Sinapic Acid Reduces Hypoxia‐Induced Cell Damage in Hep‐2 Cells by Regulating TRPM2 Channel Activation

ABSTRACT

Hypoxia (HPX) is a significant pathological factor that contributes to mitochondrial dysfunction, oxidative stress, inflammation, and calcium dysregulation in laryngeal squamous cell carcinoma (LSCC). The redox‐sensitive TRPM2 channel is crucial for mediating HPX‐induced Ca²⁺ excess and subsequent cell death. This study investigated whether sinapic acid (SPA), a natural phenolic compound with known antioxidant and anti‐inflammatory properties, can protect Hep‐2 cells from HPX‐induced injury by modulating TRPM2 activity. Hep‐2 cells were exposed to CoCl₂‐induced HPX and treated with SPA (10–150 μM). Cell viability, oxidative stress parameters (malondialdehyde [MDA], glutathione [GSH], and reactive oxygen species [ROS]), intracellular Ca²⁺ levels, mitochondrial membrane potential, and pro‐inflammatory cytokines (IL‐1β and TNF‐α), TRPM2 expression, and PI‐positive cell death were evaluated. HPX markedly decreased cell viability and increased oxidative and inflammatory responses, mitochondrial depolarization, and TRPM2‐dependent Ca²⁺ influx. SPA significantly restored GSH levels, reduced MDA and ROS accumulation, suppressed cytokine production, and stabilized mitochondrial membrane potential. Moreover, SPA attenuated TRPM2 upregulation and effectively lowered Ca²⁺ overload. Co‐treatment with the widely used TRPM2 channel inhibitor 2‐APB further enhanced SPA's protective effects and produced the greatest reduction in cell death. These findings demonstrate that SPA mitigates HPX‐induced cytotoxicity through antioxidative, anti‐inflammatory, and TRPM2‐modulating mechanisms, highlighting its potential therapeutic relevance in LSCC.

Schematic representation of the protective effect of SPA against HPX‐induced OS, TRPM2 activation, Ca²⁺ overload, and apoptosis in Hep‐2 cells.

1. Introduction

Despite improvements in diagnostic methods and multimodal treatment approaches, laryngeal squamous cell carcinoma (LSCC), one of the most prevalent and severe cancers of the upper aerodigestive tract, still presents a significant therapeutic challenge [1, 2]. The biological behavior of LSCC is strongly influenced by the tumor microenvironment, where hypoxia (HPX), oxidative stress (OS), and chronic inflammation act as major drivers of tumor progression, therapeutic resistance, and poor clinical outcomes [3, 4]. Hypoxic niches arise from the rapid, disorganized proliferation of tumor cells, leading to insufficient vascularization and oxygen deprivation. This state not only disrupts cellular metabolic homeostasis but also induces profound alterations in mitochondrial function, redox regulation, and apoptotic signaling pathways [5, 6].

HPX‐induced OS is a central contributor to cellular damage in LSCC and many other solid tumors [7]. Excessive reactive oxygen species (ROS) production in oxygen‐limited conditions damages proteins and DNA, encourages lipid peroxidation, weakens antioxidant defense mechanisms, and sets off inflammatory cascades [8]. These events collectively destabilize cellular homeostasis, enhance genomic instability, and ultimately favor malignant transformation and resistance to treatment [9]. Moreover, HPX and OS are closely linked to dysregulation of intracellular calcium ([Ca²⁺]_c) signaling, a key determinant of cell survival and death. Perturbations in Ca²⁺ homeostasis, particularly through redox‐sensitive ion channels, significantly influence mitochondrial integrity, energy metabolism, and apoptotic pathways [10, 11].

Among the ion channels involved in redox and Ca²⁺ signaling, the Transient Receptor Potential Melastatin‐2 (TRPM2) channel has emerged as a critical mediator of oxidative damage. Under stress, TRPM2 is a key pathway for Ca²⁺ influx and is highly sensitive to ROS and other oxidative metabolites [12, 13]. Activation of this channel amplifies oxidative damage, disrupts mitochondrial membrane potential, enhances the production of pro‐inflammatory cytokines, and promotes cell death by apoptosis. In cancer‐related contexts, upregulation of TRPM2 has been associated with increased cellular vulnerability to OS and altered inflammatory responses, highlighting its potential as a molecular target in diseases characterized by redox imbalance [14, 15, 16].

Natural phenolic compounds have gained increasing interest as potential cytoprotective or adjuvant therapeutic agents due to their anti‐inflammatory, antioxidant, and ion channel–modulatory properties [17, 18]. Among these, sinapic acid (SPA) is a hydroxycinnamic acid widely present in cereals, fruits, and vegetables. SPA exhibits strong free radical‐scavenging activity, maintains mitochondrial function, reduces inflammatory mediator production, and modulates redox‐regulated signaling pathways [19, 20]. Evidence from multiple experimental models indicates that SPA can mitigate oxidative damage, restore intracellular antioxidant capacity, and protect cells against stress‐induced damage [21].

Despite growing scientific interest, the cytoprotective potential of SPA in HPX‐exposed Hep‐2 cells and particularly its effect on TRPM2‐mediated Ca²⁺ signaling and oxidative–inflammatory pathways remains not fully understood. Clarifying how SPA influences TRPM2 expression, mitochondrial membrane potential, ROS accumulation, and inflammatory cytokine production under hypoxic stress is crucial for assessing its therapeutic potential. In this context, the current study investigates the effects of SPA on HPX‐induced cellular damage in Hep‐2 cells. By evaluating cell viability, OS markers, mitochondrial function, intracellular Ca²⁺ dynamics, and TRPM2 channel activity, this study aims to uncover the protective mechanisms of SPA and its capacity to modulate redox‐ and Ca²⁺‐dependent signaling pathways. Such mechanistic insights may provide the development of innovative, biology‐driven treatment‐oriented strategies targeting HPX‐related cellular dysfunction in LSCC.

2. Materials and Methods

2.1. Chemicals and Cell Culture

SPA (Cat_D7927) and other chemicals were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Hep‐2 cells (ATCC CCL‐23) were acquired from the Şap Institute in Ankara, Türkiye. The reason for choosing the HEp‐2 cell line in this research is that it is widely used in epithelial cancer models and studies on drug resistance [22, 23]. The cells were cultivated in DMEM media containing 1% penicillin/streptomycin and 10% heat‐inactivated FBS (Sigma‐Aldrich, St. Louis, MO, USA). The cells were passaged and separated into groups once they achieved 80%–85% confluence. Cell cultures were maintained in T25 flasks under incubator conditions.

2.2. Cell Viability Assay and Study Groups

Cell viability was assessed using the Cell Counting Kit‐8 (CCK‐8; Abbkine, Cat. No: KTA1020) test, a sensitive and reproducible colorimetric method commonly used to evaluate metabolically active cells. Hep‐2 cells were seeded into 96‐well plates at a concentration of 1 × 10⁵ cells per well and incubated under standard cell culture conditions until approximately 80%–85% confluence was achieved. SPA solutions were freshly prepared and applied to the cells at concentrations of 5, 10, 25, 50, 100, and 150 µM for 24 h. To create an HPX‐like cellular damage model, cells were treated with CoCl₂ (200 µM) for the same duration [17, 24]. Cells in the control group were kept under standard culture conditions without any treatment. At the end of the incubation period, CCK‐8 solution was added to each well and incubated according to the manufacturer's protocol. Absorbance values were measured at 450 nm using a Multiskan SkyHigh microplate reader (Thermo Scientific, Waltham, MA, USA) to reflect the number of viable cells, and cell viability was expressed as a percentage compared to the control group. According to the results, CoCl₂ treatment significantly decreased Hep‐2 cell viability, whereas SPA treatment reduced CoCl₂‐induced cytotoxicity in a dose‐dependent manner. Among the tested doses, 100 µM SPA showed the most significant protective effect on cell viability and was selected for subsequent experiments. Accordingly, the study groups were formed as follows: (1) The control group (CTR) consisted of cells incubated under standard culture conditions. (2) The SPA group consisted of cells treated only with SPA (100 µM). (3) The HPX group consisted of cells treated only with CoCl₂ (200 µM). (4) In the HPX + SPA group, CoCl₂ (200 µM) and SPA (100 µM) were applied to the cells together.

2.3. Cytokine, Glutathione (GSH), and Malondialdehyde (MDA) Analysis

In the study groups, MDA, GSH, interleukin‐1 beta (IL‐1β), and tumor necrosis factor alpha (TNF‐α) levels were determined using commercially available ELISA kits according to the manufacturer's instructions. The Human MDA ELISA Kit (Cat. No: E1371Hu), Human GSH ELISA Kit (Cat. No: EA0142Hu), Human IL‐1β ELISA Kit (Cat. No: E0143Hu), and Human TNF‐α ELISA Kit (Cat. No: E0082Hu) were obtained from BT Lab (Shanghai, China). Absorbance values for all assays were measured at 450 nm using a Multiskan SkyHigh microplate reader (Thermo Scientific, Waltham, MA, USA). Concentrations were calculated according to standard curves generated for each assay.

2.4. Western Blot Analysis

Overall protein concentrations in Hep‐2 cells were determined with a BCA Protein Assay Kit (Thermo Fisher Scientific, Cat. No: 23227). A total of 50 µg of protein was loaded into each well. Electrophoresis was performed using the Bio‐Rad Mini‐PROTEAN Tetra Cell gel electrophoresis system. Afterward, the gels were prepared for protein transfer. A transfer “sandwich” was assembled in the cassette in the following order from cathode to anode: filter paper, gel, nitrocellulose membrane, and filter paper. Protein transfer from the gel to the nitrocellulose membrane was carried out using the Bio‐Rad semi‐wet transfer system. After that, the membranes were incubated with primary antibodies diluted in 5% non‐fat milk for an entire night at 4°C. TRPM2 (1:1000) and β‐actin (1:1000). Subsequently, membranes were incubated for 1 h with secondary antibodies, also diluted in 5% milk (anti‐mouse: 1:5000; anti‐rabbit: 1:10,000). After incubation, membranes were treated with an enhanced chemiluminescent (ECL) reagent, and protein bands were visualized using the SYNGENE G: Box Chemi XRQ imaging system. Band intensities were quantified using ImageJ software.

2.5. Measurements of JC‐1 and ROS

JC‐1 dye was used to evaluate mitochondrial membrane potential (mΔΨ) in Hep‐2 cells. Cells were incubated with 3 µM JC‐1 dye (Cat. No: T3168, Thermo Fisher Scientific, Istanbul, Turkey) [25]. Intracellular ROS levels were determined using 3 µM CM‐H2DCFDA fluorescent dye (Cat. No: C6827, Thermo Fisher Scientific, Istanbul, Turkey). Following intracellular oxidation, CM‐H2DCFDA was converted into fluorescent dichlorofluorescein (DCF), allowing the detection of OS [26]. Fluorescence images were captured using a ZEISS Axiovert 5 inverted fluorescence microscope (Carl Zeiss, Germany). DCF fluorescence was monitored at approximately 485/525 nm. Fluorescence intensity values were analyzed as arbitrary fluorescence units. Representative fluorescence images were presented for both DCF and JC‐1 staining.

2.6. Determination of [Ca²⁺]c Fluorescence Intensity

The present study investigated HPX‐induced [Ca²⁺]c elevation in Hep‐2 cells associated with TRPM2 channel activation using a ZEISS Axiovert 5 inverted fluorescence microscope (Carl Zeiss, Germany). [Ca²⁺]c changes were evaluated using the calcium‐sensitive fluorescent dye Fluo‐4 AM (Cat. No: F14201, Thermo Fisher Scientific, Istanbul, Türkiye). Following treatment, Hep‐2 cells were incubated with 1 µM Fluo‐4 AM for 60 min at 37°C in the dark. After incubation, fluorescence images were obtained with a ×20 objective under fluorescence microscopy. Fluo‐4 AM fluorescence was monitored at excitation and emission wavelengths of approximately 494 nm and 506 nm, respectively. Fluorescence intensity values obtained from selected 15 µm² regions within the Hep‐2 cell cytosol were analyzed and expressed as arbitrary fluorescence units in green fluorescence images. To evaluate the involvement of TRPM2 channels in calcium influx, cells were treated with 100 µM 2‐aminoethoxydiphenyl borate (2‐APB), a widely used TRPM2 channel inhibitor, prior to oxidative stimulation with 1 mM H₂O₂ [16, 27].

2.7. Propidium Iodide (PI)‐Positive) Percentage

Hep‐2 cells cultured on glass‐bottom dishes were incubated with a mixture of PI (2 µg/mL, Cat. # P1304MP, Thermo Fisher Scientific) and Hoechst 33342 (4 µM, Cat. # 4082, Cell Signaling Technology, Danvers, MA, USA) prior to imaging with the Axiovert‐5 camera. The recorded and taken pictures were analyzed using ZEN Blue software (version 3.12) in red (PI) and blue (Hoechst). For PI, 535 and 617 nm were used as the excitation and emission wavelengths, respectively, while for Hoechst 33342, they were 348 and 455 nm, respectively. The percentage of PI‐positive cells was calculated by dividing the number of PI‐positive cells by the total number of cells and expressed as a percentage (%).

2.8. Statistical Analysis

All variables were presented as mean ± standard deviation (mean ± SD). The statistical analyses were carried out using SPSS software (version 23.0; SPSS Inc., Chicago, IL, USA), and the normality of the data distribution was examined using the Shapiro−Wilk test. Differences between groups were evaluated using a one‐way analysis of variance (ANOVA), and if significance was found, a Tukey's post hoc test was conducted. A p value less than 0.05 was deemed statistically significant.

3. Results

3.1. Influence of SPA on Cell Viability and TRPM2 Levels in HPX‐Induced Hep‐2 Cells

Exposure of Hep‐2 cells to 200 μM cobalt chloride (CoCl₂) for 24 h resulted in a notable reduction in cell viability compared to the unaffected control (CTR) group (p < 0.001). SPA treatment produced a concentration‐dependent improvement in viability, with a maximal protective effect at 100 μM (p < 0.05 vs. HPX; Figure 1a). Western blot technique demonstrated a notable increase in TRPM2 protein levels under hypoxic conditions. Co‐treatment with SPA significantly attenuated this elevation and restored TRPM2 levels toward baseline, indicating that SPA counteracts HPX‐induced TRPM2 overexpression and enhances cell survival (Figure 1b and S1).

Figure 1.

SPA enhances cell viability and reduces TRPM2 expression in CoCl₂‐induced hypoxic Hep‐2 cells (mean ± SD). (a) Cell viability of Hep‐2 cells treated with several dosages of SPA (10–150 μM) after being exposed to CoCl₂ (200 μM) for 24 h (*p < 0.001 vs. CTR; ^& p < 0.05 vs. HPX). (b) Western blot analysis and densitometric analysis showing TRPM2 protein expression levels (^a p < 0.05 vs. HPX; ^b p < 0.05 vs. HPX + SPA). β‐Actin served as the loading control.

3.2. Effects of SPA on OS and Proinflammatory Cytokine in HPX‐Induced Hep‐2 Cells

Hypoxic stress resulted in a significant decline in intracellular GSH levels (Figure 2a) and a concomitant rise in MDA levels (Figure 2b), signifying enhanced lipid peroxidation (p < 0.05 vs. HPX). SPA administration restored GSH content and reduced MDA accumulation, demonstrating its antioxidative efficacy. Furthermore, HPX significantly increases TNF‐α and IL‐1β levels (p < 0.05). In contrast, SPA co‐treatment significantly reduced both cytokines, indicating that SPA mitigates HPX‐driven inflammatory and OS in Hep‐2 cells (Figure 2c,d).

Figure 2.

Effects of SPA on GSH (a), MDA (b), IL‐1β (c), and TNF‐α (d) levels in CoCl₂‐induced hypoxic Hep‐2 cells (mean ± SD, and n = 8) (^a p < 0.05 vs. HPX; ^b p < 0.05 vs. HPX + SPA).

3.3. Effects of SPA on Mitochondrial Membrane Potential and ROS in HPX‐Induced Hep‐2 Cells

JC‐1 staining showed that HPX induced a prominent mitochondrial depolarization, as evidenced by a higher JC‐1 fluorescence ratio (green/red). SPA co‐treatment markedly restored the mitochondrial membrane potential toward CTR values (p < 0.05 vs. HPX) (Figure 3a). Similarly, DCFH‐DA fluorescence measurements demonstrated that CoCl₂ exposure elevated intracellular ROS levels, while SPA significantly reduced ROS accumulation (p < 0.05) (Figure 3b). Representative fluorescence micrographs confirmed these findings, showing intense green DCF and diminished orange JC‐1 signals in HPX cells, which were reversed after SPA administration (Figure 3c). These data indicate that SPA preserves mitochondrial integrity and limits ROS‐dependent damage under hypoxic conditions.

Figure 3.

Effects of SPA on JC‐1 and ROS levels in CoCl₂‐induced hypoxic Hep‐2 cells. Data are presented as mean ± SD (n = 8). (a) Quantification of JC‐1 fluorescence intensity. (b) Quantification of intracellular ROS levels measured by DCFH‐DA fluorescence. (c) Representative fluorescence microscopy images of JC‐1 and ROS staining, along with merged images demonstrating their co‐localization. (^a p < 0.05 versus HPX; ^b p < 0.05 versus HPX+SPA). Scale bar = 50 µm. a.u., arbitrary units.

3.4. SPA and TRPM2 Inactivation Attenuate H₂O₂‐Induced [Ca²⁺]_c Elevation

Fluo‐4 AM fluorescence analysis revealed that both CoCl₂ and H₂O₂‐treated cells exhibited a pronounced increase in [Ca²⁺]_c intensity (p < 0.05 vs. CTR). SPA markedly reduced this elevation, suggesting suppression of TRPM2‐mediated Ca²⁺ influx. Use of 2‐APB (100 μM), a TRPM2 inhibitor, further diminished [Ca²⁺]_c levels in HPX and HPX + SPA groups (p < 0.01), confirming the TRPM2 dependence of oxidative Ca²⁺ overload. Fluorescence micrographs corroborated these results, showing weaker green fluorescence in SPA and 2‐APB‐treated cells compared with HPX groups (Figure 4a–c).

Figure 4.

Effects of SPA and TRPM2 inhibition on H₂O₂‐induced increase of the [Ca²⁺]_c in CoCl₂‐exposed Hep‐2 cells (mean ± SD, and n = 8). (a) Quantitative analysis of [Ca²⁺]_c levels in the absence and presence of H₂O₂ (1 mM). HPX and H₂O₂ exposure markedly elevated [Ca²⁺]_c fluorescence intensity compared with the CTR and SPA groups (p < 0.05). SPA co‐treatment significantly reduced [Ca²⁺]_c accumulation in hypoxic cells (^{*, a} p < 0.05 vs. SPA [groups with +H₂O₂]; ^{*, b} p < 0.05 vs. HPX [groups with +H₂O₂]). (b) Effects of the TRPM2 inhibitor 2‐APB (100 µM) on H₂O₂‐induced Hep‐2 cells. 2‐APB markedly suppressed [Ca²⁺]_c generation in both HPX and HPX + SPA groups, indicating TRPM2‐dependent oxidative stress modulation (^{&, #, $} p < 0.01 vs. groups with +H₂O₂; ^{c, d} p < 0.001 vs. groups with H₂O₂ + 2‐APB). (c) Representative fluorescence microscopy images of Fluo‐4‐stained cells showing intracellular [Ca²⁺]_c fluorescence in CTR, H₂O₂‐treated, and 2‐APB‐treated conditions across all experimental groups. Scale bar = 20 µm. a.u., arbitrary unit.

3.5. SPA and TRPM2 Inactivation Suppress HPX‐Induced Cell Death in Hep‐2 Cells

PI and Hoechst 33342 double‐labeling demonstrated that CoCl₂ exposure markedly increased the proportion of PI‐positive (dead/apoptotic) Hep‐2 cells relative to CTR (p < 0.001). Treatment with SPA (HPX + SPA) substantially reduced PI positivity, indicating attenuation of HPX‐induced apoptosis. The combined application of SPA and 2‐APB (HPX + SPA + 2‐APB) produced the greatest reduction in cell death percentage (p < 0.001 vs. HPX), confirming that TRPM2 inhibition potentiates the cytoprotective action of SPA. Merged fluorescence images supported these findings by showing decreased red PI intensity and preserved nuclear morphology (Figure 5a,b).

Figure 5.

SPA and TRPM2 antagonist 2‐APB attenuate HPX‐induced cell death in Hep‐2 cells (mean ± SD, and n = 8). (a) Representative fluorescence microscopy images of propidium iodide (PI, red) and Hoechst 33342 (blue) staining in different treatment groups (×objective 20). (b) Quantification of PI‐positive cell percentages. (^{a, b, c} p < 0.001 vs. the other groups).

4. Discussion

HPX is a well‐known pathogenic factor contributing to metabolic reprogramming, redox imbalance, cytokine activation, and treatment resistance in solid tumors. The present study demonstrates that SPA exhibits potent cytoprotective effects against HPX‐induced oxidative, inflammatory, and calcium‐dependent damage in Hep‐2 cells. The findings of this study confirm that hypoxic stress disrupts multiple layers of cellular homeostasis, suggesting that SPA can alleviate these disturbances through multifactorial mechanisms, via the TRPM2 channel.

The significant increase in intracellular ROS levels following CoCl₂ exposure is consistent with HPX's known role as a primary trigger of OS [28]. Previous studies have also shown that HPX‐mimicking agents can cause excessive ROS production by disrupting the mitochondrial electron transport chain and activating NADPH oxidases in various cancer and neuronal models [29, 30]. Similar to previous observations, the current study documented significant ROS accumulation and elevated MDA levels in hypoxic Hep‐2 cells, suggesting increased lipid peroxidation and membrane instability. SPA's ability to markedly reduce ROS and restore GSH levels demonstrates a strong antioxidant capacity. This aligns with earlier studies describing phenolic compounds as effective free‐radical scavengers; however, the present work advances these findings by showing that SPA restores redox balance in a hypoxic LSCC model—a context where OS is particularly high [19, 31]. The restoration of GSH is especially important, as reduced GSH depletion is one of the earliest events in oxidative damage and strongly predisposes cells to apoptosis [32, 33]. The current findings, therefore, reflect a substantial strengthening of endogenous antioxidant defenses mediated by SPA.

HPX is closely intertwined with inflammation, as excessive ROS facilitates activation of redox‐sensitive transcription factors and inflammasome pathways [34, 35]. The elevated TNF‐α and IL‐1β concentrations observed in hypoxic Hep‐2 cells in this study confirm the established link between OS and inflammatory amplification [36, 37]. Persistent activation of these cytokines is known to promote cell damage, mitochondrial dysfunction, and tumor progression [38, 39]. The demonstration that SPA markedly reduces both TNF‐α and IL‐1β levels suggests that the compound modulates upstream regulators that couple OS to inflammation. Previous studies have similarly shown that phenolic acids can suppress pro‐inflammatory cascades [18, 40], but this is the first evidence that SPA exerts such an effect in LSCC cells under hypoxic conditions.

The significant upregulation of the TRPM2 channel after HPX is among the most remarkable results of the current investigation. TRPM2 is known to be activated by oxidative metabolites, such as ADP‐ribose and hydrogen peroxide, and its activation leads to sustained Ca²⁺ influx. This excessive Ca²⁺ entry plays an important role in mitochondrial depolarization, activation of caspases, and promotion of apoptotic pathways [12, 13].

In previous experimental systems, including neuronal, retinal, and immune cells, TRPM2 activation has been established as a pivotal event in OS‐induced damage [17, 41, 42]. The data presented here indicate that a similar mechanism operates in LSCC cells, where HPX markedly enhances TRPM2 protein expression. This suggests that LSCC cells rely on TRPM2‐mediated Ca²⁺ entry as part of their maladaptive response to HPX. A particularly important finding is that SPA significantly suppresses TRPM2 upregulation. Although SPA is not classified as a direct TRPM2 antagonist, its ability to reduce ROS levels may indirectly limit TRPM2 activation, since oxidative metabolites are strong activators of the channel. Recent evidence further highlights TRPM2 as a critical redox‐sensitive Ca²⁺ channel involved in HPX‐associated mitochondrial dysfunction, inflammatory signaling, and cancer cell survival. Emerging studies have demonstrated that TRPM2‐mediated oxidative Ca²⁺ influx contributes to mitochondrial instability and cytokine amplification in various pathological conditions, including tumor progression and ischemic injury [12, 13, 14, 41]. Therefore, modulation of TRPM2 activity may represent a promising therapeutic strategy for controlling HPX‐related oxidative and inflammatory damage in LSCC cells.

Mitochondrial depolarization, as assessed by JC‐1 staining, confirms that HPX causes severe mitochondrial impairment, as commonly reported in cellular models subjected to OS. Depolarized mitochondria are unable to maintain ATP production, regulate Ca²⁺ buffering, or prevent cytochrome c release, all hallmarks of the initiation of programmed cell death [43, 44]. SPA's ability to restore mitochondrial membrane potential is particularly noteworthy. This suggests a stabilization of the mitochondrial environment, likely resulting from reduced ROS, corrected Ca²⁺ imbalance, and diminished inflammatory signaling. The combined use of SPA and 2‐APB, a TRPM2 inhibitor [45], produced the strongest reduction in [Ca²⁺]_c levels and cell death [46]. This synergistic effect reinforces the conclusion that TRPM2 serves as a major conduit for Ca²⁺ overload during hypoxic stress. The dramatic decline in PI‐positive apoptotic cells in the SPA + 2‐APB group underscores the central role of TRPM2 in HPX‐driven apoptosis. Therefore, combining redox‐modulating agents such as SPA with targeted TRPM2 blockers could represent a promising strategy for future therapeutic development.

Although the present study provides comprehensive evidence of SPA's protective effects against HPX‐induced cellular injury, several limitations should be considered. The current findings were obtained under in vitro conditions using Hep‐2 cells; further studies across different experimental models may contribute to a broader understanding of the underlying mechanisms. In addition, while the results strongly support the involvement of TRPM2‐mediated pathways, future studies focusing on more detailed molecular signaling mechanisms may further clarify the protective actions of SPA under hypoxic conditions.

5. Conclusion

This study demonstrates that SPA effectively protects Hep‐2 cells from HPX‐induced damage by reducing OS, suppressing inflammatory cytokines, stabilizing mitochondrial membrane potential, and limiting TRPM2‐mediated Ca²⁺ overload. The findings suggest that TRPM2 activation is a key driver of hypoxic damage, and that SPA primarily mitigates this pathway through its antioxidant and anti‐inflammatory actions. The enhanced protection observed with TRPM2 inhibition further highlights the channel's central role in HPX‐associated cytotoxicity. Overall, SPA emerges as a promising candidate for targeting redox‐ and calcium‐dependent mechanisms in HPX‐related cellular dysfunction.

Author Contributions

Supervision: Halil İbrahim Altıner, Ramazan Çınar, and Kenan Yıldızhan. Study design: Halil İbrahim Altıner, Tarık Yağcı, Ramazan Çınar, and Kenan Yıldızhan. Literature search: All authors. Data collection: Halil İbrahim Altıner, Ramazan Çınar, and Tarık Yağcı. Data assessment: Halil İbrahim Altıner, Ramazan Çınar, Kenan Yıldızhan, and Tarık Yağcı. Western blot analyses: Ramazan Çınar. Statistical evaluation: Kenan Yıldızhan and Ramazan Çınar. Manuscript preparation: Kenan Yıldızhan and Halil İbrahim Altıner. All authors reviewed the manuscript.

Ethics Statement

There is no information about humans, living animals, or higher invertebrates.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: The raw protein band expression data of TRPM2 (A) in the Hep‐2 cells. (n = 3). The expression of TRPM2 protein levels in the four groups (CTR, SPA, HPX, and HPX + SPA) was assessed using a standard Western blot technique. β‐Actin (B) levels were used to control the loading amount.

Data Availability Statement

Data supporting the findings of this study can be obtained from the corresponding author upon reasonable request and in accordance with scientific and ethical standards.