A novel antagonistic role of natural compound icariin on neurotoxicity of amyloid β peptide

Alzheimer's disease (AD) is a neurodegenerative disease that mostly affects the elderly. Prevalence studies revealed that there were 25 million persons with AD globally in 2000, and this number is predicted to increase to 114 million by the year 2050 if new preventive or neuroprotective therapies do not emerge12. Existing treatments for AD cannot cure the disease, but can offer some people modest improvement in some symptoms with side effects. Therefore, there is a need for alternative drugs and in particular for that, which has no side effects.

One of the major characteristics of AD is the abnormal aggregation of amyloid β peptide (Aβ) into extracellular fibrillar deposits known as senile plaque3. It is accepted that abnormal production and aggregation of Aβ are initial pathogenic events in AD1456. Substantial evidence shows that Aβ-induced oxidative stress plays a key role in the pathogenesis or progression of AD78. Several studies have reported that Aβ-induced cytotoxicity is caused by intracellular accumulation of hydrogen peroxide (H₂O₂) produced during its aggregation, ultimately leading to the peroxidation of membrane lipids and to a cell death91011. Therefore, inhibition of Aβ aggregation is viewed as a potential method to slow the progression of AD.

Epimedium brevicornum Maxim is a medicinal herb which has been widely used for the treatment of impotence, infertility, osteoporosis, cardiovascular diseases, amnesia, and senile functional diseases12. The neuroprotective potential of icariin, a flavonoid compound isolated from it, against Aβ-induced toxicity in PC12 cells had been explored, which demonstrated that treatment with icariin significantly decreased Aβ_25-35-induced cytotoxicity and apoptosis rate by inhibiting tau protein hyperphosphorylation13. Icariin has been shown to improve the learning and memory abilities in rats with Aβ_25-35-induced Alzheimer's disease that can reduce production of insoluble fragments of Aβ through suppression of β-secretase expression14. However, the role of icariin in the inhibition of Aβ accumulation and aggregation has not yet been investigated. The present study was undertaken to explore the anti-amyloidogenic properties of icariin and to examine the effects of icariin on the formation of Aβ aggregates in vitro by using fluorescence spectroscopy with ThioflavinT (ThT) and transmission electron microscopy (TEM). Further, the neuroprotective effect of icariin and its mechanism in Aβ_1-42-treated human neuroblastoma SH-SY5Y cells were also explored.

Material & Methods

Aβ_1-42 was purchased from Anaspec (San Jose, USA). Icariin was isolated from the root of E. brevicornum Maxim according to the methods reported by Du et al15. The purity of Icariin was tested by nuclear magnetic resonance (NMR) and high performance liquid chromatography (HPLC) (the purity was over 98%). Dulbecco's modified Eagle/F12 medium (DMEM), foetal bovine serum, thioflavinT(ThT), glycine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were obtained from Sigma-Aldrich, USA.

Preparation of Aβ_1-42 aggregate: The treatment of Aβ_1- 42 was done as described earlier1617.

Cell culture and treatment: SH-SY5Y human neuroblastoma cells were purchased from American Type Tissue Culture (Manassas, USA), cultured in DMEM/F12 supplemented with 10 per cent heat-inactivated foetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator with 5 per cent CO₂ at 37°C. For the cytotoxicity assay, the cells were rinsed once with PBS pH 7.4 and the medium was replaced with phenol red-free and serum-free DMEM before Aβ treatment. To explore the influence of icariin on the cytotoxicity of Aβ_1- 42 aggregations, Aβ was preincubated with different concentrations of icariin for three days. Aβ_1-42 was dissolved in PBS with a final concentration of 10 mM before used. After the cells were incubated for 48 h in the presence or absence of 5.0 μM Aβ_1-42 with or without icariin, MTT assay was performed to probe the cell viability, and the same volume of PBS was taken as negative control18.

ThT fluorescence assay and TMT igaming: ThT is a dye that increases its fluorescence intensity upon binding with the Aβ aggregates and is, therefore, used to estimate the relative amount of amyloid-like aggregates19. ThT binding assay was carried out and the solution of Aβ_1-42 peptide was prepared for TEM analysis as described earlier1617.

H₂O₂ assay16: After SH-SY5Y cells were replaced into 12-well plates at 4×10⁵ cell/well and incubated overnight, the cells were washed once with PBS and the medium was replaced with phenol red-free and serum-free DMEM. Before treated with Aβ_1-42 peptide, the cells were preincubated with indicated concentrations of icariin for two hours, after that, freshly prepared Aβ_1-42 peptide with a final concentration as 5.0 μM was added and cultured for 24 h. The medium was taken to test for the level of H₂O₂ according to the protocol supplied by manufacturer (Biovision, USA). The collected medium was reacted with oxiRed™ probe for 30 min at room temperature protected from light to produce product with colour, the absorbance value was determined by microplate reader (Varioskan, USA) at 570 nm.

MTT assay16: For the cytoprotective assessment of icariin, before Aβ_1-42 peptide a final concentration of 5.0 μM icariin was added, the cells were treated with geniposide for 2 h at indicated concentration of icariin, and incubated for 48 h. Cells were incubated for 2 h at 37°C with MTT (0.5 mg/ml final concentration) and dissolved in fresh complete medium, in which metabolically active cells reduced the dye to purple formazan. Formazan crystals were dissolved with DMSO, and the absorbance was measured on a BMG microplate reader (BMG Technologies, USA), using a reference wavelength of 630 nm and a test wavelength of 570 nm.

Statistical analysis: Statistical analysis was performed using the Student's t test and the ANOVA test followed by Bonferroni correction using the software of Origin version 8.0 (Northampton, USA).

Results

Inhibition of aggregation of Aβ_1-42 peptide by icariin: To explore the role of icariin on the aggregation of Aβ_1-42, fluorescence intensity of Aβ_1-42 aggregate was determined with ThT binding assay. After incubation for three days in the presence or absence of icariin, the extent of Aβ aggregation in the samples was evaluated by taking 10 μl of Aβ_1-42 from incubated samples and transferring to a single well of a 96-well plate. To each sample, 200 μl of 10 μM ThT in 0.1M glycine buffer (pH 8.9) was added and the plate was read on a microplate reader (Varioskan, Thermo, USA) for fluroscence intensity at Ex/Em 450/482 nm. All ThT fluroscence experiments were performed in triplicate. As shown in Fig. 1, icariin inhibited the aggregation of Aβ_1-42 in a dose-dependent manner with a calculated IC₅₀ (half maximal inhibitory concentration) of 0.4813 μM (n = 6, R² = 0.9912).

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Fig. 1

Effect of icariin on the aggregation of Aβ_1-42 peptide. Data are mean ± SD from six wells of two independent experiments (n = 6, R²=0.9912).

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To visually confirm the effect of icariin on the aggregation of Aβ_1-42, TEM imaging was done to evaluate the fibril formation of Aβ_1-42 in the absence (Fig. 2A) or presence of icariin (Fig. 2B). Incubation of 50 μM solution of Aβ_1-42 in 20 μM PBS (PH 7.4) for three days at room temperature in the presence of 5.0 μM icariin markedly inhibited the aggregation of Aβ_1-42, as much smaller and fewer fibrils were observed in presence of icariin compared to its absence.

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Fig. 2

Transmission electron microscopy imaging of Aβ_1-42 aggregate in the absence (A) or presence (B) of icariin. After incubation for three days without (A) or with (B) icariin at 10 μM, 2 μl of each sample was prepared for electron microscopy analysis. Images were acquired using Hitachi 7500 transaction electron microscope at 80 kv. Magnification was 100,000 ×; Scale bar = 500 nm.

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Effect of icariin on neurotoxicity of Aβ_1-42 peptide: The results revealed that icariin prevented cell injury induced by the addition of Aβ_1-42. The effect was dose dependent and preincubation with 10 μM icariin was capable of increasing SH-SY5Y cell viability by 22 per cent as compared to the effect of Aβ_1-42 alone under control conditions (Fig. 3).

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Fig. 3

Icariin increased the cell viability of Aβ_1-42-treated SH-SY5Y cells. Data are given as mean ± SD from three independent experiments (n = 3). *P<0.05 vs the group of Aβ_1-42 treatment alone.

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It has been reported that Aβ in either oligomeric or fibril form has a stronger neurotoxicity than its monomeric form20. To confirm this, we compared the cytotoxicity of fresh prepared Aβ_1-42 and preincubated Aβ_1-42 at 37°C for four hours to obtain the Aβ aggregates. The results showed that the cytotoxicity of Aβ_1-42 aggregate was stronger than Aβ_1-42 monomer at the same concentrations, and icariin also inhibited the neurotoxicity of Aβ_1-42 aggregate. Preincubation with 10 μM icariin for two hours increased the cell viability by about 18 per cent in Aβ_1-42 aggregate-treated SH-SY5Y cells (Fig. 4).

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Fig. 4

Cytotoxicity of fresh prepared Aβ_1-42 and Aβ_1-42 aggregates in SH-SY5Y cells. Data are shown as mean ± SD from three independent experiments (n = 3).*, P<0.05 vs the group of fresh prepared Aβ1-42 (Aβ) treatment alone; *P<0.05 vs the group of Aβ_1-42 aggregate (Aβ aggregate) treatment alone.

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Icariin attenuates the level of H₂O₂ in Aβ_1-42-treated SH-SY5Y cells: To confirm the protective mechanism of icariin on Aβ_1-42 induced cell damage in SH-SY5Y cells, the level of H₂O₂ was measured in Aβ_1-42-treated cells. The results suggested that incubation with 5.0 μM Aβ_1-42 peptide increased H₂O₂ levels compared to control (P<0.001), and icariin dose-dependently inhibited the production of H₂O₂ in Aβ_1-42-treated SH-SY5Y cells (Fig. 5).

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Fig. 5

Icariin decreases the level of H₂O₂ in Aβ1-42-treated SH-SY5Y cells. Data are mean ± SD from 4 wells of two independent experiments (n = 4). ***, P<0.001 vs. the group of Aβ_1-42 alone.

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Discussion

There is evidence linking oxidative damage to the brain of Alzheimer's disease (AD) which is characterisitized by excellular amyloid plaques and neurofilbriilary tangles4. Moreover, Aβ was found to be responsible for senile plaque formation and cell damage in AD21. There are different hypotheses to explain the toxic effect of Aβ, including the formation of ion channels in cell membrane, the spontaneous fragmentation of Aβ to generate peptidyl radicals, and the direct formation of hydrogen peroxide by the peptides22. According to the latter hypothesis, Aβ generates hydrogen peroxide from molecular oxygen through electron transfer interactions involving bound redox-active metal ion. Hydrogen peroxide is readily converted to a highly reactive hydroxyl radical by Fenton reaction and both forms may be responsible for some of the oxidative damage observed during post-mortem examination of AD patient's brain tissue23.

Icariin exerts multiple therapeutic and preventive effects including antioxidant effect, anti-inflammatory effect, immunoregulation, anti-tumour activity, cardioprotective effect, stimulating osteoblasts, antidepressant-like effect, and regulatory effect on the memory deficits in aluminium-treated rats24. It was also reported that icariin inhibited Aβ_1-42-induced neurotoxicity on cortical neurons by enhancing cocaine and amphetamine-regulated transcript (CART) mRNA and protein levels25. The present study revealed that icariin inhibited the aggregation of Aβ_1-42 and prevented neurotoxicity of aggregated Aβ_1-42 in SH-SY5Y cell culture. In vitro evidence shows that Aβ as either oligomeric or fibril form has a stronger neurotoxicity than its monomeric form, which may play a critical role in the apoptosis of neurons and the impairment of cognition in AD20. Inhibition of Aβ aggregation is, therefore, viewed as a potential therapeutic approach to slow or mitigate the progression of AD. All these evidence demonstrates that icariin may be a promising compound to be further tested for the prevention of Aβ-related AD.

Icariin reduced the production of H₂O₂ in Aβ_1-42- treated SH-SY5Y cells and this effect occurred in a dose-dependent manner. The neuroprotective effects of icariin in Aβ-stressed SH-SY5Y cells may be due to the inhibition of the Aβ aggregation process and subsequently reducing the production of H₂O₂ and thus damage due to oxidative stress.

In conclusion, the results indicate towards the neuroprotective mechanism of icariin. Further studies need to be done to see whether the anti-amyloidogenic and neuroprotective effects of icariin can influence Aβ clearance and be helpful to overcome the memory deficits caused by Aβ in AD.