Neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), are primarily characterized by progressive neuronal damage and cell death, resulting in clinical manifestations such as movement disorders, cognitive decline, and behavioral abnormalities (Cho et al., 2024; Vongthip et al., 2024). Glutamate, the primary excitatory neurotransmitter within the central nervous system (CNS), is fundamental to neuronal viability and normal brain function (Esposito et al., 2013; Al-Nasser et al., 2022). However, pathologic accumulation of glutamate, frequently observed during the neurodegenerative disease progression, can induce oxidative stress and excitotoxicity, ultimately leading to neuronal death (Sheldon and Robinson, 2007; Song et al., 2019). Neurons, particularly hippocampal cells, are especially prone to producing reactive oxygen species (ROS) and are highly vulnerable to redox imbalances (Abbah et al., 2022), suggesting that suppression of glutamate-induced oxidative stress, such as the use of antioxidants, could be a promising strategy for the prevention and treatment of neurodegenerative disorders (Lee et al., 2021).

Numerous studies on the brain’s defense mechanisms against oxidative stress have demonstrated that Brain-Derived Neurotrophic Factor (BDNF) and Nuclear Factor Erythroid 2–Related Factor 2 (Nrf2) are critical signaling molecules in this protective process (Johnson et al., 2008; Yan et al., 2021). BDNF, which promotes neuronal survival and growth, is released within the CNS and exerts its effects through binding to its receptor, tropomyosin-related kinase receptor B (TrkB) (Vilar and Mira, 2016; Li et al., 2022). Elevated BDNF expression has been shown to alleviate the risk of neurodegenerative diseases by protecting neurons from oxidative stress (Gao et al., 2022). In addition, Nrf2 defends against oxidative stress by regulating the expression of various antioxidant enzymes, such as glutathione peroxidase and heme oxygenase-1 (HO-1) (Alonso-Piñeiro et al., 2021), involved in the cellular defense response. Several studies have demonstrated that the upregulation of HO-1 effectively attenuates glutamate-induced oxidative stress in HT22 cells, underscoring the neuroprotective role of Nrf2 signaling (Kim et al., 2012; Tang et al., 2015).

β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione; β-Lap), a natural naphthoquinone isolated from the bark of the lapacho tree (Tabebuia avellanedae) (Gomes et al., 2021), has been noted for its anti-cancer, anti-bacterial, and anti-inflammatory effects (de Castro et al., 2013; Macedo et al., 2013; Sitônio et al., 2013). While previous studies have reported the neuroprotective effects of β-Lap in several in vivo models of neurodegenerative diseases, (Lee et al., 2018b; Park et al., 2019; Mokarizadeh et al., 2020), its precise mechanisms of action remain to be fully clarified. The HT22 mouse hippocampal neuronal cell line, known for its high susceptibility to oxidative stress, is widely recognized as a valuable in vitro model for exploring glutamate-induced neurotoxicity associated with neurodegenerative diseases (Prasansuklab et al., 2023). In this study, we utilized this cell line to investigate whether β-Lap exerts a neuroprotective effect against glutamate-induced oxidative damage and, if so, to elucidate the underlying mechanisms of its neuroprotective effect.

Reagents

β-Lap, L-Glutamic acid (Glutamate), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide (ANA-12) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Welgene (Gyeongsan, Korea). Fetal bovine serum (FBS) and Penicillin Streptomycin were purchased from Gibco (Grand Island, NY, USA). 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Tocris Bioscience (Bristol, UK). Anti-BDNF, anti-TrkB, anti-phospho-extracellular signal-regulated kinase (ERK), anti-ERK, anti-phospho-cAMP response element-binding protein (CREB), anti-CREB, anti-HO-1, and U0126 were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-phospho-TrkB was brought from Abcam, Inc. (Cambridge, UK). Anti-Nrf2 and anti-Lamin B1 were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-β-actin was obtained from Bioss (Boston, MA, USA).

Cell culture

The HT22 cells, a mouse hippocampus-derived neuronal cell line, were purchased from Merck (Darmstadt, Hesse, Germany). HT22 cells were cultured in high-glucose DMEM with 10% heat-inactivated FBS and 10,000 U/mL penicillin-streptomycin. The cells were incubated at 37°C in 5% CO₂ atmosphere, with the media was replaced every one to two days. Cells of passages 3 to 10 were utilized in all experiments.

Cell viability assay

Cell viability was measured by MTT assay. HT22 cells were seeded at 5×10³ cells/well in 96-well plates and incubated for 24 h. Afterward, the cells were treated with or without β-Lap (1, 3, 10 nM) for 2 h. Glutamate (7.5 mM) was then added and incubated for an additional 24 h. MTT reagent (5 mg/mL) was added and incubated for 3-4 h, followed by the addition of dimethyl sulfoxide (DMSO) to dissolve the formazan crystals. Absorbance was measured at 540 nm using a Bio-Tek microplate reader (Winooski, VT, USA).

DPPH radical-scavenging assay

The reduction of DPPH radicals was assessed to confirm the antioxidant activity of β-Lap as previously described (Prasansuklab and Tencomnao, 2018). In a 96 well plate, 100 µL of varying concentration of β-Lap (1, 3, 10, and 30 nM) were combined with 100 µL of 250 µM DPPH solution. After a 30 min reaction in the dark, the absorbance at 517 nm was recorded using a microplate reader. The DPPH radical scavenging activity was presented as a percentage (%) and calculated using the formula below: [(A₀-A₁)/A₀]×100, A₀ denotes the control, and A₁ denotes the sample.

ABTS radical scavenging assay

The ABTS radical scavenging assay of β-Lap was measured as previously described (Prasansuklab and Tencomnao, 2018). A 14 mM ABTS solution and a 4.9 mM K₂S₃O₈ solution were dissolved in distilled water. The ABTS and K₂S₃O₈ solutions were subsequently combined at a 1:1 (v/v) ratio and left to react at room temperature in the dark for 16-18 h. The final concentrations in the ABTS working solution were 7 mM ABTS and 2.45 mM K₂S₃O₈. Prior to use, the ABTS working solution was diluted with ethanol to adjust an absorbance of 0.7 and 0.8 at 734 nm. Afterward, in a 96-well plate, 180 µL of the diluted ABTS working solution and 20 µL of β-Lap (1, 3, 10, and 30 nM) were combined and incubated at room temperature in the dark for 15 min. Absorbance was recorded at 734 nm using a microplate reader. The ABTS radical scavenging activity was presented as a percentage (%) and calculated using the formula below: [(A₀-A₁)/A₀]×100, A₀ denotes the control, and A₁ denotes the sample.

Measurement of reactive oxygen species

ROS generation was measured using the fluorescent dye H₂DCFDA. HT22 cells were plated at 2.5×10⁵ cells/well in a 6 well plate and incubated for 24 h. Following pre-treatment with β-Lap (1, 3, and 10 nM) and Trolox (50 μM) for 2 h, the cells were exposed to glutamate (7.5 mM) for 8 h. The cells were subsequently incubated with 10 µM H₂DCFDA at 37°C in the dark for 10 min. After incubation, the cells were washed with DPBS, suspended, and analyzed using a BD FACSAria^TM III flow cytometer (San Jose, CA, USA). Fluorescence intensity was measured at an excitation wavelength of 488 nm.

Estimation of intracellular glutathione

Glutathione (GSH) levels were determined with a Cayman Chemical Glutathione Assay Kit (Ann Arbor, MI, USA) using the DTNB method. The cell lysate was obtained as follows. HT22 cells were plated in a 60 mm dish at 3×10⁵ cells/dish and incubated for 24 h. After incubation, the cells were pre-treated with β-Lap (1, 3, and 10 nM) and Trolox (50 μM) for 2 h, then treated with glutamate (7.5 mM) for an additional 24 h. Subsequently, the cells were washed with DPBS and harvested using a scraper. The cell pellets were collected and suspended in DPBS supplemented with 1 mM EDTA, and the cell samples were centrifuged (10,000 xg, 15 min, 4°C). The cell samples were loaded into a 96-well plate, followed by the addition of the Assay cocktail. After 25 min of incubation on an orbital shaker, absorbance was recorded at 405 nm using a microplate reader.

Nuclear and cytosolic protein extraction

Using Thermo NE-PER Nuclear and Cytoplasmic Extraction Reagents (Waltham, MA, USA), nuclear and cytosolic proteins were extracted from β-Lap-treated HT22 cells.

Western blot analysis

HT22 cells were lysed in RIPA buffer, and the cell lysates were centrifuged (14,000 RPM, 15 min, 4°C). Protein concentrations were quantified with the BCA assay. For protein isolation, samples were loaded onto SDS-PAGE gels and subjected to electrophoresis. The proteins within the gel were then transferred onto polyvinylidene fluoride (PVDF) membranes and blocked in Tris-buffered saline (TBS) with 10% skim milk. The membranes were incubated with primary antibodies at 4°C overnight, including BDNF (1:1000), p-TrkB (1:700), TrkB (1:1000), p-ERK (1:1000), ERK (1:1000), p-CREB (1:1000), CREB (1:1000), Nrf2 (1:500), HO-1 (1:1000), Lamin B1 (1:200), β-actin (1:5000). Following this, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Protein bands were visualized using the Cytiva ECL Detection Reagent (Marlborough, MA, USA) and analyzed with the Amersham^TM ImageQuant 800 (Cytiva Life Sciences).

Statistical analysis

All data are provided as mean ± standard error of the mean (SEM). Statistical analysis and visualization were conducted using GraphPad Prism 8.0.2. One-way analysis of variance (ANOVA) with Tukey’s post hoc test and an Unpaired Student’s t-test were performed for statistical analysis. A p-value of less than 0.05 was considered statistically significant.

β-Lap exerts a neuroprotective effect against glutamate-induced cytotoxicity in HT22 cells

To explore the neuroprotective effect of β-Lap against glutamate-induced cytotoxicity, we assessed cell viability. As shown in Fig. 1A and 1B, HT22 cells treated with glutamate exhibited significantly reduced cell viability (39.40 ± 3.00%) compared to control. This reduction in cell viability was significantly improved by treatment with β-Lap at 3 nM (76.98 ± 1.80%) and 10 nM (81.20 ± 0.98%), demonstrating a dose-dependent effect.

Figure 1. Neuroprotective effect of β-Lap on glutamate-mediated cytotoxicity in HT22 cells. HT22 cells were pretreated with β-Lap (1, 3, and 10 nM) for 2 h, followed by exposure to glutamate (7.5 mM) for 24 h. (A) Morphology of cells (Scale bar is 50 μm). (B) Cell viability assessed through the MTT assay. Data are presented as mean ± SEM (n=8-12). *p<0.05 vs CTL, ^#p<0.05 vs Glu alone. CTL, control; Glu, glutamate; β-Lap, β-lapachone.

β-Lap exhibits antioxidant effects in glutamate-exposed HT22 cells

To investigate the underlying mechanism of β-Lap’s neuroprotective effect against glutamate-induced injury, we examined its antioxidant potential by measuring intracellular ROS and GSH levels in glutamate-exposed HT22 cells. Exposure of HT22 cells to 7.5 mM glutamate significantly elevated intracellular ROS levels to 202.35 ± 5.02% compared to the control group. This glutamate-induced increase in ROS levels was significantly attenuated by β-Lap treatment at 3 nM (142.46 ± 1.20%) and 10 nM (88.82 ± 7.06%) in a dose-dependent manner. Trolox, a well-known antioxidant, also reduced ROS levels (106.59 ± 7.68%). The GSH level was remarkably decreased in glutamate-exposed cells (2.06 ± 0.17 μM) compared to control (5.45 ± 0.07 μM), and this decrease was significantly reversed by β-Lap at 3 nM (3.95 ± 0.20 μM) and 10 nM (4.77 ± 0.25 μM), and Trolox at 50 μM (5.32 ± 0.16 μM). In addition, the cell viability of glutamate-exposed HT22 cells declined to 42.82 ± 1.61%, whereas treatment with β-Lap at concentrations of 3 nM (80.47 ± 2.27%) and 10 nM (84.81 ± 2.06%) significantly restored cell viability. Likewise, Trolox (81.03 ± 1.88%) effectively counteracted the reduced cell viability in glutamate-treated HT22 cells (Fig. 2). These results suggest that the neuroprotective mechanism of β-Lap may involve its antioxidant properties.

Figure 2. Antioxidant effects of β-Lap in glutamate-treated HT22 cells. HT22 cells were pretreated with β-Lap (1, 3, and 10 nM) or Trolox (50 μM) for 2 h prior to glutamate (7.5 mM) exposure. (A) H₂DCFDA fluorescence intensity. (B) GSH levels. (C) Cell viability assessed through the MTT assay. Data are presented as mean ± SEM (ROS and GSH, n=3-5; MTT, n=8-12). *p<0.05 vs CTL, ^#p<0.05 vs Glu alone. CTL, control; Glu, glutamate; β-Lap, β-lapachone; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; GSH, glutathione.

β-Lap exerts in vitro antioxidant activity

To investigate the underlying mechanism involved in the neuroprotective effect of β-Lap against glutamate-induced injury, its antioxidant capacity was evaluated using both the DPPH and ABTS radical scavenging assays. As shown in Fig. 3A, β-Lap exhibited significant DPPH radical scavenging activity in a concentration-dependent manner, with inhibition percentages as follows : 1 nM (9.52 ± 1.84%), 3 nM (41.14 ± 1.99%), 10 nM (52.51 ± 0.59%), 30 nM, (59.27 ± 0.66%). The IC₅₀ value of β-Lap was 3.23 nM. Likewise, in Fig. 3B, β-Lap demonstrated concentration-dependent scavenging activity against ABTS radicals, with inhibition percentages of 1 nM (7.94 ± 0.17%), 3 nM (44.98 ± 0.15%), 10 nM (60.26 ± 0.55%), 30 nM (75.85 ± 0.47%), and an IC₅₀ value was 6.18 nM. Trolox similarly exhibited concentration-dependent scavenging activity in both the DPPH and ABTS assay, with IC₅₀ value of 9.97 μM and 6.02 μM, respectively. These results suggest that β-Lap exhibits strong antioxidant properties.

Figure 3. In vitro antioxidant effects of β-Lap. The graphs were generated by plotting various concentrations of β-Lap (1, 3, 10, and 30 nM) and Trolox (1, 3, 10, and 30 μM) against the percentage inhibition of DPPH and ABTS radicals. (A) The DPPH radical scavenging activity of β-Lap and Trolox. (B) The ABTS radical scavenging activity of β-Lap and Trolox. Data are presented as mean ± SEM (DPPH, n=7-10; ABTS, n=6). *p<0.05 vs β-Lap 0 nM or Trolox 0 μM. β-Lap, β-lapachone; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; DPPH, 2,2-Diphenyl-1-picrylhydrazyl; ABTS, 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.

β-Lap activates the BDNF/TrkB/ERK/CREB signaling molecules

We examined the expression and phosphorylation levels of relevant proteins to determine whether the BDNF/TrkB/ERK/CREB signaling molecules contribute to the neuroprotective effects of β-Lap in HT22 cells exposed to glutamate. Glutamate treatment significantly downregulated the expression of BDNF (44.05 ± 4.01%), as well as the phosphorylation of TrkB (44.20 ± 2.95%), ERK (46.14 ± 2.22%), and CREB (25.30 ± 9.35%) relative to the control (Fig. 4). β-Lap treatment at 3 nM and 10 nM increased BDNF expression to 57.66 ± 3.83% and 79.49 ± 2.85%, respectively (Fig. 4A). Similarly, the phosphorylation of TrkB was enhanced to 67.62 ± 2.53% and 76.09 ± 0.09% at 3 nM and 10 nM of β-Lap, respectively (Fig. 4B). The phosphorylation of ERK was upregulated to 57.96 ± 3.15% and 72.82 ± 3.81% at these concentrations (Fig. 4C), while CREB phosphorylation increased to 58.53 ± 5.08% and 73.44 ± 4.41% with β-Lap treatment at 3 nM and 10 nM (Fig. 4D). These results suggest that β-Lap protects neuronal cells against glutamate-induced oxidative stress by regulating the activation of the BDNF/TrkB/ERK/CREB signaling molecules.

Figure 4. Effect of β-Lap on BDNF expression and the phosphorylation of TrkB, ERK or CREB. HT22 cells were treated with glutamate (7.5 mM) for 24 h after pre-incubation with or without β-Lap (3 and 10 nM) for 2 h. (A-D) Effects of β-Lap on the expression levels of (A) BDNF/β-actin, (B) p-TrkB/TrkB, (C) p-ERK/ERK, and (D) p-CREB/CREB in HT22 cells. BDNF expression were normalized to β-actin, while the phosphorylation levels of TrkB, ERK, CREB were normalized to their respective total protein levels. Western blot data were quantified and analyzed using a t-test. Data are presented as mean ± SEM (n=3-5). *p<0.05 vs CTL, ^#p<0.05 vs Glu alone. CTL, control; Glu, glutamate; β-Lap, β-lapachone; BDNF, brain-derived neurotrophic factor; TrkB, tropomyosin-related kinase receptor B; ERK, extracellular signal-regulated kinase; CREB, cAMP response element-binding protein.

β-Lap upregulates Nrf2 nuclear translocation and the expression of antioxidant enzyme

Following confirmation that β-Lap regulates the activation of the BDNF/TrkB/ERK/CREB signaling molecules, we further investigated β-Lap’s effects on Nrf2 nuclear translocation and HO-1 expression in HT22 cells exposed to glutamate. In glutamate-exposed cells, as shown in Fig. 5A, cytosolic Nrf2 levels increased to 156.76 ± 4.84%, while nuclear Nrf2 levels decreased to 41.16 ± 5.39% relative to the control group. However, treatment with β-Lap at 3 nM (63.86 ± 6.20%) and 10 nM (86.09 ± 4.49%) reduced cytosolic Nrf2 levels. In contrast, β-Lap at 3 nM (135.00 ± 5.41%) and 10 nM (62.47 ± 18.32%) increased nuclear Nrf2 levels. Moreover, glutamate treatment reduced HO-1 expression to 50.91 ± 1.47% relative to the control, but this reduction was restored by β-Lap at 3nM (66.74 ± 1.72%) and 10 nM (78.59 ± 1.71%) (Fig. 5B). These results suggest that β-Lap may play a crucial role in combating glutamate-induced oxidative stress in HT22 cells by promoting Nrf2 nuclear translocation and enhancing HO-1 expression.

Figure 5. Effect of β-Lap on Nrf2 nuclear translocation and the expression of HO-1. HT22 cells were treated to glutamate (7.5 mM) for 24 h following pre-incubation with or without β-Lap (3 and 10 nM) for 2 h. (A-B) Effects of β-Lap on the expression of (A) C-Nrf2/β-actin and N-Nrf2/Lamin B1, (B) HO-1/β-actin in HT22 cells. β-actin and Lamin B1 were utilized as markers for cytosolic and nuclear fractions, respectively. Western blot data were quantified and analyzed using a t-test. Data are presented as mean ± SEM (n=3-5). *p<0.05 vs CTL, ^#p<0.05 vs Glu alone. CTL, control; Glu, glutamate; β-Lap, β-lapachone; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1.

ANA-12 inhibits the neuroprotective effects of β-Lap

Subsequently, we aimed to further elucidate the mechanism by which β-Lap exerts neuroprotection via the activation of BDNF/TrkB/ERK/CREB and ERK/Nrf2/HO-1 pathways in glutamate-exposed hippocampal neurons. For this, we assessed the inhibitory effects of ANA-12, a TrkB inhibitor, on the neuroprotective effects of β-Lap. Glutamate-treated HT22 cells showed a significant reduction to 43.28 ± 1.20% in cell viability compared to control group, while β-Lap treatment at 10 nM significantly improved cell viability to 79.72 ± 1.18%. However, co-treatment with β-Lap and ANA-12 neutralized the β-Lap’s neuroprotective effect against glutamate-induced cytotoxicity (50.21 ± 2.13%) (Fig. 6A). In addition, treatment of HT22 cells with glutamate significantly elevated intracellular ROS levels to 225.61 ± 25.53% relative to the control. This elevation in ROS levels was significantly mitigated by β-Lap treatment at 10 nM (100.22 ± 10.29%). The inhibitory effect of β-Lap on intracellular ROS levels was counteracted by ANA-12 in glutamate-treated HT22 cells (210.13 ± 22.87%) (Fig. 6B). Glutamate treatment reduced BDNF expression to 38.47 ± 5.41%, along with the phosphorylation of TrkB, ERK, and CREB to 45.81 ± 1.97%, 50.72 ± 1.27%, and 37.28 ± 3.78%, respectively. Treatment with 10 nM β-Lap increased these reduced levels of BDNF expression (78.33 ± 4.00%) and the phosphorylation of TrkB (76.82 ± 3.93%), ERK(76.93 ± 2.52%), and CREB (77.45 ± 3.11%). Co-treatment with ANA-12 and β-Lap, however, resulted in a reduction of BDNF expression (57.76 ± 5.26%) and the phosphorylation of TrkB (62.53 ± 1.67%), ERK (66.65 ± 2.25%), and CREB (57.05 ± 3.33%), thereby negating the neuroprotective effects of β-Lap (Fig. 7). Moreover, as shown in Fig. 8A and Fig 8B, glutamate exposure elevated cytosolic Nrf2 levels (150.36 ± 14.27%) while decreasing nuclear Nrf2 levels (46.59 ± 2.69%) and HO-1 expression (54.80 ± 2.93%). In contrast, treatment with 10 nM β-Lap significantly lowered cytosolic Nrf2 levels to 59.92 ± 2.59% and enhanced nuclear Nrf2 levels and HO-1 expression to 77.15 ± 2.19% and 85.95 ± 3.17%, respectively. Additionally, ANA-12 inhibited the antioxidant effects of β-Lap, increasing cytosolic Nrf2 levels to 121.72 ± 5.24% and reducing the nuclear translocation of Nrf2 to 60.41 ± 2.45%, while also reducing HO-1 expression to 69.25 ± 3.92%. These results suggest that β-Lap exerts the neuroprotective effects by regulating the activation of BDNF/TrkB/ERK/CREB and ERK/Nrf2/HO-1 pathways.

Figure 6. Inhibitory effects of ANA-12 on the neuroprotective action of β-Lap. HT22 cells were pre-treated with or without β-Lap (10 nM), in combination with ANA-12 (10 μM) for 2 h prior to glutamate (7.5 mM) treatment. (A) Cell viability assessed through the MTT assay. (B) H₂DCFDA fluorescence intensity. Data are presented as mean ± SEM (MTT, n=8-12; ROS, n=3). *p<0.05 vs CTL , ^#p<0.05 vs Glu alone, ^$p<0.05 vs β-Lap treated group. CTL, control; Glu, glutamate; β-Lap, β-lapachone; ANA-12, N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide.

Figure 7. Inhibitory effect of ANA-12 on BDNF expression and the phosphorylation of TrkB, ERK, or CREB. HT22 cells were pre-treated with or without β-Lap (10 nM), in combination with ANA-12 (10 μM) for 2 h prior to glutamate (7.5 mM) treatment. (A-D) The inhibitory effects of ANA-12 on the expression of (A) BDNF/β-actin, (B) p-TrkB/TrkB, (C) p-ERK/ERK, and (D) p-CREB/CREB in HT22 cells. BDNF expression levels were normalized to β-actin, while the phosphorylation levels of TrkB, ERK, CREB were normalized to their respective total protein levels. Data are presented as mean ± SEM (n=3). *p<0.05 vs CTL, ^#p<0.05 vs Glu alone, ^$p<0.05 vs β-Lap treated group. CTL, control; Glu, glutamate; β-Lap, β-lapachone; ANA-12, N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide; BDNF, brain-derived neurotrophic factor; TrkB, tropomyosin-related kinase receptor B; ERK, extracellular signal-regulated kinase; CREB, cAMP response element-binding protein.

Figure 8. Inhibitory effect of ANA-12 on Nrf2 nuclear translocation and HO-1 expression. HT22 cells were pretreated with or without β-Lap (10 nM), in combination with ANA-12 (10 μM) for 2 h prior to glutamate (7.5 mM) treatment. (A-B) The inhibitory effects of ANA-12 on the expression levels of (A) C-Nrf2/β-actin and N-Nrf2/Lamin B1, (B) HO-1/β-actin in HT22 cells. Data are presented as mean ± SEM (n=3). *p<0.05 vs CTL, ^#p<0.05 vs Glu alone, ^$p<0.05 vs β-Lap treated group. CTL, control; Glu, glutamate; β-Lap, β-lapachone; ANA-12, N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1, heme oxygenase-1.

U0126 inhibits the neuroprotective effects of β-Lap

We confirmed the inhibitory effect of U0126, an ERK inhibitor, on the neuroprotective action of β-Lap. In HT22 cells exposed to glutamate, cell viability decreased to 50.63 ± 1.97%, however, this reduction was reversed by β-Lap treatment at 10 nM, increasing cell viability to 87.06 ± 1.86%. Co-treatment with β-Lap and U0126 neutralized the neuroprotective effects of β-Lap, reducing cell viability to 60.28 ± 2.66% (Fig. 9A). Glutamate exposure in HT22 cells elevated intracellular ROS levels to 234.03 ± 19.10% relative to the control, but treatment with 10 nM β-Lap reduced this glutamate-induced ROS increase to 95.06 ± 5.18%. However, U0126 interfered with β-Lap’s ability to reduce ROS levels, resulting in ROS levels at 186.95 ± 18.72% in oxidative stress-induced hippocampal neurons (Fig. 9B). Furthermore, as shown in Fig. 10A and 10B, glutamate exposure decreased the expression of BDNF (56.12 ± 4.92%) and HO-1 (47.05 ± 2.52%) in HT22 cells. Treatment with 10 nM β-Lap significantly reversed this decrease, increasing BDNF expression to 85.45 ± 0.48% and HO-1 expression to 81.60 ± 2.73%. However, co-treatment with β-Lap and U0126 inhibited the β-Lap-induced increase in BDNF (65.81 ± 1.85%) and HO-1 expression (62.64 ± 3.88%). These results suggest that β-Lap protects HT22 cells from oxidative toxicity by upregulating BDNF and HO-1 expression through activation of the BDNF/TrkB/ERK/CREB and ERK/Nrf2/HO-1 pathways.

Figure 9. Inhibitory effects of U0126 on the neuroprotective action of β-Lap. HT22 cells were pre-treated with or without β-Lap (10 nM), in combination with U0126 (10 μM) for 2 h before glutamate (7.5 mM) treatment. (A) Cell viability assessed through the MTT assay. (B) H₂DCFDA fluorescence intensity. Data are presented as mean ± SEM (MTT, n=8-12; ROS, n=3). *p<0.05 vs CTL , ^#p<0.05 vs Glu alone, ^$p<0.05 vs β-Lap treated group. CTL, control; Glu, glutamate; β-Lap, β-lapachone.

Figure 10. Inhibitory effects of U0126 on BDNF and HO-1 expression. HT22 cells were pre-treated with or without β-Lap (10 nM), in combination with U0126 (10 μM) for 2 h before glutamate (7.5 mM) treatment. (A-B) The inhibitory effects of U0126 on the expression levels of (A) BDNF/β-actin, (B) HO-1/β-actin. Data are presented as mean ± SEM (n=3). *p<0.05 vs CTL, ^#p<0.05 vs Glu alone, ^$p<0.05 vs β-Lap treated group. CTL, control; Glu, glutamate; β-Lap, β-lapachone; BDNF, brain-derived neurotrophic factor; HO-1, heme oxygenase-1.

In the present study, we first demonstrated the neuroprotective effects of β-Lap against glutamate-induced oxidative damage in HT22 hippocampal cells. β-Lap significantly enhanced cell viability in a dose-dependent manner and reduced ROS accumulation under glutamate-induced oxidative stress. These effects of β-Lap were diminished by treatment with ANA-12, a TrkB inhibitor, or U0126, an ERK inhibitor. Additionally, β-Lap upregulated BDNF protein expression and increased the phosphorylation of its downstream signaling molecules, such as TrkB, ERK, and CREB, and these effects of β-Lap were abolished by ANA-12. β-Lap also induced Nrf2 nuclear translocation and the protein expression of HO-1, which were reversed by ANA-12. Furthermore, the upregulation of BDNF and HO-1 expression by β-Lap was inhibited by U0126. Taken together, these findings suggest that β-Lap exerts neuroprotective effects against glutamate-induced oxidative injury by modulating the BDNF/TrkB/ERK/CREB and ERK/Nrf2/HO-1 pathways.

Glutamate-induced toxicity, a pivotal contributor to neuronal cell death in neurodegenerative diseases and acute brain injuries, is primarily driven by ROS (Song et al., 2019). Excessive glutamate depletes intracellular GSH level, compromising the antioxidant defense system, exacerbating oxidative stress, and ultimately leading to cell death (Franco and Cidlowski, 2009; Selvaraj et al., 2023). ROS play a critical role in the pathogenesis of various diseases and are recognized as key risk factors for brain damage in neurodegenerative disorders (Migliore and Coppedè, 2009). Among brain regions, the hippocampus is especially sensitive to oxidative stress due to its reduced levels of antioxidant enzymes, high metabolic activity, and abundance of polyunsaturated fatty acids, compared to other regions of the brain (Lee et al., 2018a). These factors, combined with its high density of glutamate receptors, further exacerbate its vulnerability to oxidative damage, which can impair hippocampus-dependent functions such as cognition and memory (Huang et al., 2015). Supporting this, β-Lap significantly increased cell viability in a dose-dependent manner in glutamate-exposed HT22 cells. Our study further revealed that β-Lap exhibits an antioxidant effects by lowering intracellular ROS levels and restoring GSH levels. These findings suggest that β-Lap protects neurons from glutamate-mediated oxidative stress, thereby exerting neuroprotective effects.

One of the mechanisms underlying the neuroprotective effect of β-Lap may be its association with upregulating BDNF expression through activation of the BDNF/TrkB/ERK/CREB pathway. BDNF is a crucial neurotrophin that promotes neuronal survival in response to various neuronal injuries across several CNS regions, including the hippocampus, cerebral cortex, and hypothalamus, with the highest expression levels observed in the cerebral cortex and hippocampus (Ibrahim et al., 2022; Correia et al., 2023). Dysregulation of BDNF is recognized as a key factor in the development of neurodegenerative diseases (Bathina and Das, 2015). BDNF primarily binds to the TrkB receptor and is widely recognized for its neuroprotective role, especially in the context of neurodegenerative diseases (Sampaio et al., 2017). The binding of BDNF to TrkB initiates the activation of three downstream signaling pathways: the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, and the phospholipase C γ (PLC γ) pathway (Autry and Monteggia, 2012). Among MAPKs, ERK activation protects cells against oxidative stress by promoting the phosphorylation of CREB, thereby regulating the expression of various genes critical for cell survival, growth, and repair (Zhen et al., 2023). Previous studies have shown that ERK-mediated phosphorylation regulates BDNF expression through CREB activation (Mao et al., 2015). CREB plays an essential role in promoting neuronal survival and resilience to oxidative stress (Zhi et al., 2020; Narasimhamurthy et al., 2022), and its dysregulation has been implicated in the progression of neurodegenerative disorders (Sharma and Singh, 2020). In this context, our findings indicate that β-Lap alleviates the reduction in BDNF expression levels in glutamate-exposed HT22 cells, protecting hippocampal neurons from oxidative stress-induced cytotoxicity by enhancing the phosphorylation of TrkB, ERK, and CREB. Moreover, the observation that ANA-12 suppresses the activation of these factors induced by β-Lap provides further evidence supporting the involvement of β-Lap in the BDNF/TrkB/ERK/CREB signaling pathway.

Another mechanism underlying the neuroprotective effect of β-Lap may involve the upregulation of antioxidant enzyme, such as HO-1, through activation of the ERK/Nrf2/HO-1 pathway. Nrf2 plays an integral role in maintaining cellular redox homeostasis and is essential for protecting neurons from oxidative damage (Bellezza et al., 2018). Nrf2 strengthens cellular defenses against oxidative stress by upregulating various antioxidant enzymes, including catalase, glutathione peroxidase, and HO-1 (Baek and Kim, 2020). Among these, HO-1 is a key gene that mitigates glutamate-induced oxidative stress in HT22 cells (Song et al., 2019). Accumulating evidence suggests that the activation of Nrf2 and HO-1 is an effective strategy for attenuating oxidative damage (Li et al., 2023). Furthermore, several natural antioxidant compounds have shown the potential to not only directly suppress ROS but also activate the Nrf2 pathway to enhance cellular defense mechanisms (Chiu et al., 2023; Piao et al., 2024). In particular, studies have demonstrated that ERK activation facilitates the dissociation of Nrf2 from Keap1, enabling its translocation into the nucleus and subsequent upregulation of antioxidant gene expression (Park et al., 2013). Consistent with these findings, our results demonstrated that β-Lap restored Nrf2 nuclear translocation in glutamate-exposed HT22 cells and elevated the expression of HO-1. The relationship between the BDNF/TrkB/ERK/CREB pathway and ERK/Nrf2/HO-1 pathways was confirmed through the suppression of Nrf2 nuclear translocation and HO-1 expression by ANA-12.

In conclusion, we demonstrated that β-Lap may play a crucial role in neuroprotection by activating BDNF/TrkB/ERK/CREB and ERK/Nrf2/HO-1 signaling pathways under glutamate-induced oxidative stress (Fig. 11). These findings suggest that β-Lap could serve as a promising therapeutic candidate capable of preventing neurodegenerative disease progression by protecting neuronal cells. It should be noted that further research is required to elucidate the neuroprotective effects of β-Lap in vivo.

Figure 11. A graphical abstract illustrates β-Lap’s neuroprotective mechanisms against glutamate-induced oxidative stress in HT22 cells.

This research was supported by the GRRC program of Gyeonggi province (GRRCAjou2023-B01).

The authors declare no conflict of interest.