Acanthus ebracteatus leaf extract provides neuronal cell protection against oxidative stress injury induced by glutamate

The plant material used in this study is the leaves of A. ebracteatus collected from the Princess Maha Chakri Sirindhorn Herbal Garden (Rayong Province, Thailand). The plant was authenticated by Professor Dr. Thaweesakdi Boonkerd and deposited with voucher specimen number A013422(BCU) at the herbarium of Kasin Suvatabhandhu (Department of Botany, Faculty of Science, Chulalongkorn University, Thailand). The extraction was carried out twice using hexane and absolute ethanol as extracting solvents. Briefly, the leaves were dried in a ventilated incubator at a temperature of at 40 °C and ground into a fine powder. Then, the extracts were prepared by macerating 35 g of the dried leaf powder in 350 mL of each solvent for 48 h under agitation at room temperature (RT), followed by filtration. The residue powder was re-extracted by a similar process, and all filtrates were subsequently combined before removing the solvent by vacuum evaporation. The yield of hexane extract (AEH) and ethanolic extract (AEE) of A. ebracteatus leaves was found to be 2.14% and 7.98% (w/w), respectively. Each resulting extract was dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 100 mg/mL, stored at − 20 °C, and protected from light until further analysis.

The total flavonoid content was determined using the aluminum chloride colorimetric method modified for a microplate format as described previously [33]. In brief, 50 μL of the extract sample (1 mg/mL) was made up to 200 μL with 95% ethanol, and mixed well with 10 μL of 10% (v/v) AlCl₃ solution and 10 μL of 1 M NaOAc solution. After the reaction was allowed to stand for 40 min in the dark, the absorbance of the reaction mixture was measured at 415 nm using a microplate reader (Perkin-Elmer). Quercetin (Sigma-Aldrich) was used as a standard to construct the calibration curve for quantification, and the content of total flavonoids was reported as mg of quercetin equivalent (QE) per g of dry weight extract.

The total phenolic content was determined using the Folin-Ciocalteu method adapted for analysis with a microplate reader, as previously described [33]. Briefly, 50 μL of the extract sample (1 mg/mL) was mixed thoroughly with 50 μL of 10-fold diluted Folin-Ciocalteu’s phenol reagent (Sigma-Aldrich). After 20 min of incubation, the mixture was neutralized by addition of 50 μL of a 7.5% (w/v) Na₂CO₃ solution and then kept in the dark at RT for a further 20 min. Finally, the absorbance was measured at 760 nm using an EnSpire® Multimode Plate Reader (Perkin-Elmer). The content of total phenolics was calculated from a standard calibration curve using gallic acid (TCI America, Portland, OR, USA), and the results are expressed as mg of gallic acid equivalent (GAE) per g of dry weight extract.

The immortalized mouse hippocampal HT22 cell line, which served as an in vitro model of neurodegeneration, was a generous gift from Prof. David Schubert at the Salk Institute (San Diego, CA, USA). The HT22 cell line was originally a glutamate-sensitive subclone of the HT-4 cell line, which was derived from the immortalization of primary mouse hippocampal neuronal tissues with a temperature-sensitive SV40 T-antigen [34]. These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine serum (Sigma-Aldrich), 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco, Waltham, MA, USA) in a humidified atmosphere of 5% CO₂ at 37 °C. The culture medium was replaced every two days. Upon reaching approximately 80% confluency, the cells were passaged by trypsinization and subcultured into fresh medium to maintain their exponential growth.

The MTT assay measures cell viability based on the metabolic activity of viable cells to reduce the yellow tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), to a purple formazan product. HT22 cells were seeded at a density of 6 × 10³ cells/well in a 96-well plate and incubated overnight prior to treatment with 5 mM glutamate (Sigma-Aldrich) alone or glutamate in combination with different concentrations of the extracts for 24 h. After the exposure period, the MTT solution (Biobasic, Markham, Ontario, Canada) was added to each well at a final concentration of 0.5 mg/mL and incubated for an additional 4 h in the dark. The generated formazan crystals were dissolved in a DMSO-ethanol mixture (1:1, v/v) after the supernatants were carefully removed from the wells. Finally, the absorbance was determined at 550 nm by using an EnSpire® Multimode Plate Reader (Perkin-Elmer, Waltham, MA, USA). The results are expressed as a percentage relative to control (untreated) cells.

The LDH assay determines cell viability based on the release of cytoplasmic lactate dehydrogenase (LDH) from damaged cells, which converts the colorless tetrazolium salt, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT), into a red formazan product. HT22 cells were seeded at a density of 6 × 10³ cells/well in a 96-well plate and incubated overnight prior exposure to 5 mM glutamate alone or glutamate in combination with different concentrations of the extracts for 24 h. After treatment, the amount of LDH released into the culture medium was measured using the CytoTox 96® assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. In brief, the culture supernatant was incubated with reconstituted substrate mix in the dark for 30 min at RT, followed by addition of the stop solution before measurement. The absorbance was then recorded at 490 nm by a microplate reader (Perkin-Elmer). The results are expressed as a percentage of maximum LDH release obtained by complete lysis of control (untreated) cells.

Apoptotic cell death was determined based on the externalization of phosphatidylserine to the outer cell surface and increased plasma membrane permeability to dye by using an FITC Annexin V apoptosis detection kit with propidium iodide (PI) (BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, HT22 cells were seeded onto a 6-well plate at a density of 1.5 × 10⁵ cells/well and incubated overnight prior to treatment with 5 mM glutamate alone or glutamate in combination with the extracts for 18 h. At the end of the exposure period, the harvested cells were washed twice with phosphate-buffered saline (PBS), re-suspended in the binding buffer, and stained by the solution of FITC-conjugated annexin V and PI for 15 min in the dark. The fluorescence intensity of stained cells, at least 10,000 cells per group, was immediately analyzed by a BD FACSCalibur™ flow cytometer (BD Bioscience, Heidelberg, Germany). The results are expressed as a percentage of annexin V-positive/PI-negative cells (early apoptosis) plus annexin V/PI-positive cells (late apoptosis).

The radical scavenging assay was performed using the DPPH and ABTS methods for evaluation of antioxidant activity of the sample based on its hydrogen atom- or electron-donating capacity to neutralize the free radicals. A working solution of stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•) (Sigma-Aldrich) was dissolved in ethanol to a final concentration of 0.2 mg/mL. The cation radical ABTS• + working solution was generated by the oxidation of 7 mM 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (Sigma-Aldrich) with 2.45 mM potassium persufhate at a 1:1 (v/v) ratio. The reaction mixture was allowed to stand for 16–18 h in the dark prior to dilution with ethanol until the absorbance reached between 0.7 and 0.8 at 734 nm. For the assay protocol, the DPPH• or ABTS• + working solution was added to the extract sample (1 mg/mL) at a ratio of 9:1 (v/v). The reaction mixture was incubated in the dark at RT for 15 min or 30 min, and the absorbance was recorded using a microplate reader (Perkin-Elmer) at 517 nm or 734 nm for the DPPH or ABTS assay, respectively. Ascorbic acid (vitamin C) (Calbiochem, San Diego, CA, USA) was used as a reference standard in both assays. Radical scavenging activity was expressed as the percent inhibition of free radicals calculated by the following equation: % Inhibition = 100 - [(Abs of sample - Abs of blank) × 100/ Abs of control]. The antioxidant capacity is expressed as vitamin C equivalent antioxidant capacity (VCEAC) in mg per g of dry weight extract.

Measurement of the intracellular ROS level was performed using oxygen-sensitive 2′,7′-dichloro-dihydrofluoroscein diacetate (H₂DCFDA) (Molecular Probes, Eugene, OR, USA) based on the ability of ROS to oxidize the cell-permeant, non-fluorescent H₂DCFDA molecule into a highly fluorescent 2′,7′-dichlorofluorescein (DCF) molecule. HT22 cells were seeded at a density of 1 × 10⁴ cells/well in a 96-well plate and incubated overnight prior to exposure to 5 mM glutamate alone or glutamate in combination with different concentrations of the extracts for 14 h. After treatment, the cells were loaded with 5 μM H₂DCFDA, incubated at 37 °C for another 30 min, and then washed three times with Hank’s balanced salt solution (HBSS) (Gibco). Fluorescence was immediately determined using an EnSpire® Multimode Plate Reader (Perkin-Elmer), and photographs were obtained using an Axio Observer A1 fluorescence microscope (Carl Zeiss, Jena, Germany), with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Data are expressed as the percentage of fluorescence intensity relative to control (untreated) cells.

At the end of treatments, the total RNA from HT22 cells in each group was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The amount of RNA was quantified by measuring absorbance at 260 nm, and then 1 μg of total RNA was reverse transcribed to cDNA using oligo(dT)17 primer and AccuPower RT PreMix (Bioneer, Daejeon, South Korea). The cDNA was used as a template for subsequent real-time PCR reactions performed on an Exicycler™ 96 real-time quantitative thermal block (Bioneer) by using the GreenStar™ qPCR PreMix (Bioneer) and the specific primers listed in Table 1. The thermal cycling conditions included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles, each consisting of 15 s of denaturation at 95 °C, 15 s at 55 °C for primer annealing, and 30 s at 72 °C for chain elongation. A melting curve analysis was performed after amplification to verify the accuracy of the amplicon. The relative expression level of each target gene was normalized to β-actin expression and analyzed using the 2^-ΔΔCT method.

HT22 cells were seeded in 12-well plate at a density of 4 × 10⁴ cells/well prior exposure to 5 mM glutamate alone or glutamate in combination with the extract for 16 h. After treatments, an immunofluorescence technique was performed to determine nuclear translocation of AIF. In brief, the cells were fixed with cold 4% (w/v) paraformaldehyde solution for 20 min, permeabilized in 0.1% (w/v) Triton X-100 for 10 min, and blocked with 5% (w/v) BSA for 30 min. Then, the cells were incubated overnight with anti-AIF antibody (1:400; Cell Signaling Technology) at 4 °C, followed by incubation for 1 h with Alexa Fluor 555-conjugated goat anti-rabbit (1:2000; Sigma-Aldrich) at RT. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) solution for 10 min at RT. Following mounting with ProLong Gold antifade mountant (Thermo Fisher Scientific), stained cells were imaged using an LSM 700 confocal laser scanning microscope (Carl Zeiss, Jena, Germany).

To identify putative phytochemical components in AE leaf extract that may be responsible for neuroprotection against glutamate-induced oxidative toxicity, we carried out LC-MS analysis as well as quantitative determination of total flavonoids and phenolics in AEE. Our results revealed a total of 95 ion chromatographic peaks of AEE detected in the positive ion mode (Fig. 1). After identification of each molecular ion peak [M + H]⁺ by comparison of observed m/z values with the calculated (theoretical) values recorded in databases and the literature, we proposed 11 phytochemical compounds that could have beneficial effects for antioxidant defense or neurological function, of which 5 have been previously reported for A. ebracteatus (Fig. 1 and Table 2). The identified peaks were annotated by number and are detailed in Table 2 as follows: peak number, retention time (Rt), observed m/z, peak area, compound name, theoretical mass, and mass error. Furthermore, the total flavonoid content and total phenolic content of AEE were found to be 20.22 ± 3.69 mg QE and 84.86 ± 3.69 mg GAE per g of dry weight extract, respectively.

To examine the neuroprotective effect of AE leaf extracts against glutamate-induced oxidative cytotoxicity, HT22 cells were exposed to various concentrations of either AEE or AEH (3.125, 6.25, 12.5, 25, and 50 μg/mL) in the presence or absence of 5 mM glutamate, and then cell viability was assessed. We first evaluated the toxic effects of AEH and AEE on HT22 cells and found that neither extract caused noticeable cell death, as shown in Fig. 2a. Treatment of glutamate alone at 5 mM caused a reduction in cell viability of approximately 50%; however, this effect could be rescued in the presence of AE leaf extracts. Our results showed that both AEH and AEE exhibited a significant protective effect by restoring the viability of glutamate-treated HT22 cells in a dose-dependent manner, as determined by the MTT reduction (Fig. 2b) and LDH leakage (Fig. 2c) assays. Morphological examination under a microscope showed normal morphology of HT22 cells upon glutamate treatment combined with AEH or AEE (Fig. 2d). These results indicate that AE leaf extracts protect against neuronal damage caused by glutamate. The concentration of 50 μg/mL AEH and AEE was chosen for subsequent experiments, as it resulted in maximal protection among the concentrations tested.

As it is known that the toxicity caused by excessive glutamate contributes to neuronal cell death via the apoptotic pathway [12], we therefore tested whether the AE leaf extracts could suppress glutamate-induced apoptotic cell death by using Annexin V-FITC/PI staining and flow cytometric analysis to further confirm the neuroprotective effect of the extracts. Our results demonstrated the induction of apoptosis in HT22 cells following glutamate exposure and that the percentage of apoptotic cell death in 5 mM glutamate-treated cells was dramatically increased to approximately 40% compared to that of the control, in which the majority of apoptotic cells were in late stage (Fig. 3a). However, co-treatment of the cells with 50 μg/mL AEH or AEE could significantly reduce the apoptotic rate of glutamate-treated cells to an extent comparable to that observed in the control cells (Fig. 3b), indicating the cytoprotective and antiapoptotic effects of AE leaf extracts against glutamate toxicity in neurons.

Previous studies have demonstrated that the main mechanism of glutamate-induced apoptotic cell death in HT22 cells is mediated by AIF translocation into the nucleus [35]. Thus, we performed immunocytochemistry and Western blot analysis to determine the effect of AE leaf extract on the subcellular distribution of AIF. Our results revealed that AIF proteins, which are mainly distributed throughout the cytosol under control conditions, were translocated into neuronal nuclei of the HT22 cells following treatment with 5 mM glutamate (Fig. 4a). Moreover, the AIF expression detected by Western blotting was found to be significantly increased in the nucleus but decreased in the cytoplasm (Fig. 4b), confirming that glutamate caused the nuclear translocation of AIF. However, exposure of glutamate-treated cells to 50 μg/mL of AEE significantly restored both nuclear and cytoplasmic expression of AIF proteins to a level similar to those of untreated cells (Fig. 4a and b). These results suggest that the protective effect of AE leaf extract against neuronal cell death may be mediated by the inhibition of glutamate-induced translocation of AIF to the nucleus.

We further explored the mechanism by which AE leaf extracts protected neurons from glutamate-induced cytotoxicity. Since enhanced oxidative stress has been considered a pivotal mechanism underlying the neurotoxic action of glutamate and it is well known that increases in oxygen free radical formation trigger an AIF-mediated pathway of apoptotic cell death [14], we thus investigated whether the AE leaf extracts could attenuate ROS accumulation induced by glutamate and evaluated the antioxidant activities of the extracts in vitro. The results of the DCFH-DA assay showed that 5 mM glutamate treatment caused significantly increased intracellular ROS formation in HT22 cells, as represented by an approximately twofold higher DCF-derived fluorescence intensity relative to untreated cells (Fig. 5a and b). However, co-treatment of the cells with 50 μg/mL of either AEH or AEE was able to restore ROS production in glutamate-treated cells to a level comparable to that observed in the control cells in a dose-dependent manner (Fig. 5b). In contrast, the DPPH and ABTS assays revealed different antioxidant activities of the extracts, as shown in Table 3. The AEE exhibited a much greater capacity for radical scavenging than AEH. These data demonstrate that AE leaf extracts possess antioxidant properties and are capable of attenuating glutamate-mediated neuronal death, possibly by lowering ROS production.

We further elucidated the mechanism of AE leaf extract in antioxidant-mediated neuroprotection against glutamate-induced toxicity. It is well known that the induction of the Nrf2/antioxidant response element (ARE) signaling pathway is a major mechanism of cellular protection against oxidative stress by controlling the expression of antioxidant-related genes whose protein products are involved in the elimination of free radicals [36, 37]. Therefore, we examined the effect of AE leaf extracts on the Nrf2 signaling pathway by using real-time reverse transcription (RT) PCR and Western blot analysis. Our results revealed that 50 μg/mL AEE significantly increased the mRNA expression levels of antioxidant-related genes under Nrf2 regulation, including excitatory amino acid transporter 3 (EAAT3), NAD(P)H:quinone oxidoreductase (Nqo1), and glutamate-cysteine ligase modifier subunit (Gclm), by approximately 2- to 4-fold over controls and samples treated with 5 mM glutamate, whereas exposure to glutamate and AEH or to glutamate alone did not result in a significant difference in their expression (Fig. 6a). These findings were also correlated with a significant increase in protein expression of EAAT3, which was confirmed by Western blots of AEE-treated cells (Fig. 6b). Thus, we next investigated whether AEE could activate transcription factor Nrf2. We found that AEE treatment caused rapid Nrf2 accumulation in the nucleus of glutamate-exposed HT22 cells, while there was no alteration in the cytoplasmic level. After an hour-long exposure of HT22 cells to 5 mM glutamate and 50 μg/mL of AEE, the nuclear Nrf2 level was significantly elevated by 3- and 2-fold the level of the control and glutamate alone groups, respectively (Fig. 6c), indicating activation of Nrf2 by AEE. Taken together, the above results suggest that AE leaf extract protected neurons from the cytotoxic effect of glutamate, possibly by effective activation of transcription factor Nrf2, promoting the expression of downstream antioxidant-related genes of the Nrf2/ARE signaling pathway.