Houttuyniae Herba protects rat primary cortical cells from Aβ 25–35 -induced neurotoxicity via regulation of calcium influx and mitochondria-mediated apoptosis

Abstract

Amyloid beta (Aβ) fibrils are believed to play a major role in the pathogenesis of Alzheimer’s disease. Although the mechanisms underlying Aβ toxicity remain largely unknown, Aβ fibrils disrupt calcium homeostasis and generate free radicals, resulting in oxidative stress, mitochondrial dysfunction, and apoptotic cell death. Houttuyniae Herba, the aerial part of Houttuynia cordata Thunb. (Saururaceae), is a commonly used herb in traditional Asian medicine. It has been reported to have various bioactivities, including antioxidant effects. In the present study, we investigated the protective effect of standardised Houttuyniae Herba water extract (HCW) against Aβ_25–35-induced neurotoxicity and its possible mechanisms in rat primary cortical cells. Pretreatment with HCW attenuated the cell damage caused by 8 μM Aβ_25–35 exposure, as evidenced by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, a lactate dehydrogenase assay, and microtubule-associated protein 2 immunostaining. Moreover, HCW inhibited the Aβ_25–35-induced elevation of the intracellular calcium level, reactive oxygen species overproduction, mitochondrial membrane potential disruption, and caspase 3 activation. These results indicate that HCW protects rat primary cortical neurons against Aβ_25–35-induced toxicity via the regulation of calcium and the inhibition of mitochondria-mediated apoptosis.

Keywords

Houttuyniae Herba neuroprotective Alzheimer’s disease amyloid beta

Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disease. Its clinical hallmarks include progressive impairment of memory, judgement, decision making, orientation to physical surroundings, and language.¹ AD is pathologically characterised by the presence of intraneuronal fibrillary tangles, extracellular senile plaques, and loss of synapses and neurons, particularly in the cerebral cortex and hippocampus.^2,3 Amyloid beta (Aβ) peptide, a peptide of 40–43 amino acids, is the main component of plaques in the AD brain, and its fibrillar forms have been shown to be neurotoxic in both in vitro and in vivo models.^3
–5 Although the precise mechanism of Aβ neurotoxicity is not well understood, it has been speculated to involve various factors including oxidative stress, excessive increases in intracellular calcium, and induction of neurotoxic cascades.⁶ More specifically, Aβ fibrils primarily lead to calcium influx, which generates reactive oxygen species (ROS).⁷ Disruption of the mitochondrial membrane potential (ΔΨm) by ROS may release apoptosis-inducing factors that activate the caspase cascade and ultimately result in apoptotic cell death.^8,9

Recently, studies on the neuroprotective effects of medicinal foods or medicinal herbal extracts in models of neurodegenerative diseases have attracted much attention because of worldwide aging. For example, the leaf and stem of Vitis amurensis Rupr. (Amur grape), the peel of Ipomoea batatas L. (sweet potato), and Monascus-fermented rice (red mould rice) have been reported to ameliorate Aβ-induced memory impairment by inhibiting oxidative stress in in vivo systems.^10
–12 Moreover, blueberry, mangosteen, green tea, and Samjunghwan have been shown to have neuroprotective effects against Aβ toxicity via their antioxidant mechanisms in in vitro AD models.^13

–16 The leaf of Laurus nobilis L. (Bay laurel) and the seed of Cassia obtusifolia L. (sicklepod), which are also known as medicinal foods, have been demonstrated to protect dopaminergic neurons in in vitro and in vivo systems through their antioxidant and antiapoptotic effects.^17,18 Because neurodegenerative diseases arise from multiple pathological or neurotoxic pathways, medicinal herbal extracts that contain various compounds with multiple bioactivities have been studied.¹⁸

Houttuyniae Herba, the aerial part of Houttuynia cordata Thunb. (Saururaceae), is widely used as a medicinal food in East Asia.¹⁹ It has been reported to have various bioactivities such as antioxidant, anti-inflammatory, antipyretic, and antileukemic effects.^19

–24 Chemically, H. cordata is composed of volatile oils, flavonoids, alkaloids, fatty acids, sterols, and polyphenolic acids,^22,25 and it contains many bioactive compounds such as chlorogenic acid, caffeic acid, quercetin, hyperin, and rutin, which have shown neuroprotective effects in various experimental models.^26

–31

In this study, we examined the effects of standardised Houttuyniae Herba water extract (HCW) on Aβ_25–35-induced neurotoxicity and the possible mechanisms of its effects in the primary cortical cells of rat, by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, immunostaining of microtubule-associated protein 2 (MAP-2), and measurements of intracellular calcium and ROS levels, ΔΨm, and caspase 3 activation.

Materials and methods

Materials

Neurobasal medium and B27 supplement were purchased from Gibco (Carlsbad, California, USA). Penicillin and Streptomycin were purchased from Hyclone Lab Inc. (Logan, Utah, USA). Chlorogenic acid, caffeic acid, poly-l-lysine (PLL), Aβ_25–35, MTT, dimethyl sulfoxide (DMSO), paraformaldehyde (PFA), glutamine, 3,3-diaminobenzidine (DAB), sodium chloride, phosphate-buffered saline (PBS), glycine, trizma base, 2,7-dichlorodihydrofluorescein diacetate (H₂DCF-DA), and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). A cytoscan LDH cytotoxicity assay kit was purchased from Bioworld (Dublin, Ohio, USA). A Fluo-4 NW calcium assay kit was purchased from Molecular Probes Inc. (Eugene, Oregon, USA). Tetramethylethylenediamine, protein assay, Tween-20, ammonium persulfate, acrylamide, enhanced chemiluminescence (ECL) reagent, and skimmed milk were purchased from Bio-Rad Lab (Hercules, California, USA). Sodium dodecyl sulfate (SDS) was purchased from Duchefa Biochemie BV (Haarlem, The Netherlands). A caspase 3 fluorometric assay kit was purchased from BioVision Inc. (Mountain View, California, USA). A rabbit anticleaved caspase 3 antibody was obtained from Cell Signaling Technology Inc. (Danvers, Massachusetts, USA). Rabbit anti-MAP-2 and rabbit anti-glial fibrillary acidic protein (GFAP) antibodies were purchased from Chemicon International Inc. (Temecula, California, USA). A mouse anti-β-actin antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). Biotinylated anti-rabbit antibody, normal goat serum, and an avidin–biotin peroxidase complex (ABC) standard kit were purchased from Vector Lab (Burlingame, California, USA). Anti-rabbit and anti-mouse horseradish peroxidase secondary antibodies were purchased from Assay Designs Inc. (Ann Arbor, Michigan, USA).

Preparation of extract and standardisation

A dried Houttuyniae Herba, the aerial part of H. cordata, was purchased from Jung Do herbal Drug Co. (Seoul, Korea). The voucher specimen (KHUOPS-MH022) was deposited in the herbarium of the College of Pharmacy, Kyung Hee University (Seoul, Korea). It was boiled in distilled water for 2 h at 100°C and the suspension was filtered and lyophilised. The powder (yield, 16.35%) was kept at −20°C and the extract was dissolved in PBS and diluted with neurobasal medium before each experiment. HCW was standardised based on the contents of chlorogenic acid and caffeic acid known as active components in H. cordata,^22,32 using reverse-phase high-performance liquid chromatography (PerkinElmer 200 HPLC system; PerkinElmer Inc., Norwalk, Connecticut) equipped with a photodiode array detector. Separation was carried out using a Shiseido Capcellpak C18 column (250 × 4.6 mm, 5 µm; Tokyo, Japan) at 25°C. The injection volume was 10 µL. The mobile phases (A: 0.1% acetic acid in methanol and B: 0.1% acetic acid in water) were 10–10% A for 0–10 min; 10–30% A for 10–40 min; and 30–100% A for 40–50 min at a flow rate of 0.8 mL/min. The detector wavelength was set at 280 nm. HCW and reference to the calibration curve obtained with chlorogenic acid and caffeic acid were analysed in triplicates. Chlorogenic acid and caffeic acid were found in HCW at mean levels of 1.60 ± 0.03 mg/g and 0.76 ± 0.02 mg/g, respectively (Figure 1).

Figure 1.

HPLC chromatogram of chlorogenic acid, caffeic acid, and HCW. Chlorogenic acid and caffeic acid were determined using reverse-phase HPLC equipped with a photodiode array detector. Chlorogenic acid and caffeic acid were detected at 23.44 min and 26.14 min, respectively. HCW also showed a chlorogenic acid peak and a caffeic acid peak at these times. HCW: Houttuyniae Herba water extract; HPLC: high-performance liquid chromatography.

Primary cultures of cortical neurons

Cortical neuronal cultures were derived from an 18-day-old embryo of Sprague-Dawley rat (Daehan Biolink Co., Eumseong, Korea). The procedures have been described previously.³³ Briefly, cerebral cortex was dissected, collected, dissociated, and seeded in PLL-precoated plates or dishes. Cultures were maintained in a humidified incubator of 5% CO₂ at 37°C in neurobasal medium with 2 mM glutamine, 2% B27, 100 units/mL penicillin, and 100 μg/mL streptomycin. Treatments were performed on the 6th to 8th day in in vitro. This cell culture protocol has been shown to be neuron inductive and a sufficiently low glial cell count (<15%) was confirmed by immunostaining using anti-MAP-2 and anti-GFAP antibodies. Aβ_25–35 was reconstituted in sterile water at a concentration of 500 μM. Aliquots were incubated at 37°C for 72 h to form aggregated amyloid.

Determination of cell viability

Cell viability was measured by the colorimetric MTT assay as described.³⁴ Cells were seeded in a 96-well plate at a density of 1.3 × 10⁴ cells/well, and on the 7th day in vitro, they were treated with HCW at 0.1–100 μg/mL 30 min before the treatment of 8 μM Aβ_25–35 for a total of 24 h. They were incubated with 0.5 mg/mL of MTT at 37°C for 3 h. MTT medium was carefully aspirated and the formazan dye was eluted using DMSO. The plate was shaken and the absorbance was measured using a spectrophotometer (Versamax microplate reader, Molecular Device, Sunnyvale, California, USA) at a wavelength of 570 nm. Cell viability was expressed as a percentage of the value in the vehicle-treated control group.

LDH release assay

Release of LDH was assessed using a cytoscan LDH cytotoxicity assay kit according to the manufacturer’s protocol. Cells were seeded in 100 mm dishes at a density of 2.0 × 10⁶ cells/dish, and on the 7th day in in vitro, they were treated with HCW at 100 μg/mL 30 min before the treatment of 8 μM Aβ_25–35 for a total of 24 h. The supernatants (100 μL) of the treated cells were centrifuged, transferred to a 96-well plate, and reacted with 100 μL of the mixture of dye solution in the dark for 30 min. Absorbance at 490 nm was measured using a spectrophotometer and was expressed as a percentage of the value in the vehicle-treated control group.

MAP-2 immunostaining

Cells were seeded on cover slips in a 24-well plate at a density of 3.0 × 10⁴ cells/well, and on the 7th day in vitro, they were treated with HCW at 100 μg/mL 30 min before the treatment of 8 μM Aβ_25–35 for a total of 24 h. The treated cells were fixed with 4% PFA at room temperature for 30 min. The fixed cells were pretreated with 1% hydrogen peroxide (H₂O₂) in PBS and incubated overnight with rabbit anti-MAP-2 antibody (1:1000 dilution) for neuronal cell detection. They were then incubated with a biotinylated anti-rabbit IgG, followed by ABC solution at room temperature. The activity was visualised with DAB for 4 min. Finally, the cortical neurons on cover slips were mounted on glass slides, air-dried, and photographed with an optical light microscope (BX51T-32F01; Olympus Corp., Tokyo, Japan). For quantification of the effect of HCW in the rat cortical neurons, MAP-2 immunoreactive (IR) neurons were counted on at least four cover slips from independent experiments for each condition and were expressed as a percentage of the value in the vehicle-treated control group.

Measurement of the intracellular calcium level

The intracellular calcium level was measured using a Fluo-4 NW calcium assay kit. The membrane-permeable Fluo-4 AM, a fluorescent calcium indicator, is converted into Fluo-4 within the cell, and calcium binding increases the green fluorescence of Fluo-4. Cells were seeded in a 24-well plate at a density of 1.0 × 10⁵ cells/well, and on the 6th day in in vitro, they were treated with HCW at 100 μg/mL for 30 min. The media were then aspirated, and Fluo-4 AM and probenecid were mixed with assay buffer (Hank’s balanced salt solution (HBSS) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) and added to the wells. The plate was incubated at 37°C for 15 min followed by 15 min of incubation at room temperature before the addition of 8 μM Aβ_25–35 for 3 h. The fluorescence intensity of Fluo-4 was measured at an excitation wavelength of 494 nm and an emission wavelength of 516 nm using a fluorescence microplate reader (SpectraMax Gemini EM; Molecular Device). The fluorescence intensity was expressed as a percentage of the value in the vehicle-treated control group. Representative images were taken using a fluorescence microscope (BX51T-32F01; Olympus Corp.).

Measurement of intracellular ROS generation

Intracellular ROS was measured using a fluorescent probe, H₂DCF-DA. Intracellular H₂O₂ or low-molecular-weight peroxides oxidise H₂DCF-DA to the highly fluorescent compound dichlorofluorescein (DCF). Cells were seeded in a black 96-well plate at a density of 1.3 × 10⁴ cells/well, and on the 6th day in in vitro, they were treated with HCW at 100 μg/mL 30 min before the treatment of 8 μM Aβ_25–35 for 2 h. Cells were incubated with 10 μM H₂DCF-DA at 37°C for 30 min, the fluorescence intensity of DCF was measured at an excitation wavelength of 480 nm and an emission wavelength of 530 nm using a fluorescence microplate reader. The fluorescence intensity was expressed as a percentage of the value in the vehicle-treated control group.

Measurement of mitochondrial membrane potential

The ΔΨm was measured using a fluorescent dye, JC-1 reagent as described.¹³ In living cells, JC-1 exists as a green fluorescent monomer at depolarised membrane potentials or as an orange-red fluorescent J-aggregate at hyperpolarised membrane potentials. Cells were seeded in 60 mm dishes at a density of 1.0 × 10⁶ cells/dish, and on the 7th day in in vitro, they were treated with HCW at 100 μg/mL 30 min before the treatment of 8 μM Aβ_25–35 for a total of 21 h. Treated cells were incubated with JC-1 reagent solution at 37°C for 15 min after HCW and/or Aβ_25–35 treatment. Cells were washed and transferred to 96-well plates. Then, the red (585/590 nm) and green (510/527 nm) fluorescence data were acquired using a fluorescence microplate reader, and the ratio of red-to-green fluorescence was expressed as a percentage of the value in the vehicle-treated control group.

Measurement of caspase 3 activation

The caspase 3 activation was measured using 7-amino-4-trifluoromethyl coumarin (DEVD-AFC) substrate and antibody against cleaved caspase 3. Cells were seeded in 60 and 100 mm dishes at densities of 1.0 × 10⁶ cells/dish and 2.0 × 10⁶ cells/dish; and on the 7th day in vitro, they were treated with HCW at 100 μg/mL 30 min before the treatment of 8 μM Aβ_25–35 for a total of 24 h. For measurement of caspase 3 activity, the treated cell lysates were incubated with reaction buffer containing 10 mM dithiothreitol (DTT) and 1 mM DEVD-AFC substrate. The mixture was incubated for 2 h at 37°C and the protease activity was detected at an excitation wavelength of 360 nm and an emission wavelength of 460 nm using a fluorescence microplate reader.

To measure the expression level of cleaved caspase 3, the treated cell lysates were separated by 15% SDS-polyacrylamide gel electrophoresis and transferred onto membranes. Membranes were incubated with 5% skimmed milk in Tris-buffered saline with Tween-20 for 1 h. They were then incubated with rabbit anticleaved caspase 3 antibody (1:1000 dilution) at 4°C, followed by incubation with horseradish peroxidase conjugated anti-rabbit IgG for 1 h. Antibody detection was carried out using an ECL detection kit and visualised using the LAS-4000 mini system (Fujifilm Corp., Tokyo, Japan).

Statistical analysis

The data are expressed as mean ± SEM. Student’s t test followed by a Mann-Whitney test was used to compare vehicle-treated control group and Aβ_25–35-only treated group with p < 0.05 being considered statistically significant. One-way analysis of variance followed by Tukey’s multiple comparison test was used to compare Aβ_25–35-only treated group and Aβ_25–35 + HCW-treated group. For all analyses, GraphPad Prism software (GraphPad Software Inc., San Diego, California, USA) was used.

Results

Protective effect of HCW against Aβ_25–35-induced neurotoxicity

To evaluate the protective effect of HCW against Aβ_25–35-induced neurotoxicity in rat cortical cells, we performed the MTT and LDH assays. HCW itself at 0.1–100 μg/mL did not cause any apparent cytotoxicity (Figure 2(a)). However, treatment with 8 μM Aβ_25–35 reduced cell viability by 68.92 ± 1.31% of the control value, whereas pretreatment with HCW at 10 and 100 μg/mL significantly prevented cell loss by 87.06 ± 2.55% and 89.49 ± 1.89% of the control value, respectively (Figure 2(b)). In the LDH assay, 8 μM Aβ_25–35 resulted in an increase in LDH release in the medium by 145.01 ± 4.45% of the control value, whereas pretreatment with HCW at 100 μg/mL significantly blocked LDH release by 89.10 ± 0.57% of the control value (Figure 2(c)).

Figure 2.

Protective effects of HCW against Aβ_25–35-induced neurotoxicity in MTT and LDH assays. (a) and (b): Cell viability was determined by MTT assay. LDH leakage was determined as an indicator of cytotoxicity and was colorimetrically measured (c). Cortical neurons were treated with HCW only for 24 h (a) or 30 min before the treatment of 8 μM Aβ_25–35 for a total of 24 h ((b) and (c)). Values are indicated as the mean ± SEM (n = 7). ***p < 0.001, **p < 0.01 compared with the control group, ###p < 0.001 compared with the Aβ_25–35-only treated group. HCW: Houttuyniae Herba water extract; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; LDH: lactate dehydrogenase; Aβ: amyloid beta.

Based on the effects of HCW, we explored the changes in neurite normality and cell survival using MAP-2 immunostaining. In the control group, many MAP-2 IR cells with normal shapes and neurite lengths were observed (Figure 3(b) and (e)), whereas the Aβ_25–35-only treated group showed most of MAP-2 IR neurons with shrunken cell bodies and shortened neurites (Figure 3(c) and (f)) and exhibited decreased survival of MAP-2 IR neurons to 58.76 ± 2.77% of the control value (Figure 3(a)). Pretreatment with HCW at 100 μg/mL normalised the neuronal morphology (Figure 3(d) and (g)) and increased the survival rate of MAP-2 IR neurons up to 165.93 ± 15.45% compared with the Aβ_25–35-only treated group (Figure 3(a)).

Figure 3.

Protective effects of HCW against Aβ_25–35-induced neuronal damage in MAP-2 immunostaining. The numbers of MAP-2 IR neurons were counted (a), and representative images are shown in (b) to (g). Control group ((b) and (e)), Aβ_25–35-only treated group ((c) and (f)), Aβ_25–35 + HCW (100 μg/mL)-treated group ((d) and (g)). Cortical neurons were treated with HCW 30 min before the treatment of 8 μM Aβ_25–35 for a total of 24 h. Values are indicated as the mean ± SEM (n = 5). ***p < 0.001 compared with the control group, ##p < 0.01 compared with the Aβ_25–35-only treated group. HCW: Houttuyniae Herba water extract; MAP-2: microtubule-associated protein-2; IR: immunoreactive; Aβ: amyloid beta.

Inhibitory effect of HCW on intracellular calcium increased by Aβ_25–35

To examine the effects of HCW on Aβ_25–35-induced intracellular calcium increase in rat cortical neurons, we used Fluo-4 AM dye. Addition of 8 μM Aβ_25–35 induced increase in intracellular calcium level up to 155.30 ± 6.21% than the control value, whereas that of HCW at 100 μg/mL pretreated group was 107.56 ± 1.21% of the control value (Figure 4).

Figure 4.

Inhibitory effects of HCW on intracellular calcium increase by Aβ_25–35. The intracellular calcium level was measured using the calcium indicator, Fluo-4 AM (a), and representative images are shown in (b) to (d). Control group (b), Aβ_25–35-only treated group (c), Aβ_25–35 + HCW (100 μg/mL)-treated group (d). Scale bar = 50 μm. Cortical neurons were treated with HCW for 30 min and reacted with Fluo-4 AM dye for 40 min, followed by Aβ_25–35 stimulation for 3 h. Values are indicated as the mean ± SEM (n = 5). ***p < 0.001 compared with the control group, ###p < 0.001 compared with the Aβ_25–35-only treated group. HCW: Houttuyniae Herba water extract; Aβ: amyloid beta.

Inhibitory effect of HCW on intracellular ROS level induced by Aβ_25–35

To evaluate the effect of HCW on Aβ_25–35-induced ROS production in rat cortical neurons, we used H₂DCF-DA. Exposure to 8 μM Aβ_25–35 led to significant ROS elevation up to 128.96 ± 3.11% of the control value. Pretreatment with HCW at 100 μg/mL markedly inhibited ROS generation by 114.60 ± 3.08% of the control value (Figure 5).

Figure 5.

Inhibitory effect of HCW on intracellular ROS level increased by Aβ_25–35. Accumulation of intracellular ROS was measured using a fluorescent probe H₂DCF-DA. Cortical neurons were treated with HCW for 30 min and incubated with 8 μM Aβ_25–35 for a further 2 h. Values are indicated as the mean ± SEM (n = 5). ***p < 0.001 compared with the control group, #p < 0.05 compared with the Aβ_25–35-only treated group. HCW: Houttuyniae Herba water extract; ROS: reactive oxygen species; H₂DCF-DA: 2,7-dichlorodihydrofluorescein diacetate; Aβ: amyloid beta.

Protective effect of HCW on mitochondrial membrane depolarisation by Aβ_25–35

To explore the effect of HCW on Aβ_25–35-induced ΔΨm depolarisation in rat cortical neurons, we used JC-1 reagent. In mitochondria with normal ΔΨm, the dye JC-1 aggregates, producing red fluorescence, whereas in depolarised mitochondria, JC-1 remains a monomer, producing green fluorescence. Exposure to 8 μM Aβ_25–35 depolarised ΔΨm, decreasing the red/green fluorescence ratio to 76.63 ± 3.30% of the control value. Pretreatment with HCW at 100 μg/mL attenuated this depolarisation of ΔΨm, with red/green fluorescence ratio of 96.75 ± 4.36% of the control value (Figure 6).

Figure 6.

Protective effect of HCW on ΔΨm depolarised by Aβ_25–35. The mitochondrial membrane potential (ΔΨm) was quantified by calculating the ratio of red to green fluorescence intensity of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1). Cortical neurons were treated with HCW 30 min before the treatment of 8 μM Aβ_25–35 for a total of 21 h. Values are indicated as the mean ± SEM (n = 5). ***p < 0.001 compared with the control group, ##p < 0.01 compared with the Aβ_25–35-only treated group. HCW: Houttuyniae Herba water extract; ΔΨm: mitochondrial membrane potential; JC-1:5,5′,6,′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanineiodide; Aβ: amyloid beta.

Inhibitory effect of HCW on caspase 3 activation induced by Aβ_25–35

To investigate the effect of HCW on Aβ_25–35-induced caspase 3 activation in rat cortical neurons, we used DEVD-AFC substrate and anticleaved caspase 3 antibody. The activity of caspase 3 was increased by exposure to 8 μM Aβ_25–35 up to 134.18 ± 1.90% of the control value. However, pretreatment of HCW at 100 μg/mL significantly attenuated the activation of caspase 3, with the fluorescence intensity of 110.59 ± 3.73% of the control value (Figure 7(a)). Western blot analyses were consistent with these results (Figure 7(b)).

Figure 7.

Inhibitory effect of HCW on caspase 3 activation induced by Aβ_25–35. The caspase 3 activity was measured by fluorescence intensity of caspase 3 substrate DEVD-AFC, and Western blot analyses were performed to measure the cleaved caspase 3 expression level. Cortical neurons were treated with HCW 30 min before the treating with 8 μM Aβ_25–35 for a total of 24 h. Values are indicated as the mean ± SEM (n = 4). **p < 0.01 compared with the control group, ##p < 0.01 compared with the Aβ_25–35-only treated group. HCW: Houttuyniae Herba water extract; DEVD-AFC: 7-amino-4-trifluoromethyl coumarin; Aβ: amyloid beta.

Discussion

We demonstrated that HCW has neuroprotective effects against Aβ_25–35-induced toxicity and that these effects occur via the regulation of calcium homeostasis, suppression of ROS overproduction, normalisation of ΔΨm, and inhibition of caspase 3 activation.

First, to evaluate the neuroprotective effects of HCW against Aβ_25–35-induced toxicity, we performed an MTT assay, an LDH assay, and a MAP-2 immunostaining. Aβ, the major component of senile plaques, is thought to play a crucial role in the pathogenesis of AD.³⁵ Three Aβ-related fragments (Aβ_1–40, Aβ_1–42, and Aβ_25–35) exhibit toxicity in multiple cell types in vitro.⁹ Among these fragments, Aβ_25–35, the shortest peptide, is the active core fragment of the full-length Aβ_1–42 peptide; yet, it has toxicity similar to that of the native full-length Aβ_1–42 peptide.³⁶ In previous studies, the treatment of primary cultured cortical neurons with Aβ_25–35 was shown to provoke significant cell death by mechanisms including intracellular calcium increases, ROS overproduction, and mitochondrial dysfunction, leading to the apoptosis pathway.^2,7 In our study, HCW significantly suppressed Aβ_25–35-induced neuronal toxicity in MTT assay and decreased Aβ_25–35-induced LDH release in LDH assay. MAP-2 immunostaining revealed that Aβ_25–35 caused cortical cell loss, with cells exhibiting shrunken bodies and shortened neurites. In contrast, HCW increased the number of MAP-2 IR neurons with normal shapes. These results suggest that HCW provides both functional and morphological protection of cortical neurons against Aβ_25–35 toxicity.

Given that the primary event provoked by Aβ fibrils is calcium influx and subsequent ROS generation, we measured intracellular calcium levels and ROS generation. Calcium signalling is essential for the fundamental functions of neurons,³⁷ and alterations in calcium homeostasis and changes in calcium signalling can result in neurodegeneration, including that of AD.³⁸ Furthermore, the increase in intracellular calcium levels induced by Aβ_25–35 leads to ROS generation.⁶ Although ROS are natural by-products of the normal metabolism of oxygen in mitochondria,⁹ excessive ROS readily damage biological molecules and ROS-induced membrane damage causes further calcium influx which contribute to final neuronal death via apoptotic mechanisms.⁷ Thus, as almost simultaneous early events induced by Aβ_25–35, the intracellular calcium and ROS increases play a role in Aβ-mediated neurotoxicity.⁷ In the present study, HCW largely inhibited the increase in intracellular calcium and the overproduction of intracellular ROS induced by Aβ_25–35.

The overproduction of intracellular ROS increases the permeability of the mitochondrial membrane, causing mitochondrial dysfunction.³⁹ Because this has been identified in a large proportion of neurodegenerative diseases such as AD,³⁵ we measured the mitochondrial membrane potential after treatment with HCW and/or Aβ_25–35. Although the underlying mechanisms of mitochondrial abnormalities are not fully understood,⁹ mitochondrial dysfunction is thought to contribute to neuronal cell death through the initiation of programmed cell death, including the release of caspases.⁴⁰ In this study, HCW remarkably improved the decrease in ΔΨm induced by Aβ_25–35.

Finally, to explore the effects of HCW on apoptosis induced by Aβ_25–35, we measured caspase 3 activity and performed Western blot analysis. Caspases are essential for the execution of apoptotic cell death in response to various stimuli.⁹ During apoptosis, mitochondria with increased permeability releases some apoptotic factors that activate caspase cascade,⁴¹ in which caspase 3, as a downstream effector, has been suggested to play a critical role.⁴² Like other members of the caspase family, caspase 3 is synthesised as an inactive proenzyme that is processed by self-proteolysis and/or cleavage by other proteases in cells undergoing apoptosis.⁴² In the present study, Aβ_25–35 induced caspase 3 activation to a degree similar to that reported in previous studies,^9,13 and pretreatment with HCW significantly reduced the degree of caspase activation. We confirmed this by Western blot analysis.

The results of the present study demonstrate that HCW protects rat primary cortical cells against Aβ_25–35-induced neurotoxicity via the regulation of intracellular calcium and the inhibition of mitochondria-mediated apoptosis. Phenol-rich plants have well-known antioxidant activity⁴³ and have been reported to exert a neuroprotective effect via antioxidant, anti-inflammatory, and antiapoptotic mechanisms.^44,45 H. cordata also possesses phenolic compounds such as chlorogenic acid and caffeic acid, which were used to standardise the HCW in this study. Chlorogenic acid has been shown to be protective against methylglyoxal-induced apoptosis in neuro-2a cells,²⁸ and caffeic acid has been reported to attenuate neuronal damage, astrogliosis, and glial scar formation in cryoinjured mouse brain.³¹ Two other phenolic compounds, quercetin and rutin, were demonstrated to protect neurons from damage induced by Aβ toxicity or transient focal ischemia.^26,29 Taken together, the phenolic compounds of HCW may contribute, at least in part, to the neuroprotective effects of HCW against Aβ_25–35 toxicity, although this remains to be further studied.

In summary, HCW significantly protected rat primary cortical cells against the reduced cell viability and altered neuronal morphology induced by Aβ_25–35 neurotoxicity by regulating intracellular calcium, preventing ROS overproduction, and inhibiting ΔΨm loss and caspase 3 activation. These results suggest that HCW is a promising candidate for a neuroprotective agent in AD. Further in vivo studies are needed to confirm the efficacy of HCW in AD treatment.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Acknowledgement

This study was performed using research funds from Kyung Hee University [20100639].

Declaration of Conflict of Interest

The authors declared no conflicts of interest.

References

Nussbaum

Ellis

. Alzheimer's disease and Parkinson's disease. N Engl J Med 2003; 348: 1356–1364.

Liang

Jiang

Hao

. Catalpol protects primary cultured cortical neurons induced by Aβ_1–42 through a mitochondrial-dependent caspase pathway. Neurochem Int 2009; 55: 741–746.

Pereira

Ferreiro

Cardoso

De Oliveira

. Cell degeneration induced by amyloid-β peptides: implications for Alzheimer's disease. J Mol Neurosci 2004; 23: 97–104.

Behl

Davis

Lesley

Schubert

. Hydrogen peroxide mediates amyloid β protein toxicity. Cell 1994; 77: 817–827.

Grace

Rabiner

Busciglio

Characterization of neuronal dystrophy induced by fibrillar amyloid β: implications for Alzheimer's disease. Neuroscience 2002; 114: 265–273.

Ekinci

Linsley

Shea

. β-Amyloid-induced calcium influx induces apoptosis in culture by oxidative stress rather than tau phosphorylation. Mol Brain Res 2000; 76: 389–395.

Cho

Ban

Kim

Jeong

Lee

Song

. Aralia cordata protects against amyloid β protein (25-35)-induced neurotoxicity in cultured neurons and has antidementia activities in mice. J Pharmacol Sci 2009; 111: 22–32.

Petit

Susin

Zamzami

Mignotte

Kroemer

. Mitochondria and programmed cell death: back to the future. FEBS Lett 1996; 396: 7–13.

Liu

Jin

Sun

. The neuroprotective effects of tanshinone IIA on β-amyloid-induced toxicity in rat cortical neurons. Neuropharmacology 2010; 59: 595–604.

10.

Jeong

Kim

Lee

Song

Bae

. Leaf and stem of Vitis amurensis and its active components protect against amyloid β protein (25-35)-induced neurotoxicity. Arch Pharm Res 2010; 33: 1655–1664.

11.

Kim

Choi

Cho

Kim

Lim

Kim

. Ipomoea batatas attenuates amyloid β peptide-induced neurotoxicity in ICR mice. J Med Food 2011; 14: 304–309.

12.

Lee

Kuo

Wang

Pan

. Red mold rice ameliorates impairment of memory and learning ability in intracerebroventricular amyloid β-infused rat by repressing amyloid β accumulation. J Neurosci Res 2007; 85: 3171–3182.

13.

Kim

Park

Seo

Jang

Hong

. Evaluation of Samjunghwan, a traditional medicine, for neuroprotection against damage by amyloid-beta in rat cortical neurons. J Ethnopharmacol 2010; 130: 625–630.

14.

Lee

Yoo

Yun

. Inhibitory effect of green tea extract on β-amyloid-induced PC12 cell death by inhibition of the activation of NF-κB and ERK/p38 MAP kinase pathway through antioxidant mechanisms. Mol Brain Res 2005; 140: 45–54.

15.

Moongkarndi

Srisawat

Saetun

Jantaravinid

Peerapittayamongkol

Soi-ampornkul

. Protective effect of mangosteen extract against β-amyloid-induced cytotoxicity, oxidative stress and altered proteome in SK-N-SH cells. J Proteome Res 2010; 9: 2076–2086.

16.

Zhu

Bickford

Sanberg

Giunta

Tan

. Blueberry opposes β-amyloid peptide-induced microglial activation via inhibition of p44/42 mitogen-activation protein kinase. Rejuvenation Res 2008; 11: 891–901.

17.

Ham

Shin

Lee

Nam

Koo

. Neuroprotective effect of the n-hexane extracts of Laurus nobilis L. in models of Parkinson's disease. Biomol Ther 2011; 19: 118–125.

18.

Kim

Choi

Ryu

Hur

Kim

. Cassiae semen, a seed of Cassia obtusifolia, has neuroprotective effects in Parkinson's disease models. Food Chem Toxicol 2010; 48: 2037–2044.

19.

Toda

. Antioxidative effects of polyphenols in leaves of Houttuynia cordata on protein fragmentation by copper-hydrogen peroxide in vitro. J Med Food 2005; 8: 266–268.

20.

Chang

Chiang

Chen

Liu

Wang

Lin

. Antileukemic activity of Bidens pilosa L. var. minor (Blume) Sherff and Houttuynia cordata Thunb. Am J Chin Med 2001; 29: 303–312.

21.

Chen

Liu

Chen

Chao

Chang

. A study of the antioxidative and antimutagenic effects of Houttuynia cordata Thunb. using an oxidized frying oil-fed model. J Nutr Sci Vitaminol 2003; 49: 327–333.

22.

Nuengchamnong

Krittasilp

Ingkaninan

. Rapid screening and identification of antioxidants in aqueous extracts of Houttuynia cordata using LC-ESI-MS coupled with DPPH assay. Food Chem 2009; 117: 750–756.

23.

Shin

Joo

Jeon

Park

Jang

Kim

. Anti-inflammatory effects of a Houttuynia cordata supercritical extract. J Vet Sci 2010; 11: 273–275.

24.

Tian

Zhao

Guo

Yang

. A comparative study on the antioxidant activities of an acidic polysaccharide and various solvent extracts derived from herbal Houttuynia cordata . Carbohydr Polym 2011; 83: 537–544.

25.

Jang

Bae

Lee

Park

Bae

. Caffeic acid and quercitrin purified from Houttuynia cordata inhibit DNA topoisomerase I activity. Nat Prod Res 2010: 1–10.

26.

Ansari

Abdul

Joshi

Opii

Butterfield

. Protective effect of quercetin in primary neurons against Aβ(1-42): relevance to Alzheimer's disease. J Nutr Biochem 2009; 20: 269–275.

27.

Chen

Wang

Ren

Yang

. Protective effect of hyperin on focal cerebral ischemia reperfusion injury in rats. Zhong Xi Yi Jie He Xue Bao 2006; 4: 526–529.

28.

Huang

Chuang

Yen

. Cytoprotective effects of phenolic acids on methylglyoxal-induced apoptosis in Neuro-2A cells. Mol Nutr Food Res 2008; 52: 940–949.

29.

Khan

Ahmad

Ishrat

Khuwaja

Srivastawa

Khan

. Rutin protects the neural damage induced by transient focal ischemia in rats. Brain Res 2009; 1292: 123–135.

30.

Shi

Zhao

Zhu

Yew

Yao

. Protective effects of Ginkgo biloba extract (EGb761) and its constituents quercetin and ginkgolide B against β-amyloid peptide-induced toxicity in SH-SY5Y cells. Chem Biol Interact 2009; 181: 115–123.

31.

Zhang

W-P

Chen

K-D

Qian

X-D

Fang

S-H

Wei

E-Q

. Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury. Life Sci 2007; 80: 530–537.

32.

Meng

Leung

KSY

Dong

Zhou

Jiang

Zhao

. Simultaneous quantification of eight bioactive components of Houttuynia cordata and related Saururaceae medicinal plants by on-line high performance liquid chromatography-diode array detector-electrospray mass spectrometry. Fitoterapia 2009; 80: 468–474.

33.

Cheng

Hsieh

Tsai

Chiu

Lee

. Neuroprotective effect of luteolin on amyloid β protein (25–35)-induced toxicity in cultured rat cortical neurons. Phytother Res 2010; 24: S102–S108.

34.

Siddiqui

Kashyap

Al-Khedhairy

Musarrat

Khanna

Yadav

. Protective potential of 17β-estradiol against co-exposure of 4-hydroxynonenal and 6-hydroxydopamine in PC12 cells. Hum Exp Toxicol 2010; 30: 860–869.

35.

Eckert

Keil

Marques

Bonert

Frey

Schüssel

. Mitochondrial dysfunction, apoptotic cell death, and Alzheimer's disease. Biochem Pharmacol 2003; 66: 1627–1634.

36.

Lai

CSW

Yuen

Chang

RCC

. Characterizing the neuroprotective effects of alkaline extract of Lycium barbarum on β-amyloid peptide neurotoxicity. Brain Res 2007; 1158: 123–134.

37.

Mattson

. Calcium and neurodegeneration. Aging Cell 2007; 6: 337–350.

38.

Hung

CHL

Chang

RCC

. Modulation of mitochondrial calcium as a pharmacological target for Alzheimer's disease. Ageing Res Rev 2010; 9: 447–456.

39.

Huang

Lin

Chiang

. Protective effects of Angelica sinensis extract on amyloid β-peptide-induced neurotoxicity. Phytomedicine 2008; 15: 710–721.

40.

Cardoso

Santana

Swerdlow

Oliveira

. Mitochondria dysfunction of Alzheimer's disease cybrids enhances Aβ toxicity. J Neurochem 2004; 89: 1417–1426.

41.

Benn

Woolf

. Adult neuron survival strategies—slamming on the brakes. Nat Rev Neurosci 2004; 5: 686–700.

42.

Cheng

Hsieh

Tsai

Chiu

Lee

. Neuroprotective effect of luteolin on amyloid β protein (25-35)-induced toxicity in cultured rat cortical neurons. Phytother Res 2010; 24: S102–S108.

43.

Kim

Shim

Kim

Lee

Huh

. Mulberry fruit protects dopaminergic neurons in toxin-induced Parkinson's disease models. Br J Nutr 2010; 104: 8–16.

44.

Joseph

Shukitt-Hale

Brewer

Weikel

Kalt

Fisher

. Differential protection among fractionated blueberry polyphenolic families against DA-, Aβ₄₂- and LPS-induced decrements in Ca²⁺ buffering in primary hippocampal cells. J Agric Food Chem 2010; 58: 8196–8204.

45.

Zhao

. Natural antioxidants protect neurons in Alzheimer’s disease and Parkinson's disease. Neurochem Res 2009; 34: 630–638.

Houttuyniae Herba protects rat primary cortical cells from Aβ 25–35 -induced neurotoxicity via regulation of calcium influx and mitochondria-mediated apoptosis

Abstract

Keywords

Introduction

Materials and methods

Materials

Preparation of extract and standardisation

Primary cultures of cortical neurons

Determination of cell viability

LDH release assay

MAP-2 immunostaining

Measurement of the intracellular calcium level

Measurement of intracellular ROS generation

Measurement of mitochondrial membrane potential

Measurement of caspase 3 activation

Statistical analysis

Results

Protective effect of HCW against Aβ25–35-induced neurotoxicity

Inhibitory effect of HCW on intracellular calcium increased by Aβ25–35

Inhibitory effect of HCW on intracellular ROS level induced by Aβ25–35

Protective effect of HCW on mitochondrial membrane depolarisation by Aβ25–35

Inhibitory effect of HCW on caspase 3 activation induced by Aβ25–35

Discussion

Footnotes

Funding

Acknowledgement

Declaration of Conflict of Interest

References

Protective effect of HCW against Aβ_25–35-induced neurotoxicity

Inhibitory effect of HCW on intracellular calcium increased by Aβ_25–35

Inhibitory effect of HCW on intracellular ROS level induced by Aβ_25–35

Protective effect of HCW on mitochondrial membrane depolarisation by Aβ_25–35

Inhibitory effect of HCW on caspase 3 activation induced by Aβ_25–35