P-Hydroxybenzaldehyde from Gastrodia elata Blume Reduces Hydroxyurea-Induced Cellular Senescent Phenotypes in Human SH-SY5Y Cells via Enhancing Autophagy

TL;DR

P-Hydroxybenzaldehyde from Gastrodia elata reduces signs of cellular aging in human cells by boosting autophagy.

Contribution

This study identifies P-HBA as a novel compound from Gastrodia elata that mitigates cellular senescence via autophagy enhancement.

Findings

TME and P-HBA reduce hydroxyurea-induced DNA damage and SA-β-Gal activity in SH-SY5Y cells.

P-HBA's anti-senescent effect is diminished by the autophagy inhibitor chloroquine.

P-HBA enhances autophagy to counteract cellular senescence.

Abstract

Background/Objectives: The rhizome of Gastrodia elata Blume (Tianma) is a functional food with medicinal value in China, used to improve the health of the central nervous system and reported to exhibit anti-cellular senescent activity. P-hydroxybenzaldehyde (P-HBA) is a key aromatic compound isolated from Tianma; however, its potential to mitigate cellular senescence remains unclear. Methods: We employed ultra-performance liquid chromatography-mass spectrometry to identify the chemical characterization of Tianma extract. Cell viability assay, senescence-associated-β-galactosidase (SA-β-Gal) assay, and immunofluorescence staining and autophagy analysis were used to evaluate the anti-senescent activity of P-HBA and other Tianma components. Results: Our findings demonstrate that Tianma methanol extract (TME) and P-HBA significantly reduce cellular senescent inducer hydroxyurea (HU)-induced DNA damage, SA-β-Gal activity increase, and autophagic dysfunction in human SH-SY5Y cells. Notably, an autophagy inhibitor, chloroquine, can reduce anti-cellular senescent activity of P-HBA. Conclusions: These results suggest that P-HBA exhibits the effect of reducing cellular senescent phenotypes, and its effect is achieved by enhancing autophagy.

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1. Introduction

The rhizome of Gastrodia elata blume (Tianma in Chinese) is a medicinal herb first recorded in Shennong Ben Cao Jing [1]. According to traditional Chinese medicine (TCM), it is believed to have health-improving properties for the central nervous system. Currently, it is used clinically for the treatment of spasms, dizziness, headache, epilepsy, migraine, amnesia, and hypertension [2]. Previous studies indicated that Tianma had potential in anti-brain aging [3,4]. Several bioactive components of Tianma have been identified, including gastrodin, p-hydroxybenzaldehyde (P-HBA), vanillin, parishin A, parishin B, and parishin C. Among them, gastrodin was found to reduce oxidative stress-induced endothelial cell senescence via activating the Nrf2 antioxidant defense system [5]. Gastrodin could also attenuate IL1-β-induced chondrocyte senescence via up-regulating SIRT3 expression [6]. However, gastrodin did not reduce BeSO_4_ and D-galactose (D-gal)-induced SH-SY5Y cell senescence [3]. Other active components in Tianma may also have anti-senescence effects; thus, it is worth identifying such components besides gastrodin.

Brain aging is the process by which the brain changes with age, closely linked to the senescence of neural lineage-related cells [7,8,9]. Cellular senescence is characterized by cycle arrest, telomere shortening, morphological changes, chromatin remodeling, increased lysosomal β-galactosidase, mitochondrial dysfunction, and DNA damage [7,8,10]. In the nervous system, senescence-related changes are not restricted to terminally differentiated neurons but also affect other neural lineage-related cells, such as neuroblasts. Neuroblasts are a type of immature nerve cell capable of differentiating into neurons in the brain, and they play a key role in brain cell neurogenesis [11]. However, accumulating evidence suggests that these cells can undergo senescence-associated functional decline under aging or stress conditions, thereby contributing to impaired brain homeostasis [12]. Therefore, preventing or alleviating senescence-associated phenotypes in neural lineage cells may be beneficial for maintaining brain function during aging.

Autophagy is an essential intracellular metabolic process that maintains cellular homeostasis and adapts to environmental changes by degrading and reusing damaged or obsolete intracellular components [13]. Studies have demonstrated that autophagy plays an important regulatory role in the aging process. With age, the autophagic activity of cells gradually decreases, leading to the accumulation of intracellular waste materials, including impaired organelles, protein aggregates, and metabolites. The accumulation of waste materials leads to a decline in cellular function and an increase in inflammatory responses, thus promoting the onset of cellular senescence [14]. The autophagy process can remove oxidatively damaged proteins, aggregates, and functionally impaired organelles in the cell, which can help to slow down the senescence process [15]. Therefore, promoting autophagy is expected to be a potential strategy to delay cellular senescence and the overall aging process of the organism.

Previous studies demonstrated that hydroxyurea (HU) could be used as an inducer of cellular senescence in many cell types, such as fibroblasts, peripheral blood mesenchymal stromal cells, neural stem cells, and dopaminergic neurons [16,17,18,19]. Blocked DNA replication by HU leads to replication fork collapse, thereby generating DNA damage such as double-strand breaks [19,20,21,22,23,24]. Given the driver role of DNA damage in cellular senescence, an HU-treated cell model is an alternative model for investigating cellular senescence. P-HBA is a polar aromatic compound isolated from Tianma and reported to have antioxidant, anti-aging, and neuroprotective effects [25]. However, it is unknown whether P-HBA can suppress cellular senescence. In this study, we demonstrated that HU induced various senescent phenotypes in human SH-SY5Y neuroblast-like cells, whereas Tianma methanol extracts (TMEs) and P-HBA significantly reduced them. We also investigated the role of autophagy in the bioactivity of P-HBA.

2. Results

2.1. HU Induces Senescence Phenotype in Human SH-SY5Y Cells

To assess whether HU induces cellular senescence phenotypes in SH-SY5Y cells, we first tested the cytotoxicity of HU using a CCK-8 assay. As shown in Figure 1A, treatment with HU at concentrations above 0.25 mM for 48 h resulted in a significant decrease in the viability of SH-SY5Y cells, which might result from increased cell death or decreased cell proliferation, or a combination of both. We further tested the effects of 0.25 mM HU on SA-β-Gal activity using an SA-β-Gal assay. The results showed that 0.25 mM HU treatment for 48 h could increase SA-β-Gal activity in SH-SY5Y cells (Figure 1B,C). γ-H2AX serves as a major marker of DNA damage. Immunofluorescence analysis revealed that 0.25 mM HU treatment for 48 h could increase the number of γ-H2AX-positive cells (Figure 1D,E). Previous research indicated that autophagy impairment is strongly related to cell senescence [26]. Thus, we infected SH-SY5Y cells with an mcherry-GFP-LC3 adenovirus to identify autophagosomes before autolysosome formation. The mcherry-GFP-LC3 proteins display both red and green fluorescence in autophagosomes. After entering lysosomes, GFP signals were quenched in the acidic environment of lysosomes, and only red fluorescence persisted. The imaging results showed that the control group displayed more yellow signals (red-green colocalization) than the HU group (Figure 1F,G). The decrease in yellow signals in the HU group was due to the blockage of autophagosome formation, which led to the reduction of autophagic flux. These results suggest that HU can induce a senescence phenotype in SH-SY5Y cells.

2.2. TME Counteracts HU-Induced Increase in SA-β-Gal Activity and DNA Damage

To understand the anti-senescence activity of TME, we first tested the cytotoxicity of TME. The result of CCK-8 showed that the treatment with TME alone from 0.0625–4 mg/mL for 48 h did not cause any cytotoxicity and stimulated the proliferation of SH-SY5Y cells (Figure 2A). In addition, 0.25, 0.5, 1, and 2 mg/mL TME could alleviate the decrease in cell viability induced by HU (Figure 2B). The increase in SA-β-Gal activity is one of the HU-induced senescence phenotypes. As shown in Figure 2C,E, HU could increase the number of SA-β-Gal-positive cells, which were significantly reduced by co-treatment with TME for 48 h in a concentration-dependent manner. We further investigated whether TME could reduce HU-induced DNA damage in SH-SY5Y cells. As shown in Figure 2D,F, immunofluorescence staining showed that HU treatment for 48 h dramatically increased the number of γ-H2AX-positive cells, which could be decreased by co-treatment with 0.5, 1, or 2 mg/mL TME.

2.3. Chemical Characterization of TME

To comprehensively profile the complex chemical composition of TME, we employed UPLC-MS analysis in both positive and negative ionization modes. The use of dual ionization modes provides broader metabolite coverage, with negative ion mode being particularly effective for detecting acidic compounds, thereby enabling a more complete characterization of the sample. We selected seven bioactive compounds (parishin A, parishin B, parishin C, gastrodin, P-HBA, vanillin, and protocatechuic acid) that were reported to exist in TME as references. As shown in Table 1 and Figure 3, 38 chemical components were identified, and all of the seven bioactive components were detected. Among them, P-HBA (chemical structure shown in Figure 4A) was reported to have antioxidant and neuroprotection effects [25], but its role in cell senescence has not been elucidated yet. Therefore, we continued to explore the anti-senescence activity and the underlying mechanism of P-HBA.

2.4. P-HBA Counteracts HU-Induced Increase in SA-β-Gal Activity and DNA Damage

To evaluate the effects of P-HBA on HU-induced cell senescence phenotypes, we first tested the cytotoxicity of P-HBA using a CCK-8 assay. As shown in Figure 4B, the treatment with P-HBA alone from 0.03 to 100 µM for 48 h did not show any cytotoxicity in SH-SY5Y cells. In order to assess whether P-HBA can recover cell viability impaired by HU, we co-treated SH-SY5Y cells with 0.25 mM HU and 1, 3, and 10 µM P-HBA for 48 h. The results showed that all tested concentrations of P-HBA could not rescue the impaired cell viability caused by HU (Figure 4C). The increase in SA-β-Gal activity is one of HU-induced senescent phenotypes. As shown in Figure 4D,F, HU could increase the number of SA-β-Gal-positive cells, which were significantly reduced by co-treatment with P-HBA for 48 h in a concentration-dependent manner. DNA damage is one of the major drivers of HU-induced cell senescence effects; thus, we investigated whether P-HBA could reduce HU-induced DNA damage in SH-SY5Y cells. Immunofluorescence staining showed that HU dramatically increased the number of γ-H2AX-positive cells, whereas co-treatment with 3 and 10 µM P-HBA for 48 h could reduce it (Figure 4E,G). These results indicated that P-HBA was able to reduce senescence-related phenotypes in HU-treated SH-SY5Y cells.

2.5. Enhanced Autophagy Is Involved in Anti-Cellular Senescence Activity of P-HBA

A previous study demonstrated that P-HBA was able to induce autophagy in macrophages [27]. Therefore, we further investigated the effects of P-HBA on HU-induced autophagy dysfunction. In Ad-mcherry-GFP-LC3-transfected SH-SY5Y cells, the yellow signal in the P-HBA co-treated cells was significantly increased compared to HU-treated cells (Figure 5A,B), suggesting that P-HBA enhances autophagic function in HU-treated cells. Furthermore, we used the autophagy inhibitor chloroquine (CQ) to evaluate whether enhanced autophagy is involved in anti-cellular senescence activity of P-HBA. As shown in Figure 5C,D, the results of the SA-β-Gal assay showed that P-HBA did not reduce HU-induced SA- β-Gal activity increase in the presence of CQ, suggesting that enhanced autophagy is involved in the anti-cellular senescent effects of P-HBA.

2.6. Comparison of Anti-Cellular Senescence Activities of Main Tianma Active Components

To compare anti-cellular senescence activities of other Tianma major components, six bioactive compounds related to the structure of P-HBA (parishin A, parishin B, parishin C, gastrodin, vanillin, and protocatechuic acid) were tested (Figure 6A). The CCK-8 assay showed that none of these compounds from 0.3 to 100 μM affected the viability of human SH-SY5Y cells (Figure 6B). According to SA-β-Gal staining, 10 μM of gastrodin, P-HBA, and vanillin exhibited similar inhibitory effects on HU-induced SA-β-Gal activity increase (Figure 6C). In contrast, parishin A at 10 μM exhibited a weaker inhibitory effect, while 10 μM of parishin B, parishin C, and protocatechuic acid showed no significant activity.

3. Discussion

Previous studies indicated that P-HBA exhibited anti-oxidative stress in different disease models [25,28,29]. In addition, P-HBA could bypass the blood–brain barrier and reduce brain injury in cerebral ischemia animal models [30]. In this study, we reported for the first time that P-HBA effectively alleviated the HU-induced senescent phenotype in SH-SY5Y cells, including reduced SA-β-gal activity, levels of the DNA damage marker γ-H2AX, and autophagic dysfunction. Notably, although P-HBA failed to reverse the HU-induced decrease in cell viability, it successfully mitigated the changes associated with senescence, suggesting that P-HBA’s major effect lies in enhancing cellular tolerance to stress, which leads to senescence and maintaining cellular homeostasis, rather than promoting proliferation or reversing acute cell death.

In our study, SA-β-gal activity and persistent DNA damage marked by γH2AX are used as the cell senescence markers. Although they are widely accepted and commonly used indicators of stress-induced cellular senescence, they do not encompass the full spectrum of senescence-associated features. Cellular senescence is a heterogeneous and multifactorial state, typically accompanied by additional hallmarks such as the upregulation of cell-cycle inhibitors (e.g., p21 or p16), alterations in nuclear lamina composition (including changes in lamin A/C or lamin B1), and the expression or secretion of senescence-associated secretory phenotype (SASP) factors [31]. These molecular and structural changes were not examined in the present study and therefore limit the breadth of senescence characterization. An additional consideration in defining a bona fide senescent state is the simultaneous presence of multiple senescence-associated markers within individual cells. In the present study, senescence-associated β-galactosidase activity and persistent DNA damage marked by γH2AX were assessed as parallel indicators of HU-induced stress responses, but their co-localization at the single-cell level was not examined. The absence of multi-marker co-detection limits the ability to conclusively distinguish senescence from partially overlapping or independent cellular stress responses. Future studies incorporating other complementary markers and combining SA-β-gal staining with DNA damage markers, cell-cycle inhibitors (such as p21 or p16), or nuclear lamina components (e.g., lamin A/C or lamin B1) within the same cells would provide a more stringent validation of the senescent phenotype induced by HU in this system and help to more comprehensively evaluate the anti-senescence effects of TME and P-HBA.

Autophagy dysfunction is considered one of the key drivers of brain aging [26]. Using the mCherry-GFP-LC3B dual fluorescence reporter system, we visually observed that HU treatment led to a decrease in the number of yellow spots (undegraded autophagosomes), whereas co-treatment with P-HBA increased them, indicating that P-HBA counteracts HU-induced autophagy dysfunction at the population level. Given the close relationship between defective autophagy, the accumulation of cellular damage, and the development of senescence-associated phenotypes, our findings support a functional involvement of autophagy in the modulation of HU-induced senescence-associated features by P-HBA. However, because autophagy and senescence markers were assessed in parallel rather than at the single-cell level, the precise cellular relationship between autophagy restoration and senescence suppression remains to be further clarified. In addition, the upstream signaling pathways through which P-HBA regulates autophagic activity warrant further investigation.

Aging is a main driver for the development of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s diseases [7,32]. Therefore, the ability of Tianma and P-HBA to mitigate senescence effects exhibits potential in treatment for neurodegenerative diseases to preserve neurogenic potential and cognitive function in aging populations. The therapeutic effects of P-HBA on neurodegenerative disease are under-researched. However, a study has elucidated that P-HBA could delay paralysis, improve mobility, and promote anti-stress ability in a nematode model of AD [25]. Future studies should prioritize evaluating P-HBA in transgenic animal models of Alzheimer’s disease or Parkinson’s disease, focusing on its ability to mitigate cognitive or motor deficits, reduce pathological protein burden, and modulate senescence and autophagy markers in the brain. The efficacy in vivo will be the critical next step in evaluating P-HBA’s potential as a neuroprotective agent targeting cell senescence-related pathways.

In conclusion, this study identifies P-HBA as an active component of Tianma that attenuates HU-induced senescence-associated phenotypes in human SH-SY5Y cells. Our results indicate that the protective effects of P-HBA are associated with the modulation of autophagy, as evidenced by partial restoration of autophagic flux and the functional requirement of autophagy for its anti-senescence activity. The ability of P-HBA to cross the BBB further supports its pharmacological relevance for central nervous system targets. Although the precise molecular mechanisms and upstream signaling pathways remain to be fully elucidated, these findings provide a basis for further investigation into the role of P-HBA’s potential application to neurodegeneration-induced cellular senescence.

It should be noted that the conclusions of this study are based on internally consistent results obtained from multiple technical replicates within a defined experimental framework. Accordingly, the findings are interpreted as evidence for reproducible senescence- and autophagy-associated trends under the specific conditions tested, rather than as definitive or universally generalizable effects. Further validation using independent biological experiments will be important to extend the applicability of these observations.

4. Materials and Methods

4.1. Materials

Hydroxyurea (Cat# S1732) was purchased from Beyotime Biotechnology (Shanghai, China). Gastrodin (Cat# T2999), Parishin A (Cat# T4S1820), Parishin B (Cat# T3S1816), Parishin C (Cat# TMA1603), P-Hydroxybenzaldehyde (Cat# T2S1814), Vanillin (Cat# T6717), and Protocatechuic acid (PCA) (Cat# T0562) were purchased from TargetMol (Boston, MA, USA). Phospho-Histone H2AX (Ser139) antibody (Cat# AF5836) was purchased from Beyotime Biotechnology (Shanghai, China).

4.2. Preparation of Tianma Methanol Extract

The amount of 10 g of Tianma powder was accurately weighed and dissolved in 300 mL of methanol with sonication for 30 min at room temperature. The mixture was then filtered through qualitative filter paper. The filtered liquid was concentrated using rotary evaporation at 40 °C for concentration and then lyophilized to obtain the Gastrodia elata Blume methanol extract powder.

4.3. Cell Culture

The human neuroblastoma SH-SY5Y cell line (ATCC Number: CRL-2266) was obtained from the American Type Culture Collection. Cells were cultured in DMEM/F12 (1:1) medium containing penicillin/streptomycin (100 U/mL; 100 μg/mL) and 10% fetal bovine serum in a 37 °C, 5% CO_2_ incubator. Prior to experimental procedures, cells were allowed to attach to the plate for 24 h.

4.4. Cell Viability Assay

Cell viability was assessed with the CCK-8 assay (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, SH-SY5Y cells were plated in 96-well plates at a density of 3 × 10^4^ cells/well and subjected to experimental treatments. After appropriate treatment, the cells were incubated in 10% CCK-8 reagent at 37 °C for 4 h. Absorbance at 450 nm was obtained using a SpectraMax M5 microplate reader (Molecular Devices). All values were normalized to the control group.

4.5. Senescence-Associated-β-Galactosidase Assay

Cellular senescence was assessed using a SA-β-gal staining kit (Beyotime, Shanghai, China) following the manufacturer’s instructions. After appropriate treatment, the cells were fixed and incubated with the working solution at 37 °C for 24 h. For quantitative analysis, 10 different fields were evaluated for each sample. The data were presented as the percentage of blue positive cells to the total number of cells.

4.6. Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC–MS) Analysis

The sample solution was prepared by dissolving 0.1 g of the Tianma extracts in 1 mL 80% methanol. Then, ultrasonication for 20 min, followed by centrifugation at 4 °C, 12,000 rpm for 15 min. The supernatant was filtered through a 0.22 μm microporous filter. The mixed control solutions were prepared as follows: 8 reference compounds were dissolved in 80% methanol, respectively, to prepare 1 mg/mL of stock solution. The 8 stock solutions were diluted and mixed with 80% methanol to obtain the mixed control solution (20 μg/mL).

UPLC analysis was performed on the Thermo Scientific Dionex Ultimate 3000 RS (Thermo Fisher Scientific Co., Ltd., Waltham, MA, USA). Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 µm) was used with a mobile phase of 0.1% formic acid aqueous solution (A) and acetonitrile (B) for gradient elution (0–3 min, 95% A; 38 min, 95%90%A; 812 min, 90–80%A; 1218 min, 80–50%A; 1821 min, 50–45%A; 2125 min, 45–5% A; 25~26 min, 5%95% A; 2630 min, 95% A); flow rate: 0.28 mL/min; injection volume: 2 µL; column temperature: 40 °C.

MS analysis was performed on the Q-Exactive Focus Orbitrap MS equipped with an electrospray ionization source in positive and negative ion scanning mode. The parameters were as follows: resolution: 70,000; sheath gas flow rate: 30 arb; auxiliary gas flow rate: 10 arb; spray voltage: 3.5 kV (+), 3.0 kV (−); capillary temperature: 320 °C; auxiliary gas heater temperature: 350 °C; scanning range: m/z 100~1500; data acquisition method: Full MS-ddMS^2^.

4.7. Immunofluorescence Staining

SH-SY5Y cells were plated in confocal dishes at a density of 5 × 10^4^ cells. After appropriate treatment, cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. After three washes with PBS, cells were permeabilized and blocked with a solution containing 5% donkey serum and 0.3% Triton™ X-100 (Beyotime, Shanghai, China) in PBS for 1 h at room temperature. The samples were then incubated with primary antibodies at 4 °C overnight. After washing three times with PBS, the samples were incubated with DAPI for nuclear visualization and fluorophore-conjugated secondary antibodies for 1 h at room temperature in the dark. After three washes with PBS, the image was acquired using a Leica TCS SP8 confocal laser scanning microscopy system.

4.8. Analysis of LC3B Punctate Dots

SH-SY5Y cells were seeded into 12-well plates, where they were allocated at a density of 1 × 10^5^ cells per well and reached 60–70% confluence at the time of infection. Added were 2 μL of Ad plus-mCherry-GFP-LC3B adenovirus infected for 48 h. After appropriate treatment in infected cells, the red and green fluorescence were observed using a laser scanning microscope (Leica TCS SP8 Confocal Laser Scanning Microscope). Autophagosome formation was evaluated by calculating the number of yellow puncta.

4.9. Statistical Analysis

The statistical analysis was conducted using GraphPad Prism 9.0 statistical software (GraphPad Software, Inc., San Diego, CA, USA). All experiments were performed in triplicate at least, and data were presented as mean values ± standard error of the mean (SEM). For multiple group comparisons, statistical analysis was carried out using a one-way analysis of variance, followed by Tukey’s multiple comparison. An unpaired t-test was performed for comparison between the two groups. A p-value < 0.05 was considered statistically significant.