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

INTRODUCTION 

Parkinson's disease (PD) is a common neurodegenerative disease occurring in elderly people with the pathological manifestations of loss of dopaminergic neurons in the substantia nigra (SN) due to degeneration. The severity of the disease is correlated with dopamine (DA) neuronal cell loss in the SN, which is consistent with the view that the neurodegenerative process progresses over many years before any symptoms appear (Sawle and Myers, 1993). The progressive nature of the disease suggests interesting possibilities for therapeutic intervention by blocking the underlying neurodegenerative process. The search for therapy-induced potent and specific actions of neurotrophic factors on DA neuron survival is therefore of considerable interest.

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Neurotrophic factors are essential proteins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF), which promote nerve growth, neurological development, axonal guidance, and neuronal function. Among all of the neurotrophic factors that protect and promote the repair of dopaminergic neurons, GDNF has the strongest effects (Hong et al., 2008; Rangasamy et al., 2010; Allen et al., 2013). GDNF has been shown to possess potent neurotrophic effects on DA neurons in vitro (Lin et al., 1993) and to exert neuroprotective effects in vivo. GDNF has been shown to rescue nigral DA neurons from lesion-induced cell death after surgical- or toxin-induced axotomy in rats (Beck et al., 1995; Kearns and Gash, 1995; Sauer et al., 1995) and partially also after systemic administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice (Tomac et al., 1995). The increased incidence of neuronal apoptosis and reduced protective effects of neurotrophic factors, potentially triggered by various pathological factors, underlie the degeneration of dopaminergic neurons (Holden et al., 2006).

C. tubulosa is an herbal medicine originating from several plants of the genus Cistanche. It is a major therapeutic option for kidney deficiency syndrome which is closely related to androgen hormones in traditional Chinese medicine (TCM). To date, lots of clinical and basic research on C. tubulosa has shown the activities of neurodegenerative diseases. The identification of TCM kidney-tonifying prescriptions in PD treatment may thus provide an alternative clinical treatment for PD. Echinacoside (ECH) is a major bioactive component found in the medicinal herb C. tubulosa. Studies have shown the therapeutic effects of glycosides of Cistanche and ECH, verbascoside (VER), and icariin (ICA) on Alzheimer's disease (AD), PD, and other vascular dementia patients (Urano and Tohda, 2010; Wang et al., 2013; Wu et al., 2014). Wu et al. (2014) suggested that C. tubulosa extracts that contained enough ECH and acteoside ameliorated the cognitive dysfunction caused by Aβ-42 via blocking amyloid deposition and reversing cholinergic and hippocampal dopaminergic neuronal function. Tao et al. (2015) found that phenylethanoid glycosides from C. tubulosa (Ph Gs-Ct) prevented high-altitude cerebral edema by decreasing the protein and mRNA expression of AQP4 in the brain tissue of rat models.

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Previous studies have shown that Chinese herbal compounds, including the three ingredients of C. tubulosa, epimedium, and rhizoma polygonati, alleviated damage to dopaminergic neurons and increased levels of dopamine by regulating the expression of neurotrophic factors (Wu et al., 2013). Thus, it is not yet known whether the C. tubulosa-induced neuroprotective effects are long-lasting and to what extent rescue of nigral DA neurons by administration of GDNF can afford significant preservation of motoric behaviors of relevance for the symptomatology of PD animals. This study used a TCM kidney-tonifying recipe, C. tubulosa nanopowder, that has received a national patent (patent number: 2011103028541) in China and has previously shown a certain therapeutic effect in PD. The present study, therefore, was designed to examine the neuroprotective and regenerative effects of C. tubulosa treatment and to investigate the apoptosis on the MES23.5 cells and behavioral definitive rats, and the regulation of GDNF, as measured by a battery of target tests.

MATERIALS AND METHODS 

Materials, Reagents, and Equipment 

C. tubulosa was purchased from Beijing Tong Ren Tang Group, Co., Ltd., Beijing, China. ECH, VER, and ICA came from the National Institutes for Food and Drug Control, China. The MES23.5 dopaminergic neuronal cell line was a gift from Professor Biao Chen, Laboratory of Neurobiology, Capital Medical University, Beijing, China. Fifty C57BL/6 male mice (weighing 20–25 g each) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (license number: SCXK2012-0002), Shanghai, China.

MPP+, MTT, and glutamine were purchased from Sigma-Aldrich (Carlsbad, CA, USA); DMEM/F12 medium and fetal bovine serum were purchased from Gibco Co. (Life Technologies, Carlsbad, CA, USA); and MPTP, DA standard and homovanillic acid (HVA) standard were purchased from Sigma-Aldrich (Carlsbad, CA, USA). β-actin, Bax, Bcl2, GDNF, GDNF family receptor alpha (GFRα1) and Ret antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA); 3,3'-diaminobenzidine (DAB) staining reagent kit was purchased from Fuzhou Maixin Biotech., Ltd. (Fujian, China); and SDS-PAGE gel sample preparation kit, ultrasensitive enhanced chemiluminescence (ECL) detection kit and bicinchoninic acid (BCA) assay were purchased from Beyotime Institute of Biotechnology (Beijing, China).

This study used the following instruments: ELX800 microplate reader (Bio Tek Winooski, VT, USA); CO2 incubator (Heraeus, Hanau, Germany); Gel DOC 2000 gel imaging analysis system, electrophoresis cell and electrophoresis tank (Bio-Rad, Hercules, CA, USA); DU-650 protein analyzer (Beckman Coulter, Inc., Fullerton, CA, USA); 5417R high-speed refrigerated centrifuge (Eppendorf, Hamburg, Germany); SXQM dual planetary ball mill (Changsha, Tencan Powder Technology Co., Ltd, Hunan, China); MM400 mixer mill (Retsch GmbH, Haan, Germany); Agilent 1200 high-performance liquid chromatography (HPLC; Agilent Technologies, Santa Clara, CA, USA); PowerPac Basic electrophoresis, PowerPac Basic transmembrane transfer system, Universal Hood II chemiluminescence imager, S1000 Thermal Cycler RNA reverse transcription system (Bio-Rad); Motic Med 6.0 tissue and cell image analysis system (Motic China Group, Co., Ltd, Xiamen, China). 

Preparation of C. tubulosa Nanopowder 

C. tubulosa was weighed, then purified and dehydrated. After conventional pulverization, C. tubulosa fine powder was passed through a 200-mesh sieve and freeze-dried. A temperature-controlled vacuum and high-energy ball mill were used to prepare the C. tubulosa nanopowder. First, raw C. tubulosa nanopowder was placed in a vacuum ball-milling tank that was loaded with carbide grinding balls. The ratio between the grinding balls and C. tubulosa nanopowder ranged from 15:1 to 5:1. To obtain a fine powder, the speed and duration of the high-energy ball mill were set to 300 rpm and 20 min, respectively. The fine powder was weighed for the processing of nanoscale materials and processed in the mixer mill with a frequency of 25/s and oscillation of 20s for three repetitions. PBS was used to dissolve and prepare a 25 mg/mL stock solution, followed by 30 min of ultrasonication, autoclaving, and finally storage at −20◦C. 

Quality Control of the Active Components of C. tubulosa by HPLC 

The ECH and VER contained gradient elution with octadecylsilane-bonded silica as filler, methanol as mobile phase A, and 0.1% formic acid solution as mobile phase B. The detection wavelength was 330 nm. The ICA contained gradient elution with octadecylsilane-bonded silica as filler and acetonitrile-water (30:70) as the mobile phase. The detection wavelength was 270 nm. The sample, control, and negative control were measured as 10 µL each for the test. 

Cell Culture and MTT Assay to Measure Viability of MPP+-Treated Cells 

MES23.5 cells were inoculated with 5% fetal calf serum, 1% glutamine, 2% 50× Sato's solution, and DMEM/F12 medium with 2% penicillin/streptomycin. They were incubated at 37◦C in a 5% CO2 incubator with saturated humidity. The cells were isolated and passaged with 0.25% trypsin and the cell suspension was harvested in the logarithmic growth phase. Isolated cells with a density of 1 × 105 were seeded into polylysine-coated 96-well plates, followed by the addition of different final concentrations (6.25, 12.5, 25, 50, 100, 200, 400 and 800 µmol/L) of MPP+ media. MES23.5 cells that were incubated with normal culture medium for 24 h and 48 h were used as negative controls in the in vitro experiments. The cells from the different treatment groups were incubated with MTT reagent for 4 h. The solution in the wells was subsequently discarded and 150 µL of DMSO was added and oscillated for 10 min. The absorbance of each sample at a wavelength of 570 nm was measured using an automatic microplate reader. The percentage of cell viability (%) = mean absorbance of the experimental group/mean absorbance of the negative control group × 100%. 

Relevant concentrations of MPP+ medium were added for the 24-hour treatment in MES23.5 cells using the same approach as for the in vitro culture. After the treatment, the solution in the well was discarded. Media containing different concentrations (10, 50, 100, 200, 250, 500, and 1000 µg/mL) of C. tubulosa nanopowder was added to the MES23.5 cells in different wells and left to incubate for 24 h and 48 h. The MES23.5 cells that were incubated with normal culture medium for 24 h and 48 h were used as negative controls. The MES23.5 cells that were incubated with MPP+ medium were used as the vehicle. The measurements were done in triplicate for each sample. The absorbance of the corresponding treatment and control groups was measured to calculate cell viability.

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TH Expression Measured by Immunocytochemistry 

When C. tubulosa nanopowder was in 100, 200, and 250 µg/ml, the cell survival rate increased significantly (Figure 2G). Thus, in subsequent experiments, we tested the three concentrations: low-dose, middle-dose, and high-dose groups. Three replications were tested for each of the C. tubulosa groups. Sterilized, polylysine-coated coverslips were placed into 6-well plates. Next, 5 × 104 cells were seeded in each well and incubated for 24 h. Conventional fresh medium was replaced in the normal control group and a final concentration of 100 µmol/L MPP+ medium was replaced in the remaining treatment groups to incubate for 24 h. Conventional fresh media were then replaced in the normal control and vehicle groups, and final concentrations of 100, 200, and 250 µg/mL C. tubulosa nanopowder were incubated with the cells for 24 h in the low-, moderate- and high-dose C. tubulosa treatment groups, respectively. The MES23.5 cells in the different groups were washed three times in PBS to remove the supernatant and further fixed in 4% paraformaldehyde for 15 min. After washing with PBS, the cells were incubated with a peroxidase blocker at 37◦C for 30 min and then washed again with PBS. A 0.2% Triton X-100 solution was used for cell permeabilization for 10 min, followed by washing with PBS. Normal goat serum was added to each sample and incubated at room temperature for 30 min. The normal goat serum was then removed and the primary antibody diluted 1:400 in PBS was added to each sample and incubated at 4◦C overnight. The cells in the negative control group were incubated with PBS at 4◦C overnight. After washing with PBS, the cells were incubated with biotin-labeled secondary antibodies in the moisture chamber at 37◦C for 20 min. Each sample was then washed in PBS and labeled with horseradish peroxidase-streptavidin conjugates (working solution C) at 37◦C for 20 min. After washing with PBS, the cells were stained with DAB reagent in the dark for approximately 1–10 min and the development of a brown color was monitored under light microscopy. Each sample was then washed twice in distilled water for 1–2 min and the nuclei were counterstained with hematoxylin solution for 0.5–1 min. After thoroughly rinsing each sample in water, the cells were immersed in 1% hydrochloric acid alcohol for differentiation and 1% aqueous ammonia, followed by thoroughly washing the cells in water. The cells from each sample were then dehydrated in 70% ethanol for 2 min, 80% ethanol for 2 min, 90% ethanol for 2 min twice, 95% ethanol for 2 min twice, and 100% ethanol for 2 min twice. The cells were then immersed in xylene solution for 2 min twice and mounted on a glass slide with neutral resins. Under light microscopy, each sample underwent image capturing and random selection of 5–10 effective visual fields to determine the expression of selected proteins in dopaminergic neurons indicated by the intensity of the brown particles and to semi-quantify the protein content by its average gray value. 

Apoptosis Rate of MES23.5 cElls Measured by Flow Cytometry 

Adherent cells were washed once with PBS. For cell isolation, an appropriate amount of EDTA-free trypsin solution was added at room temperature and the solution was gently pipetted to allow adherent cells to detach. A cell culture medium was then added to stop the trypsinization. The mixture was transferred to a new centrifuge tube and then centrifuged for 5 min at 1500 rpm to collect the isolated cells. After discarding the supernatant, the cell pellet was gently resuspended using PBS, and the cells were counted. Approximately 1 × 105 to 5 × 105 resuspended cells were centrifuged for 5 min at 1500 rpm and the supernatant was discarded. Five microliters of Annexin V-FITC binding solution were added to the cell pellet to gently resuspend the cells. Another 5 µL of Annexin V-FITC was added and mixed thoroughly. Five microliters of propidium iodide solution were used for cell staining by incubating at room temperature for 10 min shortly before the flow cytometry. 

Western Blot Analysis for in vitro 

The cells were divided into a normal group, MPP+ treatment group, low-dose C. tubulosa treatment group, moderate-dose C. tubulosa treatment group, and high-dose C. tubulosa treatment group. The expression of Bcl2 and Bax was measured in each. A total of 1 × 105 cells per well in the 6-well plate were used for modeling and treatment in each group before cell harvesting. A mixed lysate, containing RIPA buffer, protease inhibitor, and phosphatase inhibitor, was added to lyse the cells for 30 min on ice. After centrifugation, the supernatant was used for protein analysis. The total protein was quantified using a BCA assay and separated by 10% SDS-PAGE. The separated proteins were transferred to a membrane and incubated with a 5% skim milk-blocking buffer at room temperature for 2 h. The membrane was then incubated in primary antibody (Bcl-2 0.34 mg/ml, Bax 0.11 mg/ml, 1:200 dilution) at 4◦C overnight. After a washing step, the membrane was incubated in a secondary antibody (0.5 mg/ml, 1:5000 dilution) at 4◦C for 1 h and then in ECL developer for 2 min for conventional development. Quantity One software was used for the semi-quantitative analysis of protein expression. 

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Experimental Animal Modeling and Drug Administration 

Fifty specific-pathogen-free 8-week-old male mice were randomly divided into five groups: a normal group, MPTP treatment group (Vehicle), low-dose C. tubulosa treatment group, moderate-dose C. tubulosa treatment group, and high-dose C. tubulosa treatment group. The animals were housed at 20–22◦C with free access to food and water. The mice in the normal group were intraperitoneally injected with an equal volume of normal saline for seven consecutive days. The mice in the other treatment groups were intraperitoneally injected with MPTP (30 mg/kg/d) for seven consecutive days to establish vehicles. 

During PD modeling, the mice in the low-dose, moderate-dose, and high-dose C. tubulosa treatment groups were intragastrically administered equivalent clinical volumes of 4 g/kg/d, 8 g/kg/d, and 16 g/kg/d C. tubulosa nanopowder, respectively, for 14 consecutive days. The mice in the control and vehicle groups were intragastrically administered equivalent volumes of normal saline for 14 consecutive days. All experimental procedures were approved by the Ethical Committee of Fujian University of TCM and were performed according to the internationally accepted principles for laboratory animal use and care. All efforts were made to minimize animal suffering in this study.