Introduction
Alzheimer disease (AD) is a debilitating neurodegenerative disorder that primarily affects the elderly population. It is the most common cause of dementia, accounting for approximately 60%–80% of all dementia cases worldwide [1-3]. The hallmark neuropathological features of AD include accumulation of beta-amyloid (Aβ) plaques and neurofibrillary tangles in the brain [4-6]. Aβ-induced neuronal cell death plays a crucial role in the pathogenesis of AD [7].
Currently available treatments for AD, including acetylcholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists, provide only modest symptomatic relief and do not address the underlying causes of AD or prevent/slow down neurodegenerative process [8-10]. No disease-modifying treatment for AD has been approved to date. Developing disease-modifying therapeutics, which can slow, prevent, or reverse neurodegenerative process, is a significant challenge [11,12]. Despite extensive research efforts to address these challenges in AD treatment, effective therapeutic interventions for AD remain limited. Therefore, there is a growing interest in exploring natural compounds with neuroprotective properties as potential candidates for AD treatment [13,14].
Thyme (Thymus spp.) is an aromatic herb widely used in traditional medicine for its various health benefits [15-17]. Thyme essential oil (EO), derived from the leaves and flowers of Thymus spp. plants, is known to contain bioactive components, including thymol, carvacrol, rosmarinic acid, and flavonoids [18,19]. These constituents exhibit diverse pharmacological activities, such as antimicrobial, antioxidant, anti-inflammatory, and protective effects, under a range of pathophysiological conditions, including respiratory symptoms, infections, arthritis, cancers, and cardiovascular diseases [6,19-23]. In addition, several studies have highlighted the potential neuroprotective properties of thyme EO in different neurodegenerative disorders [13]. However, therapeutic effect of thyme EO against Aβ-induced neuronal cell death, a key event in AD pathogenesis, has not been extensively investigated. Understanding the impact of thyme EO on Aβ-induced neuronal cell death and elucidating the underlying molecular mechanisms could provide valuable insights into its therapeutic potential for AD treatment.
In vitro cellular models provide valuable tools for investigating the mechanisms underlying neurodegenerative diseases and evaluating the potential of natural compounds as therapeutic agents. The SH-SY5Y human neuroblastoma cell line has been widely used as a cellular model to study AD-related pathologies due to its ability to differentiate into neuronal-like phenotype and respond to Aβ-induced cytotoxicity [24,25]. Given the potential neuroprotective effects of thyme EO and its constituent compounds, there is a need to explore its impact on Aβ-induced neuronal cell death using cellular models [5]. Therefore, this study aims to investigate the effects of thyme EO on Aβ-induced neuronal cell death in SH-SY5Y cells.
In this study, we aimed to elucidate the potential mechanisms underlying the neuroprotective effects of thyme EO by evaluating cell viability, oxidative stress markers, apoptotic pathways, and cellular adaptive responses.
The findings of this study may contribute to the growing body of evidence supporting the use of natural compounds as alternative or adjunctive therapies for neurodegenerative disorders. Moreover, it may shed light on the specific mechanisms through which thyme EO exerts its neuroprotective effects, potentially paving the way for the development of novel therapeutic strategies for AD.
Methods
Materials
Thyme EO (Aromatics), Aβ25–35, 3-(4,5-dimetyhylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT), anti-actin antibody, and other chemicals were purchased from Sigma Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin antibiotics were obtained from BRL. Dichlorofluorescein diacetate (DCF-DA) was purchased from Invitrogen.
Primary antibodies against cleaved caspase-3 and superoxide dismutase 1 (SOD1) were purchased from Cell Signaling Technology. Primary antibody against phosphorylated nuclear factor erythroid 2-related factor 2 (p-Nrf2) was obtained from Abcam and antibodies against Nrf2, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were supplied by Santa Cruz Biotechnology. Anti-heme oxygenase 1 (HO-1) antibody was obtained from Enzo Life Sciences.
Cell culture
SH-SY5Y human neuroblastoma cells were cultured in DMEM supplemented with 10% heat-inactivated FBS, penicillin (100 U/mL), and streptomycin (100 U/mL). The cells were incubated in a humidified incubator at 5% CO2 and 37℃. The cells were grown in 100 psi for one day (24 hours) in the presence or absence of thyme EO (50, 100, or 200 μg/mL). The cells were then plated at an appropriate density according to each experimental scale. For treatment with Aβ25–35, the cells were switched to serum-free medium and incubated in the presence or absence of Aβ25–35 for indicated durations.
MTT reduction assay
MTT reduction method was used for the measurement of cell viability. The cells were treated with vehicle or thyme EO (50, 100, or 200 μg/mL) for 24 hours and seeded at density of 8 × 104 cells/300 μL in a 48-well plate. When the cells were stably attached after 24 hours, the existing medium was replaced with serum-free medium in the presence or absence of Aβ25–35 (10 μM). After incubation for 22 hours, MTT solution was added and the cells were incubated for an additional 2 hours. Formazan crystals formed in living cells were dissolved using dimethyl sulfoxide (DMSO), and optical density at 540 nm was measured using a microplate reader (Molecular Device, LLC., San Jose, CA, USA). Relative cell viability (%) was calculated based on the absorbance of the vehicle-treated control group as 100%.
Western blot analysis
The expression levels of proteins were measured by western blot analysis. Protein samples were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich) on ice. After centrifugation at 14,000 × g for 15 minutes, the supernatant was transferred into a fresh tube. Protein concentrations were determined by the bicinchoninic acid protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates (25 μg protein) were separated by 10%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) at 320 mA for 2 hours. The membranes were blocked by phosphate-buffered saline (PBS) with 0.2% Tween 20 (PBST) containing 5% non-fat milk for 1 hour at room temperature. After three times washing with PBST, the membranes were incubated with primary antibodies in PBS containing 3% non-fat milk at 4℃ overnight. After three times washing with PBST, the membranes were reacted with horseradish peroxidase-conjugated anti-rabbit (Sigma-Aldrich) or anti-mouse secondary antibody (Santa Cruz Biotechnology Inc.). Specific bands were detected by enhanced chemiluminescence western blotting detection reagent (Thermo Fisher Scientific), and the images were obtained using an image analyzer, ImageQuant LAS 4000 mini (GE Healthcare Life Sciences, Marlborough, MA, USA).
Measurement of apoptosis
To measure apoptosis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was conducted using In Situ Cell Death Detection Kit (Roche Diagnostics GmbH). SH-SY5Y cells were cultured at a density of 1 × 105 cells/400 μL on a 4-well chamber slide and treated with Aβ25–35 for 24 hours in the presence or absence of thyme EO. After treatment, the cells were fixed in 4% paraformaldehyde in PBS pH 7.4 and then incubated with 3% H2O2 in methanol for 10 minutes at room temperature. The cells were incubated in 0.1% Triton X-100 in 0.1% sodium citrate for 2 minutes on ice and then reacted with TUNEL reaction mixture for 60 minutes at 37°C according to the manufacturer’s protocol. Anti-fluorescein antibody (converter-POD) and 3,3-diaminobenzidine (Vector Lab) were added for 10 minutes to visualize TUNEL-positive cells. Apoptotic cells were analyzed under a light microscope (Leica Co., Welzlar, Germany).
Measurement of reactive oxygen species
SH-SY5Y cells were plated at a density of 6 × 104 cells/300 μL in a 48-well plate or 1 × 105 cells/500 μL on a 4-well chamber slide and treated with Aβ25–35 in the presence or absence of thyme EO. After 6 hours of treatment, the cells were incubated with 15 μM DCF-DA for 15 minutes at 37°C. For cells incubated in 48-well plate, the cells were washed with PBS and solubilized in DMSO, and the relative fluorescence intensity was measured using a microplate reader with excitation at 485 nm and emission at 535 nm. For cells incubated on 4-well chamber slide, after 15 minutes of incubation with DCF-DA, the images of the cells were monitored using a fluorescence microscope (Leica Co.).
Results
Effect of thyme essential oil on Aβ25-35-induced cytotoxicity in SH-SY5Y cells
To evaluate the neuroprotective effect of thyme EO against Aβ25–35-induced cytotoxicity, SH-SY5Y cells were pretreated with thyme EO (50, 100, or 200 μg/mL) for 1 hour, followed by exposure to Aβ25–35 (10 μM) for 24 hours. MTT assay results (Fig. 1A) revealed a significant decrease in cell viability to 40.9% ± 2.7% in Aβ-treated cells compared with the control (p < 0.01). Pretreatment with thyme EO significantly restored viability to 49.6% ± 3.1%, 53.1% ± 2.9%, and 52.0% ± 2.5% at concentrations of 50, 100, and 200 μg/mL, respectively (##p < 0.01 vs. Aβ-treated group). Thyme EO alone showed no cytotoxic effect at these concentrations.
To assess apoptosis, TUNEL staining was performed (Fig. 1B). Aβ treatment significantly increased the percentage of TUNEL-positive cells to 35.8% ± 3.5% compared with 5.2% ± 1.4% in the control group (p < 0.01). Thyme EO pretreatment markedly reduced TUNEL-positive cells in a dose-dependent manner: 26.7% ± 2.9% at 50 μg/mL, 19.3% ± 2.1% at 100 μg/mL, and 13.6% ± 1.8% at 200 μg/mL (##p < 0.01 vs. Aβ-treated group). These results demonstrate that thyme EO effectively alleviates Aβ-induced cytotoxicity and apoptosis in neuronal cells.
Effect of thyme essential oil on Aβ25-35-induced apoptotic signals in SH-SY5Y cells
To elucidate the anti-apoptotic effects of thyme EO, we examined the expression of apoptosis-related proteins in SH-SY5Y cells exposed to Aβ25–35. As shown in Fig. 2A, Aβ treatment markedly increased the Bax/Bcl-2 ratio to 1.79 ± 0.14 compared with the control group (p < 0.01), indicating enhanced pro-apoptotic signaling. Pretreatment with thyme EO significantly reduced the Bax/Bcl-2 ratio in a dose-dependent manner: 1.68 ± 0.11 at 50 μg/mL, 1.11 ± 0.09 at 100 μg/mL, and 0.59 ± 0.07 at 200 μg/mL (##p < 0.01 vs. Aβ group at 100 and 200 μg/mL).
Similarly, Aβ exposure increased the level of cleaved caspase-3, a key executioner of apoptosis, to 1.43 ± 0.12-fold relative to the control (p < 0.01; Fig. 2B). This induction was significantly reduced by thyme EO pretreatment: 1.28 ± 0.10 at 50 μg/mL, 1.25 ± 0.08 at 100 μg/mL, and 1.12 ± 0.07 at 200 μg/mL (#p < 0.05 vs. Aβ group at 200 μg/mL). These results suggest that thyme EO suppresses Aβ-induced apoptotic signaling by restoring the balance of pro- and anti-apoptotic proteins and limiting caspase activation.
Effect of thyme essential oil on Aβ25-35-induced accumulation of intracellular reactive oxygen species in SH-SY5Y cells
To investigate whether thyme EO exerts antioxidative effects in Aβ25–35-induced oxidative stress, accumulation of intracellular reactive oxygen species (ROS) was assessed using the DCF-DA fluorescence assay. As shown in Fig. 3A, SH-SY5Y cells exposed to Aβ25–35 (10 μM) displayed a substantial increase in green fluorescence intensity, indicating elevated ROS levels compared with the control group. Pretreatment with thyme EO notably attenuated DCF fluorescence in a concentration-dependent manner, with markedly reduced signal observed in the 200 μg/mL group.
Quantitative analysis of DCF-DA fluorescence intensity (Fig. 3B) revealed approximately 6.8-fold increase in ROS production in Aβ-treated cells relative to the control. This Aβ-induced ROS generation was significantly suppressed by thyme EO pretreatment: approximately 3.4-fold, 2.1-fold, and 1.7-fold at 50, 100, and 200 μg/mL, respectively. These findings indicate that thyme EO effectively reduces intracellular oxidative stress triggered by Aβ exposure, suggesting a potent antioxidant function that may contribute to its neuroprotective mechanism.
Effect of thyme essential oil on phosphorylation of redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 in Aβ25-35-treated SH-SY5Y cells
To examine whether thyme EO activates antioxidant signaling pathways, we assessed the phosphorylation status of Nrf2, a key redox-sensitive transcription factor. As shown in Fig. 4A, treatment with Aβ25–35 alone did not significantly alter the levels of total Nrf2 and p-Nrf2 compared with the control group. However, pretreatment with thyme EO at concentrations of 50, 100, and 200 μg/mL resulted in a dose-dependent increase in p-Nrf2 levels.
Quantitative analysis of the p-Nrf2/Nrf2 ratio (Fig. 4B) revealed a significant elevation in response to thyme EO treatment, with values increasing to 1.33 ± 0.09 at 100 μg/mL (#p < 0.05) and 1.47 ± 0.13 at 200 μg/mL (##p < 0.01) compared with the Aβ-only treatment group. These results indicate that thyme EO enhances Nrf2 activation through phosphorylation, suggesting that its antioxidative effect may be mediated via Nrf2 signaling. This activation may serve as an upstream event leading to the induction of downstream antioxidant defenses.
Thyme essential oil-induced upregulation of antioxidant enzymes in SH-SY5Y cells
To further elucidate the antioxidant mechanism of thyme EO, the expression levels of downstream antioxidant enzymes—zinc-containing superoxide dismutase (ZnSOD) and HO-1—were analyzed in Aβ25–35-exposed SH-SY5Y cells with or without thyme EO. As shown in Fig. 5A, Aβ treatment alone significantly reduced ZnSOD protein levels to 0.72 ± 0.05-fold relative to the control (*p < 0.05). Pretreatment with thyme EO reversed this suppression in a concentration-dependent manner, restoring ZnSOD expression to 0.84 ± 0.06 (50 μg/mL), 0.92 ± 0.05 (100 μg/mL), and 1.14 ± 0.07-fold (200 μg/mL; #p < 0.05 vs. Aβ group).
Similarly, HO-1 expression was downregulated following Aβ exposure (0.66 ± 0.04-fold vs. control; Fig. 5B). Thyme EO treatment significantly elevated HO-1 expression to 1.15 ± 0.06 (50 μg/mL), 1.18 ± 0.05 (100 μg/mL), and 1.29 ± 0.06-fold (200 μg/mL), with statistical significance observed at all concentrations (#p < 0.05, ##p < 0.01 vs. Aβ group). These data indicate that thyme EO enhances cellular antioxidant capacity by upregulating Nrf2-dependent enzymes ZnSOD and HO-1, supporting its role in counteracting Aβ-induced oxidative stress.
Discussion
The present study investigated the potential protective effects of thyme EO on Aβ-induced neuronal cell death in SH-SY5Y cells. Our findings indicate that treatment with thyme EO significantly attenuated Aβ-induced neuronal cell death in the cells. MTT reduction assay demonstrated that thyme EO effectively protected the cells against cytotoxic effects of Aβ, evidenced by the increased cell viability compared with the Aβ-treated group. These results suggest that thyme EO possesses neuroprotective properties against Aβ-induced neurotoxicity.
The observed neuroprotective effect of thyme EO can potentially be attributed to its anti-apoptotic properties. Thyme EO has been reported to exhibit anti-apoptotic effects in various cellular and animal models. Apoptosis or programmed cell death plays a crucial role in maintaining tissue homeostasis and eliminating damaged or unwanted cells. Dysregulation of apoptosis can contribute to the development and progression of various diseases, including neurodegenerative disorders, cardiovascular diseases, and cancer. Studies have demonstrated that thyme EO can modulate key molecular pathways involved in apoptosis regulation, including the inhibition of pro-apoptotic proteins, activation of anti-apoptotic factors, and modulation of apoptotic signaling cascades. These findings suggest that thyme EO has the potential to interfere with apoptotic pathways by reducing DNA fragmentation observed in TUNEL assay, restoring the Bax/Bcl-2 ratio, and suppressing cleavage of caspase-3, thereby promoting cell survival and protecting against apoptosis-induced tissue damage. However, further research is needed to elucidate specific mechanisms by which thyme EO exerts its anti-apoptotic effect and explore its therapeutic potential in various diseases.
Furthermore, the antioxidant effect of thyme EO may have contributed to its neuroprotective effect. ROS generated by Aβ contribute to oxidative stress, leading to neuronal damage and cell death. Thyme EO contains several bioactive compounds, including thymol and carvacrol, which are known for their antioxidant activities [26,27]. These compounds may scavenge ROS and inhibit lipid peroxidation, thereby reducing oxidative damage and promoting cell survival. In the present study, pretreatment with thyme EO significantly reduced Aβ-induced intracellular accumulation of ROS. This finding is in accordance with a study that showed that thyme EO upregulated the antioxidant enzymes peroxidase, catalase, and SOD in lipopolysaccharide-activated macrophages [21].
Notably, the major bioactive constituents of thyme EO, particularly thymol and carvacrol, have been independently reported to exhibit neuroprotective effects in various AD models. For instance, Azizi et al. [28] demonstrated that thymol and carvacrol significantly improved learning and memory deficits and reduced neuronal degeneration in Aβ25–35-infused rats, and the effects were attributed to modulation of cholinesterase activity, oxidative stress, and inflammation. Similarly, Celik Topkara et al. [29] reported that carvacrol treatment attenuated Aβ-induced toxicity in SH-SY5Y cells and improved behavioral performance in mice, suggesting its in vivo neuroprotective potential. These findings reinforce the hypothesis that thymol and carvacrol, as principal constituents of thyme EO, contribute significantly to the observed neuroprotection in Aβ-exposed neuronal cells.
In this study, Aβ25–35 was selected as a neurotoxic agent to mimic key pathological features of AD. The use of Aβ25–35 is supported by its well-documented aggregation behavior and biological activity. As the shortest fragment that retains neurotoxic properties, Aβ25–35 rapidly forms β-sheet-rich aggregates under physiological conditions. Millucci et al. [30] showed that Aβ25–35 spontaneously aggregates in aqueous solution at pH 7.4, producing stable fibrils within minutes. Aβ25–35-derived aggregates induce cytotoxicity in various cell types, reflecting the pathological effects of full-length Aβ. Additionally, its presence in senile plaques and degenerating neurons in AD brains reinforces its translational value. With rapid aggregation, consistent toxicity, and experimental simplicity, Aβ25–35 is a robust in vitro model for Aβ-induced neurodegeneration.
Notably, the exact mechanisms underlying the neuroprotective effect of thyme EO in the context of Aβ-induced toxicity are multifaceted and warrant further investigation. Future studies should focus on elucidating the specific molecular pathways involved, including modulation of signaling cascades, gene expression patterns, and apoptotic pathways. Additionally, identification and quantification of bioactive compounds present in thyme EO responsible for the observed effects would provide valuable insights into its potential therapeutic applications.
Thyme EO’s activation of the Nrf2 pathway may be mediated by several upstream signaling mechanisms. Nrf2 is a redox-sensitive transcription factor whose activity is tightly regulated by intracellular signaling pathways, including MAPKs, PI3K/Akt, and ROS levels. Components of thyme EO—most notably thymol and carvacrol—have been reported to activate Nrf2 through these signaling axes. One proposed mechanism involves electrophilic modification of cysteine residues in Keap1, which leads to Nrf2 stabilization and nuclear translocation [31]. In addition, Nrf2 activation can be mediated by upstream kinases such as PI3K/Akt and MAPKs. While direct evidence for kinase activation by thyme EO is limited, previous reports support that such pathways may contribute to Nrf2 phosphorylation and activity [32]. A recent study also suggests that carvacrol acetate disrupts the Keap1-Nrf2 complex, promoting antioxidant gene expression and mitophagy in neuronal models [33]. Further studies using pharmacological and genetic tools are needed to dissect these mechanisms.
Although our findings highlight the potential of thyme EO as a neuroprotective agent against Aβ-induced neuronal cell death in a cellular model of AD, further studies are required to validate these results in animal models and ultimately in clinical trials. To this end, we have initiated preliminary in vivo experiments using a scopolamine-induced amnesia mouse model. In our pilot study, intraperitoneal administration of thyme EO significantly improved memory performance in the Y-maze, passive avoidance, and fear conditioning tests (data not shown). These initial results suggest that the neuroprotective effect of thyme EO may extend to behavioral and cognitive functions in vivo, warranting further investigation in transgenic models of AD.
In conclusion, this study provides evidence that thyme EO exhibits neuroprotective effect against Aβ-induced neuronal cell death in SH-SY5Y cells. The observed benefits may be attributed to its antioxidant and anti-apoptotic properties and potentiation of cellular adaptive response, which provide cellular resistance and resilience against oxidative stress. Thyme EO holds promise as a natural therapeutic agent for AD; however, further research is necessary to fully understand its mechanisms of action and evaluate its potential clinical applications in the management of AD.
PDF Links
PubReader
ePub Link
XML Download
Full text via DOI
Download Citation
