Studies indicate that Transient Receptor Potential Melastatin-2 (TRPM2), a nonselective Ca²? permeable ion channel, contributes to OS, leading to intracellular Ca²? overload and cell death in neurons⁵. TRPM2 is subsequently activated by ROS, leading to ADP-ribose production (often via PARP-1 activity) and intracellular Ca²?, creating a feedback loop that increases calcium influx and oxidative damage^6,7. Indeed, TRPM2-mediated Ca²? influx has been shown to exacerbate ROS accumulation, mitochondrial membrane permeability, and caspase cascade activation in various neuronal and non-neuronal systems^8,9. Recent studies have further highlighted the dual regulatory role of TRPM2, which depends on the cellular redox status; in this context, modulation can either alleviate or exacerbate oxidative damage¹⁰.

Currently, there is increasing interest in natural phytochemicals with antioxidant, anti-inflammatory, and cytoprotective properties. Among these, zingerone (ZGN) (4-(4-hydroxy-3-methoxyphenyl)-2-butanone), a pungent compound derived from ginger (Zingiber officinale), is emerging as a promising candidate. Recent preclinical studies and systematic reviews have documented the ability of ZGN to prevent neuroinflammation, OS, and behavioral deficits in models of cognitive impairment, heavy metal toxicity, and mood disorders^11,12. For example, it has been found to attenuate cadmium-induced oxidative damage and cognitive decline and also to attenuate inflammation in pain models through modulation of Ca²? signaling and neuronal excitability (13, 14). However, its direct effects on TRPM2-mediated pathways in neuronal cells under proconvulsant stress have not yet been addressed in the literature.

The present study investigated the neuroprotective effects of ZGN in SH-SY5Y cells exposed to PTZ through inhibition of the PARP-1/TRPM2 signaling cascade. Cell viability, OS markers (MDA and GSH), proinflammatory cytokine expression (IL-1? and TNF-?), apoptotic mediators (caspase-3 and -9), and TRPM2 and PARP-1 protein expression were examined. The study aimed to elucidate the mechanistic link between the antioxidant/anti-inflammatory effects of ZGN and the modulation of ionic signalling in a neuronal toxicity model.

Cell Culture: The SH-SY5Y cells were sourced from the ATCC. The cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 ?g/mL) (Sigma?Aldrich). They were incubated at 37°C in a humidified atmosphere containing 5% CO?. To maintain optimal bioavailability, the culture medium was renewed every other day, and the cells were seeded at suitable densities according to each experimental design.

Cell Viability Assay and Study Groups: Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Abbkine, Cat_KTA1020), a sensitive and reproducible colorimetric assay. SH-SY5Y cells were seeded in 96-well plates at a density of 1 × 106 /mL per well in a total volume of 100 ?L. Cells were treated with ZGN (5, 10, 15, 20, 25 ?M) at 85% confluency for 24 h. According to the results obtained, the 25 ?M ZGN dose was determined as the highest safe dose that did not cause a significant decrease in cell viability; cytotoxic effects were observed at higher concentrations. Absorbance values indicating the number of viable cells were measured at 450 nm using a BioTek EL808? microplate reader according to the manufacturer's instructions. The assay was performed independently in triplicate for each experimental group, and results were expressed as a percentage relative to the untreated control group. The study groups were formed as follows: the control group consisted of cells incubated under standard culture conditions without the application of any substance. ZGN group: Cells were treated with only zingerone (25 ?M) for 24 hours. PTZ group: Cells were exposed to PTZ (30 ?M) for 24 hours⁴. PTZ+ ZGN group: Cells were treated with PTZ (30 ?M) and ZGN (25 ?M) together for 24 hours. The experiment was performed independently three times for each experimental group.

Biochemical Analysis: Commercial ELISA commercial kits were used to measure the amounts of MDA, GSH, IL-1?, TNF-?, Caspase 3, and Caspase 9 in the cell supernatants that were collected. Analyses were performed according to the manufacturer?s instructions (Sun. Red Biotech China). The absorbance values were measured using an ELISA spectrophotometer (BioTek EL808?).

Western Blotting Analysis: The total protein concentration in the cells was measured spectrophotometrically using the BCA kit (Thermo Fisher Scientific, 23227). Each well was loaded with 50 µg of protein. Electrophoresis was performed using the Bio-Rad Mini-Protean Tetra Cell Gel Electrophoresis System. Afterwards, the gels were prepared for transfer. A sandwich model was created by layering a sponge, filter paper, gel, nitrocellulose membrane, filter paper, and sponge in the transfer cassette, arranged from cathode to anode. Proteins in the gel were then transferred to a nitrocellulose membrane using the Western blotting technique with the Bio-Rad semi-wet transfer system. The membranes were incubated overnight with primary antibodies diluted in 5% milk powder: TRPM2 (1:1000), PARP-1 (1:1000) and Beta-actin (1:1000). Following this, the membranes were incubated for one hour with secondary antibodies, which were also diluted in 5% milk powder (Anti-Mouse Secondary Antibody: 1:5000, Anti-Rabbit Secondary Antibody: 1:10000). The membranes were treated with a chemiluminescent conjugate (ECL), and band images were captured using the SYNGENE G: Box Chemi XRQ imaging device. The intensities of the bands in the images were analyzed using ImageJ software. Protein expression levels were normalized to beta-actin, which served as an internal control, and compared to the control group.

Statistical Analysis: All quantitative data obtained from cell viability assays, ELISA measurements, and Western blot analyses were expressed as mean ± standard deviation (SD). Prior to experimental procedures, an a priori power analysis was conducted using G*Power software to estimate the minimum sample size required to detect biologically meaningful differences. Assuming a medium effect size, an alpha value of 0.05, a statistical power of 0.80, and three experimental groups, the analysis indicated that at least eight independent samples per group were necessary. Therefore, all experiments were performed with n= 8 independent biological replicates. Data distribution was evaluated using the Shapiro?Wilk test for normality, and homogeneity of variances was assessed using Levene?s test. When parametric assumptions were met, one-way ANOVA followed by Tukey?s post-hoc test was applied. If these assumptions were not satisfied, the Kruskal?Wallis test with appropriate post-hoc comparisons was used as a non-parametric alternative. All statistical analyses were performed using SPSS software (version 17.0, USA). A p-value <0.05 was considered statistically significant.

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Figure 1: Effect of ZGN on PTZ-induced cytotoxicity in SH-SY5Y cells. Cell viability was assessed by MTT assay after exposure to PTZ (30 µM, 24 h) and/or ZGN (5-25 µM, 24 h). PTZ treatment significantly reduced cell viability compared with the control group, whereas ZGN pretreatment attenuated PTZ-induced cytotoxicity in a dose-dependent manner. Data are expressed as mean ± SD (n = 8). (ap<0.05 vs. control; bp<0.05 vs. PTZ).

ZGN Attenuates PTZ-Induced OS: To assess the oxidative status of SH-SY5Y cells, intracellular MDA (Figure 2a) and GSH (Figure 2b) levels were quantified. PTZ treatment significantly increased MDA levels and reduced GSH concentrations relative to control (p<0.05), suggesting enhanced lipid peroxidation and impaired antioxidant defence. ZGN pretreatment (25 µM) effectively counteracted these changes by lowering MDA and restoring GSH levels (p<0.05 vs. PTZ). These results demonstrate that ZGN mitigates PTZ-induced oxidative damage by reinforcing the antioxidant defence system of neuronal cells.

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Figure 2: Effects of ZGN on PTZ-induced OS parameters in SH-SY5Y cells. Malondialdehyde (MDA) and reduced glutathione (GSH) levels were analysed as indicators of lipid peroxidation and antioxidant defence, respectively. PTZ (30 µM, 24 h) significantly elevated MDA levels and decreased GSH levels (ap<0.05 vs. control), indicating OS induction. Pretreatment with ZGN (25 µM) markedly reduced MDA and restored GSH levels toward normal values (bp<0.05 vs. PTZ), suggesting that ZGN mitigates PTZ-induced oxidative damage. Data are expressed as mean ± SD (n = 8).

ZGN Suppresses PTZ-Induced Proinflammatory Responses: The inflammatory response triggered by PTZ was evaluated by measuring the cytokine levels of IL-1β (Figure 3a) and TNF-α (Figure 3b). PTZ exposure markedly increased both IL-1β and TNF-α levels compared with the control group (p<0.05), reflecting an acute neuroinflammatory process. Pretreatment with ZGN (25 µM) significantly reduced the elevated cytokine concentrations (p<0.05 vs. PTZ), demonstrating the anti-inflammatory potential of ZGN in preventing PTZ-induced inflammation in SH-SY5Y cells.

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Figure 3: Effects of ZGN on PTZ-induced inflammatory cytokine levels in SH-SY5Y cells. The concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were quantified by ELISA to evaluate the inflammatory response. PTZ (30 µ ,M, 24 h) markedly increased IL-1β and TNF-α levels compared with the control group (ap<0.05 vs. control). ZGN (25 µM) pretreatment significantly attenuated these elevations (bp<0.05 vs. PTZ), indicating its anti-inflammatory potential against PTZ-induced neuroinflammation. Data represent mean ± SD (n = 8).

ZGN Inhibits PTZ-Induced Apoptotic Pathways: To explore whether ZGN modulates PTZ-induced apoptotic signalling, the activities of caspase-3 (Figure 4a) and caspase-9 (Figure 4b) were measured. Both enzymes exhibited a significant increase in activity following PTZ exposure compared with control (p<0.05), confirming the induction of intrinsic apoptotic pathways. ZGN pretreatment (25 µM) markedly decreased caspase-3 and caspase-9 activities (p<0.05). These data suggest that ZGN protects neuronal cells from PTZ- mediated apoptosis by inhibiting caspase-dependent pathways.

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Figure 4: Effects of ZGN on PTZ-induced apoptosis-related enzyme activities in SH-SY5Y cells. Caspase-3 and caspase-9 activities were measured to assess apoptotic signalling. PTZ (30 µM, 24 h) significantly increased both caspase-3 and caspase-9 activities compared with the control group (ap<0.05 vs. control), indicating activation of intrinsic apoptotic pathways. Pretreatment with ZGN (25 µM) markedly suppressed PTZ-induced caspase activation (bp<0.05 vs. PTZ), demonstrating the anti-apoptotic effect of ZGN. Data are presented as mean ± SD (n = 8).

ZGN Regulates TRPM2 and PARP-1 Expression in PTZ-Treated Cells: Western blot analysis was conducted to evaluate the expression of TRPM2 (Figure 5a) and PARP-1 (Figure 5b) proteins, which are known mediators of OS and DNA damage. PTZ exposure resulted in a significant upregulation of both TRPM2 and PARP-1 compared to the control group (p<0.01). ZGN pretreatment markedly downregulated these proteins (p<0.01 vs. PTZ), normalising their expression levels toward control values. These results indicate that the neuroprotective effect of ZGN is mediated, at least in part, through the suppression of TRPM2 channel activation and PARP-1 associated apoptotic mechanisms.

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Figure 5: Effects of ZGN on PTZ-induced TRPM2 and PARP-1 expression levels. Western blot analysis showing TRPM2 and PARP-1 protein expression, with β-actin used as a loading control. Densitometric analysis demonstrated that ZGN treatment reduced the PTZ-induced upregulation of the TRPM2 channel and PARP-1 expression. Data are expressed as mean ± SD. (ap<0.01 vs. control; bp<0.01 vs. PTZ group).

In studies, PTZ exposure significantly reduced SH-SY5Y cell viability, consistent with its ability to induce excitotoxicity, mitochondrial dysfunction, and OS^3,4. Pretreatment with ZGN significantly restored cell viability in a concentration-dependent manner, demonstrating a potent cytoprotective profile. In a study using a similar model, PTZ was shown to cause neurotoxicity and increase inflammatory cytokine (TNF-?, IL-1?, and IL-6) and MDA levels, while decreasing GSH and GPx levels. Gallic acid treatment has been reported to exert a protective effect against PTZ-induced toxicity in SH-SY5Y cells¹⁵. Similar findings have been reported in neuronal models of rotenone, cadmium, and amyloid-?-induced cytotoxicity, with ZGN improving survival through modulation of redox signalling and mitochondrial integrity^16,17. These results confirm the capacity of ZGN to counter neurotoxic insults through both antioxidant and mitochondrial stabilizing mechanisms ¹⁸. In this study, consistent with literature data, PTZ caused cytotoxicity in SH-SY5Y cells by causing a significant decrease in cell viability. However, ZGN treatment significantly increased cell viability in a dose-dependent manner, demonstrating a protective effect (Figure 1). These results demonstrated a potent cytoprotective effect of ZGN against PTZ-induced neuronal damage.

In this study, PTZ caused a significant increase in lipid peroxidation (MDA) and consequent GSH depletion, leading to severe OS. ZGN pretreatment reversed these changes and restored redox balance (Figure 2). This is consistent with recent reports that ZGN enhances endogenous antioxidant systems (SOD, CAT, GPx) and suppresses ROS accumulation in neuronal and hepatic tissues^13,19,20. Mechanistically, the phenolic hydroxyl group acts as a free radical scavenger, while the methoxy moiety provides membrane permeability, allowing direct ROS neutralization^21,22. These biochemical properties may explain the observed reduction in PTZ-induced oxidative load.

In the current study, the significant increase in IL-1? and TNF-? following PTZ treatment suggests activation of neuroinflammatory cascades (Figure 3). ZGN significantly modulates both cytokines, suggesting an inhibitory effect on the inflammatory axis. Previous studies have documented that ZGN suppresses NF-?B nuclear translocation and NLRP3 inflammasome activation^23,24. Given that TRPM2 activation is associated with proinflammatory cytokine release^25,26, it is likely that the anti-inflammatory activity of ZGN is at least partially due to modulation of TRPM2-related signalling.

As seen in Figure 4, PTZ-induced cytotoxicity was accompanied by increased caspase-3 and caspase-9 activities, hallmarks of mitochondrial (intrinsic) apoptosis. ZGN pretreatment significantly suppressed both enzymes, confirming its anti-apoptotic potential. This is consistent with evidence that ZGN protects hippocampal and cortical neurons from toxin-induced apoptosis by preserving mitochondrial membrane potential and reducing cytochrome c release^14,18,27. The concomitant decrease in caspase-9 and caspase-3 suggests that ZGN likely acts upstream by stabilising mitochondrial integrity and preventing ROS-induced activation of the intrinsic death pathway.

Upregulation of TRPM2 and PARP-1 expression levels in PTZ-exposed cells (Figure 5) supports the concept that oxidative DNA damage and ADP-ribose overproduction contribute to Ca²-dependent cell death^28,29. ZGN significantly reduced the levels of both proteins, indicating a downregulatory effect on the PARP-1/ADPR-TRPM2 axis. PARP-1 is known to increase TRPM2 activation via ADP-ribose synthesis, and overactivation of this loop promotes Ca²? overload and apoptosis^28,29. ZGN can prevent ADPR accumulation by inhibiting PARP-1, thereby blocking TRPM2-mediated Ca²? influx and subsequent apoptotic signalling. These findings are consistent with recent evidence indicating that TRPM2 channel activation serves as a molecular hub sensitive to oxidative stress in neuronal damage^30-33.

This study provides compelling evidence that ZGN mitigates PTZ-induced OS, neuroinflammation, and apoptosis in SH-SY5Y cells through the downregulation of the PARP-1/TRPM2 signalling pathway. By restoring antioxidant capacity, suppressing pro-inflammatory cytokines, and inhibiting caspase-dependent apoptosis, ZGN confers broad neuroprotective effects against chemical-induced cytotoxicity. These findings highlight TRPM2 channel modulation as a mechanistic cornerstone of ZGN?s action, suggesting that natural vanilloid derivatives may represent viable therapeutic adjuvants for OS-related neurological disorders, such as epilepsy, ischemic injury, and neurodegeneration. Future studies employing in vivo PTZ seizure models and specific TRPM2 antagonists will be crucial to confirm causality and translational potential.

1) Singh A, Kukreti R, Saso L, et al. Oxidative stress: A key modulator in neurodegenerative diseases. Molecules 2019; 24: 1583.

2) Wang Y, Qin Z-h. Molecular and cellular mechanisms of excitotoxic neuronal death. Apoptosis 2010; 15: 1382-1402.

3) Taskiran AS, Ergul M. The modulator action of thiamine against pentylenetetrazole-induced seizures, apoptosis, nitric oxide, and oxidative stress in rats and SH-SY5Y neuronal cell line. Chemico-Biological Interactions 2021; 340: 109447.

4) Ahlatcı A, Yıldızhan K, Tülüce Y, et al. Valproic acid attenuated PTZ-induced oxidative stress, inflammation, and apoptosis in the SH-SY5Y cells via modulating the TRPM2 channel. Neurotoxicity Research. 2022; 40: 1979-1988.

5) Nazıroğlu M. TRPM2 cation channels, oxidative stress and neurological diseases: Where are we now? Neurochemical Research 2011; 36: 355-366.

6) Takahashi N, Kozai D, Kobayashi R, et al. Roles of TRPM2 in oxidative stress. Cell Calcium 2011; 50: 279-287.

7) Yazğan Y, Yıldızhan K. Chrysin boosts the cell death and anticancer actions of doxorubicin by stimulating the TRPM2 channel in glioblastoma cells. Molecular Biology 2025; 59: 784-794.

8) Osmanlıoğlu HÖ, Yıldırım MK, Akyuva Y, et al. Morphine induces apoptosis, inflammation, and mitochondrial oxidative stress via activation of TRPM2 channel and nitric oxide signaling pathways in the hippocampus. Molecular Neurobiology 2020; 57: 3376-3389.

9) Nazıroğlu M, Öz A, Yıldızhan K. Selenium and neurological diseases: focus on peripheral pain and TRP channels. Current neuropharmacology. 2020;18: 501-517.

10) Shitaw EE, AlAhmad M, Sivaprasadarao A. Inter-organelle crosstalk in oxidative distress: A unified TRPM2-NOX2 mediated vicious cycle involving Ca2+, Zn2+, and ROS Amplification. Antioxidants 2025; 14: 776.

11) Bashir N, Ahmad SB, Rehman MU, et al. Zingerone (4-(four-hydroxy-3-methylphenyl) butane-two-1) modulates adjuvant-induced rheumatoid arthritis by regulating inflammatory cytokines and antioxidants. Redox Report 2021; 26: 62-70.

12) Ahmad B, Rehman MU, Amin I, et al. A review on pharmacological properties of zingerone (4?(4?Hydroxy?3?methoxyphenyl)?2?butanone). The Scientific World Journal 2015; 2015: 816364.

13) Oviosun A, Oviosun EC, Nto NJ, et al. Zingerone attenuates cadmium-induced neuroinflammation, oxidative stress and cognitive deficit on the prefrontal cortex of adult wistar rats. Journal of Experimental Pharmacology 2025: 323-341.

14) Olasehinde TA, Olaokun OO. Zingerone as a neuroprotective agent against cognitive disorders: A systematic review of preclinical studies. International Journal of Molecular Sciences 2025; 26: 6111.

15) Yazğan Y. Effect of gallic acid on PTZ-induced neurotoxicity, oxidative stress and inflammation in SH-SY5Y neuroblastoma cells. Acta Med Alanya 2024; 8: 8-12.

16) Nageshwar Rao B, Satish Rao B. Antagonistic effects of Zingerone, a phenolic alkanone against radiation-induced cytotoxicity, genotoxicity, apoptosis and oxidative stress in Chinese hamster lung fibroblast cells growing in vitro. Mutagenesis 2010; 25: 577-587.

17) Lee C, Park GH, Kim C-Y, et al. [6]-Gingerol attenuates ?-amyloid-induced oxidative cell death via fortifying cellular antioxidant defense system. Food and Chemical Toxicology 2011; 49: 1261-1269.

18) Rashid S, Wali AF, Rashid SM, et al. Zingerone targets status epilepticus by blocking hippocampal neurodegeneration via regulation of redox imbalance, inflammation and apoptosis. Pharmaceuticals 2021; 14: 146.

19) Mani V, Arivalagan S, Siddique AI, et al. Antioxidant and anti-inflammatory role of zingerone in ethanol-induced hepatotoxicity. Molecular and cellular biochemistry. 2016; 421: 169-181.

20) Türk E, Güvenç M, Cellat M, et al. Zingerone protects liver and kidney tissues by preventing oxidative stress, inflammation, and apoptosis in methotrexate-treated rats. Drug and Chemical Toxicology 2022; 45: 1054-1065.

21) Chen J, Yang J, Ma L, et al. Structure-antioxidant activity relationship of methoxy, phenolic hydroxyl, and carboxylic acid groups of phenolic acids. Scientific Reports 2020; 10: 2611.

22) Parcheta M, ?wis?ocka R, Orzechowska S, et al. Recent developments in effective antioxidants: The structure and antioxidant properties. Materials 2021; 14: 1984.

23) Eriten B, Caglayan C, Gür C, et al. Hepatoprotective effects of zingerone on sodium arsenite-induced hepatotoxicity in rats: Modulating the levels of caspase-3/Bax/Bcl-2, NLRP3/NF-?B/TNF-? and ATF6/IRE1/PERK/GRP78 signaling pathways. Biochemical and Biophysical Research Communications 2024; 725: 150258.

24) Hsiang CY, Cheng HM, Lo HY, et al. Ginger and zingerone ameliorate lipopolysaccharide-induced acute systemic inflammation in mice, assessed by nuclear factor-?B bioluminescent imaging. Journal of Agricultural and Food Chemistry 2015; 63: 6051-6058.

25) Yağcı T, Çınar R, Altıner Hİ, et al. The role of TRPM2 channel in doxorubicin-induced cell damage in laryngeal squamous cancer cells. Doklady Biochemistry and Biophysics 2025: 520: 123-129.

26) Yıldızhan K, Nazıroğlu M. Glutathione depletion and parkinsonian neurotoxin MPP+-induced TRPM2 channel activation play central roles in oxidative cytotoxicity and inflammation in microglia. Molecular Neurobiology 2020; 57: 3508-3525.

27) Vaibhav K, Shrivastava P, Tabassum R, et al. Delayed administration of zingerone mitigates the behavioral and histological alteration via repression of oxidative stress and intrinsic programmed cell death in focal transient ischemic rats. Pharmacology Biochemistry and Behavior. 2013; 113: 53-62.

28) Yıldızhan K, Nazıroğlu M. Protective role of selenium on MPP+ and homocysteine-induced TRPM2 channel activation in SH-SY5Y cells. Journal of Receptors and Signal Transduction 2022; 42: 399-408.

29) Mortadza SS, Sim JA, Stacey M, et al. Signalling mechanisms mediating Zn2+-induced TRPM2 channel activation and cell death in microglial cells. Scientific Reports 2017; 7: 45032.

30) Kashio M, Tominaga M. Redox-sensitive TRP channels: TRPA1 and TRPM2. Redox: Principles and Advanced Applications 2017; 203.

31) Wang G, Cao L, Liu X, et al. Oxidant sensing by TRPM2 inhibits neutrophil migration and mitigates inflammation. Developmental Cell 2016; 38: 453-462.

32) Yazğan Y, Keleş ÖF, Bayir MH, et al. Selenium reduces cadmium-induced cardiotoxicity by modulating oxidative stress and the ROS/PARP-1/TRPM2 signalling pathway in rats. Toxics 2025; 13: 611.

33) Keleş ÖF, Bayir MH, Çiçek HA, et al. The effect of selenium against cadmium-ınduced nephrotoxicity in rats: The role of the TRPM2 channel. Toxics 2025;13: 87.