found that combination therapy using Madopar and kidney-tonifying recipes alleviated the motor dysfunctions of PD patients. The treatment result was better than a single therapy using Madopar. The treatment efficacy of TCM monotherapy or compound prescription in PD has been confirmed in PD animal models and clinical applications. TCM applications for tonifying kidneys and promoting blood circulation reduced the dosage of monotherapy for PD using Madopar. Some studies have suggested that TCM improved symptoms of PD and protected dopaminergic neurons, which might have been closely related to the promotion of endogenous GDNF expression (Hong-Juan et al., 2011; Qiao et al., 2012).

The kidney-tonifying compound used in this study, C. tubulosa nanopowder, contained Cistanche, epimedium, and Rhizoma polygonati. Modern research suggests that the chemical composition of Cistanche is ECH, which protects dopaminergic neurons in the SN in the MPTP-induced PD mice and inhibits the reduction of DA and the DA transporter (Zhao et al., 2010). In addition, it prevents 6-OHDA-induced reduction in DA and protects striatal dopaminergic neurons (Chen et al., 2007). Epimedium inhibits the activation of caspase-3 and exerts neuroprotective roles (Liu et al., 2011). Epimedium flavonoids effectively promote neural stem cell proliferation and differentiation (Yao et al., 2010).

In this study, C. tubulosa nanopowder antagonized the increase of MPP+-induced apoptosis in a dose-dependent manner. It significantly improved TH expression in the in vitro vehicle and had significant anti-apoptotic effects in dopaminergic neurons. The MPTP-induced vehicle mice showed behavioral disorders and significantly reduced TH expression in the midbrain tissues and DA levels, which are typical pathological features of PD. Different dosages of C. tubulosa nanopowder shortened the stationary duration, enhanced autonomous activities, improved behavioral disorders, elevated DA levels in the brain, and increased TH expression in vehicles. These results suggested that C. tubulosa nanopowder exerted protective effects in dopaminergic neurons, thereby improving behavioral disorders of vehicles. Different dosages of C. tubulosa nanopowder increased the expression of GDNF protein and its receptor proteins in the brain of vehicle mice. High-dose C. tubulosa treatment significantly upregulated Bcl2 expression and reduced Bax expression, which suggested that C. tubulosa nanopowder might promote GDNF expression and secretion in the MPTP-damaged mouse brain. In addition, it might exert neuroprotective effects in dopaminergic neurons and minimize neuronal apoptosis through the neurotrophic support roles of GDNF.

This study demonstrated that C. tubulosa nanopowder exerted protective effects in dopaminergic neurons in both in vitro and in vivo and increased TH expression to improve DA content. It also improved behavioral disorders in MPTP-induced vehicle mice, regulated protein expression of GDNF and its receptor proteins in the SN, and had anti-apoptotic effects in the PD mice. The mechanism underlying the clinical effects of C. tubulosa nanopowder in PD may involve increasing the content of endogenous GDNF in the brain and thereby reducing the damage to dopaminergic neurons. 

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