Part 1 Neuroprotective Effects Of Phenylethanoid Glycosides in An in Vitro Model Of Alzheimer's Disease

Is the neuroprotective effect of Cistanche real?

JIANHUA YANG1*, BOWIE JU1,2*, YAO YAN1, HUAN HUAN XU1,

SHANSHAN WU1, DANDAN ZHU1, DANDAN CAO1, and JUMPING HU2

1 Department of Pharmacy, The First Affiliated Hospital; 2 Department of Natural Medicines,

College of Pharmacy, Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China

Received January 8, 2016; Accepted January 6, 2017

Abstract.

The present study aimed to investigate the neuroprotective effects of phenylethanoid glycosides (PhGs) on H2O2- and β-amyloid peptide (Aβ)1-42-induced injury of PC12 cells as an in vitro model of Alzheimer's disease (AD). The optimal induction conditions were established through screening of various incubation times and concentrations. PC12 cells were treated with 0.5 µM Aβ1-42 and H2O2 in the presence of PhGs for 24 h and the cell viability was then evaluated by an MTT assay; lactate dehydrogenase (LDH) release and malondialdehyde (MDA) content were also measured. The optimal conditions for establishing the AD model were the treatment of PC12 cells with 0.5 µM Aβ1-42 for 48 h, or with 25 µM H2O2 dissolved in DMEM with PBS. PhGs at concentrations of 5, 25, and 50 µg/ml increased the viability and decreased LDH and MDA release by PC12 cells injured with Aβ1-42 or H2O2. In conclusion, the model of Aβ1-42- and H2O2-induced PC12 cell injury was successfully established. PhGs were shown to have a significant neuroprotective effect against Aβ1-42- or H2O2-induced cell injury.

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Introduction

Alzheimer's disease (AD), a neurodegenerative disorder with the clinical characteristics of progressive memory loss and cognitive function impairment (1), is the most common cause of dementia worldwide (2). The financial costs are immense due to the prevalence of AD (3). Hence, it is urgent to develop appropriate means for the management and prevention of AD.

The pathogenesis of AD is closely associated with the accumulation of neurofibrillary tangles and senile plaques (SPs) in affected brain regions (4,5). β-amyloid peptide (Aβ), the major component of SPs, has been reported to have a causative role in the progression of AD as has a toxic effect on neuronal cells (6). Aβ fragments, including Aβ1-40, Aβ25-35, and Aβ1-42, have been generated through the split of amyloid precursor protein (7). The neurotoxicity of Aβ1-42 was found to be significantly higher than that of Aβ25-35 and Aβ1-40, and Aβ1-42 is able to induce an AD model (1,8-10). Oxidative stress may be involved in the pathogenesis of AD and is the major mechanism underlying Aβ-induced neurotoxicity (11-13). Several studies suggested that Aβ1-42 caused intracellular accumulation of reactive oxygen species (ROS), leading to lipid and protein oxidation, DNA damage, and activation of cell cycle checkpoint signaling (1,14,15). Excessive amounts of H2O2 may lead to oxidative damage and induce apoptosis of PC12 cells (16). Therefore, targeting oxidative stress may be a promising approach for the development of therapeutic strategies for inhibiting Aβ-induced neurotoxicity in AD. Herba (H.) Cistanche, a Chinese herbal medicine commonly used in mainland China for nourishing the kidneys and replenishing essence and blood, has been used to treat memory loss and senile constipation (17). Phenylethanoid glycoside (PhG), one of the major constituents in H. Cistanche, improves the impairment of neuronal apoptosis caused by Aβ25-35 via its anti-oxidant effects (18,19). A previous study identified five major components from total PhGs, namely acteoside, 2'-acetylacteoside, echinacoside, cistanosides, and isoacteoside (20). Among these components, acteoside and echinacoside have been reported to have neuroprotective effects on Aβ25-35- or H2O2-induced neurotoxicity (21-23). For instance, Wu et al (24) suggested that acteoside and echinacoside ameliorated cognitive dysfunction caused by Aβ1-42. The present study aimed to investigate the protective effects of PhGs in an in vitro rat cell model of AD.

Materials and methods

Preparation of PhGs. Total PhGs were extracted from H. Cistanche as previously described (25). The air-dried stem of H. Cistanche was powdered and extracted by percolation with 80% EtOH. The percolate was evaporated under reduced

pressure, followed by re-suspension in an appropriate amount of H₂O₂ (100 µmol/l). The mixture was isolated on an SP-825 macroporous resin column (Mitsubishi Chemical, Tokyo, Japan) and eluted with 0, 30, 50, 70, and 90% EtOH in water. To obtain the PhG-rich fraction, the 30-50% EtOH eluents were concentrated and dried under reduced pressure. Ultraviolet (UV) spectrophotometry was performed to determine the total PhGs. The content of echinacoside and acteoside was determined by high‑pressure liquid chromatography according to a previous protocol (26). A Hypersil ODS-2 column (4.6x250 mm, 5 µm; Dalian Elite Analytical Instruments, Co., Ltd., Dalian, China) was used and maintained at room temperature. The mobile phases were methyl cyanides and water containing 0.4% phosphoric acid (v/v; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The flow rate was 0.75 ml/min and the wavelength was set to 333 nm.

Cell culture and drug treatment. The PC12 rat pheochromocytoma cell line was provided by Dr. He Chunhui, the Medical School, Xinjiang Medical University (Urumqi, China). Cells were cultured in high‑glucose Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Sangon Biotech Co. Ltd., Shanghai, China), 100 U/ml penicillin, and 100 U/ml streptomycins in an incubator at 37˚C containing 5% CO₂ and 95% air. Upon reaching 80% confluence, the cells were treated with 0.25% trypsin and passaged.

In order to eliminate the interference of the drug itself on the growth of PC12 cells, a toxicity experiment was performed. In brief, PC12 cells (3x10⁴ cells/ml) were seeded in 96-well plates at 100 µl/well and incubated at 37˚C overnight. After discarding the supernatant, 200 µl complete DMEM was added in the blank group, while cells in the intervention groups were treated by PhGs at various doses (5, 25, 50, 75, 100, 125, 150, 175, and 200 µg/ml). Following incubation of the cells at 37˚C for 48 h, 20 µl MTT solution (Sigma‑Aldrich; Merck KGaA) was added to each well. After incubation for 4 h, the supernatant was discarded and 150 µl dimethylsulfoxide was added per well, followed by agitation for 10 min. The optical density (OD) values at 490 nm were detected using an ELISA plate reader.

Aβ1‑ 42‑induced PC12 cell injury. Aβ1-42 peptide purchased from Bioss Biotech (Beijing, China) was dissolved in water (100 µg/ml). Subsequently, the mixture was incubated at 37˚C for 4 days and stored at 4˚C prior to use.

PC12 cells were seeded in 96-well plates (3xl0⁴ cells in 100 µl per well). After culture for 24 h for adherence, 50 µl Aβ1-42 at various final concentrations (0, 0.25, 0.5, 1, 1.5 or 2 µM) dissolved in serum-free DMEM was added, followed by incubation for 24, 48, 72 or 96 h. Cell viability was evaluated by an MTT assay. The optimal Aβ1-42 concentration was 0.5 determined to be µM. PC12 cells (3xl0⁴ cells per well) were treated with various doses of PhGs (0, 0.5, 5, 25, or 50 µg/ml) in the presence of 0.5 µM Aβ1-42 for 24 h. Cell viability was evaluated by an MTT assay.

H₂O₂‑induced PC12 cell injury. PC12 cells were plated seeded in 96-well plates (3xl0⁴ cells in 100 µl per well). After culturing

for 24 h for adherence, 100 µl H₂O₂ at various final concentrations (0, 25, 50, 100, 200, 300, 400 and 500 µM) dissolved in DMEM with or without PBS (0.01 mol/l) was added, followed by incubation for 24 h. Cell viability was evaluated by an MTT assay. The optimal H₂O₂ concentration and solvent were determined to establish the in vitro model of AD.

PC12 cells (3xl0⁴ cells per well) were treated with various doses of PhGs (0, 0.5, 5, 25, and 50 µM). After culturing for 24 h for adherence, PC12 cells were treated with 100 µl H₂O₂ dissolved in DMEM with PBS in the presence of PhGs for 24 h. The cell viability was evaluated by an MTT assay.

Lactate dehydrogenase (LDH) release assay. Cell injury was assessed by measuring the LDH activity in the supernatant of PC12 cells using an LDH kit according to the manufacturer's protocol (cat. no. 20150604; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). In brief, double-distilled H₂O, 0.2 µmol/ml pyruvic acid, matrix buffer, and coenzyme I buffer were added in sequence at 48 h after drug treatment. After incubation at 37˚C for 15 min, 2,4‑dinitro‑phenylhydrazine was added. Subsequently, 250 µl of a 0.4 M NaOH solution was added to each well. The supernatant was collected after incubation for 30 min at room temperature. The absorbance at 450 nm was then measured with a microplate reader.

Measurement of malondialdehyde (MDA). MDA was measured in the supernatant of PC12 cells using a commercial kit (cat. no. 20150604; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol In brief, dehydrated alcohol and other reagents were added in order, followed by incubation in a water bath at 95˚C for 40 min. The mixture was centrifuged at 1,006 x g and 25˚C for 10 min after cooling. The supernatant was then used to determine the MDA content. Absorbance was subsequently measured with a microplate reader at 532 nm.

Assessment of protective effects of echinacoside and acteoside against AD in vitro. PC12 cells were seeded at a density of 3xl0⁴ cells/well in 96-well plates (100 µl/well). Cells were incubated with drugs including echinacoside (cat. no. 111670-200503; National Institutes for Food and Drug Control, Beijing, China) and acteoside (cat. no. 111530-200505; National Institutes for Food and Drug Control) at various concentrations (0.5, 25 and 50 µg/ml). Subsequently, the cells were treated with Aβ1-42 or H₂O₂ for 24 h and the cell viability was measured by an MTT assay.

Statistical analysis. Values are expressed as the mean ± standard deviation. The student's t-test was used for inter-group comparisons. Statistical analyses were performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

Quantification of PhGs from H. Cistanches. The UV spectra of the PhGs extracted as well as standard solutions of echinacoside and acteoside were recorded, and the results showed that the UV spectra were consistent (Fig. 1). UV spectrophotometry

showed that the PhG content was 87.6%. The HPLC results showed that the contents of echinacoside and acteoside were

37.7 and 17.8%, respectively (Fig. 2).

Determination of the ideal PhG concentration. Compared with the blank group, PhG at 75, 100, 125, 150, 175 and 200 µg/ml had a significant inhibitory effect on PC12 cells (P<0.05), while PhG at 5, 25 and 50 µg/ml showed low toxicity on PC12 cells, and the cell viability was >80% (Fig. 3). Thus, PhGs at the concentration of 5, 25, and 50 µg/ml were used for treating PC12 cells in subsequent experiments due to not affecting the cell viability.

Aβ1‑42‑induced PC12 cell injury. Compared with that in the control group, the cell viability in the 0.5 µM Aβ1-42 injury group was 63% (P<0.05). The cell viability was decreased by Aβ1-42 in a concentration-dependent manner, and the viability was <50% in the 1, 1.5, and 2 µM Aβ1-42 injury groups (Fig. 4). Thus, treatment with 0.5 µM Aβ1-42 for 48 h was determined to be the optimal condition for establishing the in vitro AD model.

The activity of PC12 cells treated with 0.5 µM Aβ1-42 in the presence of safe doses of PhGs (5, 25, and 50 µg/ml) for 24 h was also determined. Compared with the model group (P<0.01), PhGs showed a significant neuroprotective effect on PC12 cells. The cell viability was rescued by PhGs in a dose-dependent manner (Fig. 5).

H₂O₂‑induced PC12 cell injury. The viability of PC12 cells treated with 25 µM H₂O₂ dissolved in DMEM with PBS was 56.43%. The viability of PC12 cells treated with 200 µM H₂O₂ dissolved in DMEM without PBS was 71.64% (Table I). Thus, PC12 cells treated with 25 µM H₂O₂ dissolved in DMEM with PBS were selected as the optimal condition for establishing the AD model.

Compared with the control group, the cell viability in the model group was 48.8% (P<0.05). Compared with the model group, PhGs had a significant neuroprotective effect on PC12 cells. The cell viability was dose-dependently increased by

PhGs and the viability of PC12 cells treated with PhGs at concentrations of 5, 25 and 50 µg/ml were 54, 57, and 64%, respectively (Table II).

PhGs inhibit injury‑induced LDH release by PC12 cells. Compared with the control group, the LDH content of the supernatant of injured PC12 cells was increased, which was inhibited by PhGs in a concentration-dependent manner. This result indicated that PhGs have a significant neuroprotective effect on PC12 cells (Fig. 6).

inhibited by PhGs in a concentration-dependent manner. This result indicated that PhGs has a significant neuroprotective effect on PC12 cells (Fig. 7). PhG and its components echinacoside and acteoside rescue the viability of injured PC12 cells. Compared with the model group, treatment with acteoside significantly increased the viability of Aβ1-42-injured PC12 cells in a dose-dependent manner. PhGs and echinacoside also significantly increased the viability of Aβ1-42-injured PC12 cells at all concentrations tested (Fig. 8A).

Compared with the model group, acteoside significantly increased the viability of PC12 cells treated with H₂O₂. PhGs also increased the viability of PC12 cells treated with H₂O₂, while the effect was not significant at concentrations of 5 and 25 µg/ml (Fig. 8B). In addition, echinacoside increased the cell viability at 25 µg/ml.

In conclusion, acteoside, PhGs and echinacoside exerted significant neuroprotective effects on PC12 cells subjected to injury with Aβ1-42 or H₂O₂.