Alzheimer's disease (AD) is a common kind of dementia in older people. It is characterized by the gradual worsening in cognitive functions, behavioral changes, and a deterioration of daily living activities. AD is characterized by the presence of senile plaques, which mainly consist of extracellular beta-amyloid (Aβ) peptide deposits and intracellular neurofibrillary tangles (NFTs), which are abnormal hyperpho-sphorylated tau protein aggregates. These pathological findings are associated with a loss of cholinergic fibers and inflammatory responses such as reactive gliosis (1,2).
The major pathological hallmark of AD is the abnormal aggregation of Aβ peptides in the brain. Aβ monomers tend to aggregate and produce extracellular Aβ deposits (3). Studies have indicated that the aggregated form of Aβ is toxic to neurons. Oxidative stress and mitochondrial dysfunction, which lead to apoptotic cell death, inflammatory responses, and the disturbance of energy metabolism homeostasis, are some mechanisms that progress AD. Moreover, Aβ peptide has a pivotal role in initiating these mechanisms (4).
The destruction of cholinergic neurons, followed by a decrease in the amount of the acetylcholine (Ach) neurotransmitter, is one of the hallmarks of AD. One approach for the treatment of AD is inhibiting the acetylcholinesterase (AChE) enzyme. AChE inhibitors raise the level of acetylcholine in the brain and relieve AD symptoms but cannot cure or prevent the disease (9). Due to the complex nature of AD, monotherapy is not effective in AD treatment. Therefore, the current research focused on multi-targeted drugs as disease-modifying drugs that interfere with the disease progression through various mechanisms (10). Medicinal plants contain several biologically active compounds and are useful in the treatment of complex diseases. Therefore, medicinal plants are the focus of researchers’ attention as a source of new AD therapies (11).
A. scoparia Spach and C. microcarpa (C. A. Mey.) Boiss. are plants from the Rosaceae family. Plants from the Rosaceae family have been used as ancient and traditional medicines in many countries (12). These plants show a variety of biological effects. They are rich in phenolic compounds that prevent the progression of AD (13). A. scoparia Spach is endemic to Iran. Various parts of this plant, including its oil, leaves, and gum, are frequently used in traditional medicine and industries. Almond syrup relieves coughs, the leaves can be used as laxatives, and its decoction is a remedy for liver and gallbladder failure. The resin from this plant has been used in medicinal products as antipyretics and for appetite stimulation. Also, its anti-diabetic effect for hexan extraction has been reported (14-16). Furthermore, studies show that this plant inhibits AChE enzyme (17).
C. microcarpa grows in Iran. This plant contains alkaloids, tannins, and polyphenol compounds. The roots and fruit of this plant are traditionally used as an anti-flatulent and for pain relief in the digestive system (14). Although this plant has been widely used in Iran, its pharmacological effects have not been adequately studied, and there are only a few studies that show the antioxidant and AChE inhibitory effects of C. microcarpa (17). Furthermore, in a previous study, we indicated the protective effect of this extract against Aβ-induced toxicity in PC12 cells (18). In the present study, the protective effects of Amygdalus scoparia and Cerasus microcarpa in primary neuron culture and their possible mechanisms were investigated.
The primary culture of cerebellar granule neurons (CGNs) is a proper in vitro model in neuroscience for studying molecular and cellular mechanisms of neural cell death and survival. This model has been frequently used for the assessment of mechanisms involved in neurodegeneration and neuroprotection. These cells develop after birth; then they can be easily isolated and cultured. After plating, these cells differentiate to neurons and produce a homogeneous population of neurons, which makes this a good study model (19). The CGNs culture has been used for studying the protective effect of a new compound against Aβ-induced neurotoxicity (20,21).
Materials
Dulbecco’s modified Eagle’s Medium (DMEM), penicillin-streptomycin (10,000 U/mL), fetal bovine serum (FBS), and trypsin (%0.25) were purchased from GIBCO (USA). Poly-D-lysine (PDL) was obtained from Santa Cruz Biotechnology (USA). Beta-amyloid peptide (AB 25-35) was taken from Enzo life sciences (USA). All other materials were obtained from Sigma (USA).
Plant Material
Aerial parts of C. microcarpa and A. scoparia were collected from Hamadan and Kohgiluyeh Va Boyer Ahmad provinces of Iran, respectively. The plants were recognized by the botanists, and their voucher specimens were placed at Herbarium of Traditional Medicine and Materia Medica Research Center (TMRC), Shahid Beheshti University of Medical Sciences (TMRC) (No. 3259 TMRC and No. 2343 TMRC, for Amygdalus scoparia and Cerasus microcarpa, respectively).
Plants Extraction
The total extract of the aerial parts of the collected plants was obtained using the maceration method after drying in shade and grounding. 100 mL of methanol:water (80:20) was added to 10 g of the plant powder, and then every 24h, the mixture was filtered, and the fresh solvent was added. This procedure was continued for three days. Finally, all extracts were pooled and their solvent was evaporated by a rotary evaporator, then the resultant concentrate was dried using a freeze drier.
Measurement of Total Phenolic Compounds
The Folin-Ciocalteu spectrophotometric method was used for the measurement of the total phenolic contents of the extracts, and Gallic acid was used as the standard. Folin- Ciocalteu’s reagent was mixed with a methanolic solution of the extracts or Gallic acid and incubated at 22°C for 5 minutes. After the addition of sodium carbonate (7%), the reaction mixture was incubated at 22°C for 90 minutes. Then the absorbance of the mixture was measured at 725 nm. The amount of total phenolic compounds of extracts was stated as Gallic acid equivalent, which has been expressed in milligram per 100 g dried extract.
Primary Culture of Cerebellar Granule Neurons
We isolated cerebellar granule cells from postnatal mice (BALBc) and cultured those, as previously described (22). Briefly, cerebella were dissected from mice neonates (6-7 days old, BALB/c) and digested with trypsin. Then the single-cell suspension was obtained by triturating. After that, the single cells were cultured in cell culture plates coated by PDL. The culture medium was DMEM containing 4.5 g/L glucose, 25 mM KCl, insulin (100 mU/L), and 10% FBS. We added the cytosine b-D-arabinofuranoside (Ara-C) 48 h after cell seeding, to the cell culture medium (20µM final concentration in medium), and thereby non-neuronal cell growth was inhibited. Because cerebellar granular cells condition the medium during growth and differentiation, the medium was not changed. After seven days of in vitro (DIV 7), more than 95% of cells in culture were identified as neurons characterized by MAP2 protein immunostaining. All animal experiment procedures were approved by The Medical Ethics Committee of Tarbiat Modares University (Ethical code: IR.TMU. REC.1394.178).
Treatment
To measure the protective effect of extracts against Aβ-induced toxicity, we cultured CGNs onto PDL coated 96 well plate (1 × 105cell/well) and incubated the mature neurons on DIV 7, with 10 µM Aβ in aggregated form alone or in combination with different concentrations of extracts (5-100µg/mL) for 24 hours; then the cell viability was measured by MTT assay. The powder of Aβ 25–35 peptide was reconstituted in sterile water to obtain a final concentration of 1 mM. Aliquots of this solution were incubated at 37°C for 72 h to establish the aggregated form of Aβ peptide. We prepared the stock solutions of the extract by dissolving them in DMSO, and then the stock solution was diluted with culture medium so that the final concentration of DMSO in the medium was 0.5% or less (20).
MTT Assay
The methyl tetrazolium salt (MTT) reduction assay is a common assay for measurement of the percent of surviving neurons. The assay is based on the reduction of yellow tetrazolium salt (MTT) by the mitochondrial dehydrogenases of live cells, which produced the formazan metabolite with purple color. After incubation time, the culture medium was discarded, then the fresh medium containing MTT (0.5 mg/mL final concentration) was added. After 4h incubation in a CO2 incubator at 37°C, the medium was removed, and 100 µL DMSO was added, and the plate was shaken for 5 minutes for dissolving the formazan crystals. We measured the absorbance at 570 nm against 630 nm as the reference wavelength. The viability of treated cells is expressed as a percentage of control (23).
Measurement of ROS
The fluorescent probe 2, 7 dichlorodihy-drofluorescein diacetate (DCFH-DA) is frequently used for the measurement of ROS produced during the oxidative stress. This probe easily penetrates into cells and is deacylated by deacetylase enzymes to produce DCFH. Following that, ROS could reduce DCFH to DCF that has measurable fluorescent intensity (Excitation: 488 nm, Emission 525 nm). Mature neurons on DIV 7, seeded onto PDL coated 96-well plate, were incubated with 10 µM Aβ alone or in combination with each extract at the concentration of 75 µg/mL. After the incubation time ended, we removed the culture medium from the all wells and added a fresh medium containing DCFH-DA (10 µM), then the culture was further incubated for 17 minutes. We measured the DCF fluorescence intensity using fluorescence plate reader (BioTek, United States) (24).
Measurement of Glutathione Peroxidase (GPx) Activity
We detected the GPx activity in CGNs by the colorimetric kit (BioVision). We plated CGNs on PDL coated 6-well plates (3 ×106 cell/well) and incubated them with Aβ peptide (10 µM) alone or in combination with each extract (75 µg/mL), on DIV 7, for 24 h. We removed the medium after the incubation time, harvested cells, and then homogenized the collected cells in PBS. We centrifuged the homogenate at 1000g for 10 min and kept the supernatant for enzyme activity and protein assay. The protein of samples were quantified using the Bradford method (25).
Acetylcholinesterase Activity Assay
For evaluating the effect of extracts on AChE activity of CGNs, mature CGNs on DIV 7 were incubated with different concentrations of each extract for 24 h. Also, the AChE activity of CGNs was measured after incubation of mature CGNs on DIV 7 with Aβ alone or in combination with each extract (75μg/mL) for 24 h. After the incubation, we collected the cells, then homogenized them in PBS and centrifuged the homogenate at 1000 g for 10 min. We kept the supernatant for enzyme activity and protein assay. We used Elman’s method for measuring AChE activity (26). Briefly, Elman’s reagent was prepared by mixing phosphate buffer (pH=8, 0.1 M), ATCh 75 mM as a substrate, and Di- thio- nitro-benzoic acid (DTNB) 10 mM in a ratio of 150:2:5. This reagent (100 µL) was transferred to the each well of 96-well plate. After adding 50 µL supernatant, the absorbance was immediately measured at 405 nm for 15 min, and whereby the rate of enzyme activity was calculated.
LD50 Estimation
The LD50 was estimated by the limit test method according to the OECD protocol (27, 28). As stated by this protocol, one female mouse was orally administered a single dose of 2000 mg/kg of each extract by gavage, and the animal was monitored for 48 hours. Since the first mouse survived, four additional mice received the same dose of 2000 mg/kg by gavage, sequentially. If three or more animals survived, the LD50 value was estimated over 2000 mg/kg, and the test substance was considered safe. We evaluated all animals for 14 days in terms of mortality, apparent health, and weight.
Statistical Analysis
The statistical analysis was done by Graph-Pad Prism7 software. Each data represented the Mean ± SEM of three separate experiments. Statistical differences were estimated by one-way ANOVA analysis followed by Tukeyʼs Multiple Comparison Test. We considered P<0.05 as a significant level.
Amount of Total Phenolic Compounds in the Extracts
The data for measuring the total phenol content of the extracts were obtained from the calibration curves of Gallic acid (y=2.6x-0.038, r2=0.99), which was established by the Folin-Ciocalteu method. The amount of total phenolic compounds in the extracts were 14.6±0.26 and 6.1±0.15 mg Gallic acid in 100 gr dried extract for C. microcarpa and A. scoparia, respectively.
Protective Effects of C. microcarpa and A. scoparia Extracts on Aβ- induced Cytotoxicity
Figures 1A and 1B show the Effects of C. microcarpa and A. scoparia extracts on Aβ- induced cytotoxicity in cultured CGNs, respectively. Incubation of CGNs with Aβ (10 µM) for 24 h significantly decreased the cell viability (P<0.001). Aβ-induced cytotoxicity was dose-dependently reduced by co-treatment of the CGNs with different concentrations of extracts and was entirely resolved at a concentration of 75µg/mL or above. In Figure 1C, the protective effect of extracts on the Aβ-induced cytotoxicity in CGNs has been compared with the protective effects of memantine, BHT, and donepezil. The results revealed that both extracts had a more protective effect than memantine, BHT, and donepezil. Treatment of CGNs with extracts alone at a concentration range of 5-100µg/mL did not affect the cell viability (data not shown).
Effect of C. Microcarpa and A. scoparia on ROS Production
The generation of ROS was measured by the fluorimetric method. Figure 2 gives information about ROS production in the studied groups. A significant increase in fluorescence intensity following Aβ treatment indicated that Aβ induced ROS production in CGNs. When CGNs were co-incubated with Aβ and C. Microcarpa or Aβ and A. scoparia extract, the fluorescence intensity significantly decreased, indicating the protective effect of extracts on Aβ-induce ROS production in CGNs.
Effect of C. microcarpa and A. scoparia on GPx Activity
GPx is the most important antioxidant enzyme in neural cells. Results indicated that the incubation of CGNs with Aβ significantly reduced the GPx activity comparing to that of the control group. While the cells were co-incubated with Aβ and extracts, reduction of GPx activity by Aβ was prevented, and the decrease in GPx activity was not observed in these groups (Figure 3).
Effect of C. microcarpa and A. scoparia on AChE Activity
Figures 4A and 4B show the effect of different concentrations of C. microcarpa and A. scoparia extracts (5-100 µg/mL) on AChE activity in CGNs, respectively. Both extracts dose-dependently decreased the AChE activity, but C. microcarpa extract decreased the AChE activity more effectively than A. scoparia.
Also, AChE activity in CGNs after incubation with Aβ alone and in combination with extracts was measured. The results are shown in Figure 4C. Treatment of CGNs with Aβ for 24h significantly increased the AChE activity, and co-treatment with extract attenuated AChE activity increment.
LD50 TEST
The LD50 was estimated by the limit test method according to the OECD protocol. All the five mice treated with 2000 mg/kg of each extract survived. Treated animals were observed for 14 days. Signs of toxicity were not observed during the monitoring period. Also, significant abnormal body weight changes were not observed between extract-treated and control groups. Based on these findings, the LD50 value was estimated at over 2000 mg/kg for both extracts; therefore, extracts were considered safe.
Discussion
Previous studies have shown that oxidative stress is a well-known mechanism initiated by Aβ and an essential mechanism in Aβ-induced cell death (6). The results of our study also showed that the incubation of CGNs with Aβ reduced the viability of cells, which was associated with increased ROS levels and decreased GPx activity as an antioxidant enzyme. Thus, it is suggested that cell death after CGN exposure with Aβ is mediated through an oxidative stress mechanism. Furthermore, our results indicated that Aβ-induced oxidative stress and cell death were attenuated by the co-treatment of C. microcarpa and A. scoparia methanolic extracts. Therefore, the antioxidant activity of these extracts is likely involved in their neuroprotective activity.
Although C. microcarpa and A. scoparia are widely used in Iranian traditional and folk medicine, their pharmacological and therapeutic effects have not been studied very much, and there is not much information about their constituents. Our previous study indicated that C. microcarpa extract protects against Aβ-toxicity in PC12 cells (18), and a study by Hajimehdipoor et al. showed that the methanolic extracts of these plants exhibit antioxidant and anticholinesterase activity (20). These plants belong to the Rosacea family, and plant species in this family are rich in polyphenolic and flavonoid compounds with potent antioxidant activity. The results of the present study showed that C. microcarpa and A. scoparia extracts have 14.6±0.26 and 6.1±0.15 mg/g dried extract total phenol content, respectively, which indicates a high level of phenolic compounds for these extracts.
Polyphenols are potent free radical scavenging agents, meaning that they reduce ROS levels in cells, thus protecting the cells from oxidative insults. However, recent studies have involved other mechanisms in polyphenol antioxidant activity and neuroprotection beyond direct free radical scavenging activity (29). Some natural polyphenols exhibit mitoprotective activity and directly interact with mitochondrial biomolecules. In this way, they prevent mitochondrial membrane permeabilization and, in turn, inhibit the apoptotic process initiated from mitochondria. The mitochondrial apoptosis pathway is involved in Aβ-induced toxicity and is ameliorated by polyphenols such as rosmarinic acid (30).
In addition, polyphenols such as resveratrol increase mitochondrial biogenesis and protect neurons against Aβ-induced toxicity (31). Polyphenols also promote the survival of neurons via neurotrophic factor (NTF)-like activity. Lipophilic polyphenols such as curcumin and resveratrol can cross the blood-brain barrier and induce NTF gene expression (e.g., brain-derived neurotrophic factor (BDNF)) (29). It has been reported that BDNF protects neurons against Aβ-induced toxicity (32). Also, some polyphenols activate NTF signal transduction by binding to NTF receptors or to downstream proteins and enzymes. Other mechanisms that are involved in polyphenol neuroprotective activity include the activation of prosurvival signal pathways such as PI3K/AKT and PKC (33). Overall, studies have indicated that polyphenols protect neurons through different mechanisms that inhibit death signals.
The results of our study showed that CGN exposure with different concentrations of C. microcarpa and A. scoparia extracts for 24 h reduced AChE activity. The results indicated that both extracts inhibit AChE activity in CGNs, as the previous study reported in vitro AChE inhibitory activity for these extracts (17). Several plant secondary metabolites with diverse chemical structures—including alkaloids, terpenes, and polyphenolic compounds—have AChE inhibitory effects (34). Some polyphenolic compounds such as rosmarinic acid, quercetin, and rutin, which are found in the rosacea family, also have AChE inhibitory effects (35). C. microcarpa and A. scoparia extracts are rich in phenolic compounds, which can contribute to the AChE inhibitory effect of these extracts.
Studies have indicated that natural AChE inhibitors improve memory deficits in animal models of AD and prevent Aβ-induced toxicity in cell cultures. The inhibition of AChE increases Ach levels in the brain and improves the cholinergic deficit, thus improves the cognitive function of AD patients (36). On the other hand, bounded AChE protein with Aβ peptide has been found abundantly in amyloid plaques, and AChE activity increases nearby the amyloid plaques in the AD brain (37). It has been shown that Aβ binds to the peripheral anionic site of AChE proteins (38). Attaching AChE proteins to Aβ promotes Aβ aggregation. Also, the AChE-Aβ complex is more neurotoxic than Aβ alone (39), and so the compounds that bind to the peripheral anionic site of AChE proteins inhibit Aβ binding and prevent Aβ aggregation and toxicity. Polyphenolic compounds such as flavonoids act as non-competitive inhibitors of AChE by binding to the peripheral anionic site (35). It is therefore suggested that phenolic compounds prevent neurons against Aβ-induced toxicity by inhibiting AChE and preventing Aβ-AChE complex production.
The present study indicated that the treatment of CGNs with Aβ increased AChE activity and that co-treatment with both C. microcarpa and A. scoparia extracts could attenuate it. In vitro studies have indicated that AChE activity increases in cultured neuronal cells that have been exposed to Aβ peptides (20). Similarly, the injection of Aβ peptides in the brain of experimental animals increased AChE activity associated with learning and memory deficits (40).
The mechanisms by which Aβ increases AChE activity have not been clearly identified. Aβ-induced oxidative stress and the disruption of calcium homeostasis might be involved (41). It has been reported that oxidative stress increases AChE activity and up-regulates AChE gene expression, which was ameliorated by antioxidant compounds (42). Some medicinal plant extracts (without the AChE inhibitory effect but with potent antioxidant activity) attenuate Aβ-induced elevated AChE activity by improving the oxidative stress condition in the cell culture or the brain (40, 43). Thus, it is suggested that C. microcarpa and A. scoparia extracts decrease elevated AChE activity by coping with oxidative stress.
The LD50 values of extracts were estimated by the limit test. The results indicated that the LD50 values of both extracts were greater than 2000 mg/kg. Weight loss and signs of illness were not observed during the 14 days of monitoring mice after extract treatment. Also, based on the OECD protocol, they were considered non-toxic compounds.
Conclusion
In this study, we indicated that C. microcarpa and A. scoparia extracts improve Aβ-induced cytotoxicity and oxidative stress. According to our findings, the protective effect of both extracts is due to their antioxidant activity. Also, the inhibition of AChE enzyme by the extracts might be involved. Both extracts attenuated Aβ-induced cell death by ameliorating oxidative stress. Based on these findings, C. microcarpa and A. scoparia extracts may have therapeutic effects on Alzheimer’s disease. However, further investigations are recommended.
Acknowledgements
This study was a part of an MSc thesis financially supported by Tarbiat Modares University.
Conflicts of Interest
Authors declared no conflict of interest.
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