Introduction

Seizure is a temporary dysfunction of the brain caused by an excessive and abnormal discharge of cortical neurons. Epilepsy is a central nervous system disorder characterized by repetitive seizures that usually occur without provocation due to a genetic predisposition or an underlying chronic pathological condition (Tastemur et al. 2020). It is one of the most common neurological disorders and affects about 1% of the world's population (Taskıran et al. 2018). Epileptic seizures are related to a change in ion transport mechanisms in neuronal networks and directly cause the imbalance of the excitation–inhibition and indirectly cause oxidative stress and neuroinflammation (Staley 2015). The frequent incidence of epileptic seizures significantly declines the learning and memory capacity in patients with epilepsy. It has been demonstrated that pentylenetetrazole (PTZ)-induced seizures can lead to memory impairment in rodents (Lamberty and Klitgaard 2000). The cortex and hippocampus are both effective on memory and learning, and also on the onset, generalization, and termination of seizures. Epileptic seizures cause neuronal death by exaggerating calcium influx into cells. In this way, the brain neurodegenerates, especially in the hippocampus, and changes the function of changeable synapses that store information, which can possibly be an explanation of the observed learning disorder following seizure (Ramos et al. 2012).

Experimental epilepsy models have been developed to understand the basic mechanisms involved in epileptic seizures. PTZ, a selective antagonist of the GABAA receptor, is a chemical agent used to create experimental seizures. PTZ exerts adverse effects on the neuronal membrane, affects potassium and calcium channels, releases intracellular calcium ion reserves, and reduces the neurotransmitter-induced chloride conductance (Potschka 2012). This model is clinically regarded as a response to temporal lobe epilepsy (TLE) and complex partial epilepsy (Potschka 2012).

Proton pump inhibitors (PPIs), such as pantoprazole, are benzimidazole-derivative drugs. They have antiulcer activity and are usually used for peptic ulcer treatment worldwide. PPIs suppress acid secretion by irreversible binding to hydrogen-potassium ATPase (H+/K+-ATPase) enzymes, which are located in the terminal of the parietal cells (Nakagawa et al. 2012). As well as their inhibitory effects on gastric acid secretion, PPIs have some other positive effects in the organisms, such as being anti-inflammatory, an antioxidant, and having apoptotic cell death control activities (Rai et al. 2011a, b). Moreover, in vitro studies have shown that PPIs also inhibit the release of tumor necrosis factor-alpha and interleukin-6 from the microglial cell and monocytes. These also contribute to the neuroprotective effect of PPIs against microglial, astrocyte, and monocytic toxicities (Hashioka et al. 2011). Furthermore, it has been suggested that PPIs reduce interferon-induced neurotoxicity by inhibiting the signal transducer and activator of the transcription protein 3 (STAT3) pathway (Hashioka et al. 2011). However, the effects of PPIs on epileptic seizures and neuronal cell death after seizures are still unclear. In this study, it was intended to investigate the effect of pantoprazole on pentylenetetrazole-induced epileptic seizures in rats, and pentylenetetrazole-induced neurotoxicity in the SH-SY5Y cell line.

Materials and Methods

The study was performed in in vivo and in vitro parts (Fig. 1).

Fig. 1
figure 1

Experimental protocols of study (created by BioRender)

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In Vivo Studies

Animals

Eighteen male adult Wistar Albino rats with bodyweights of 230–250 g were included in the experiment (n = 18). The animals included in the experiment were kept at an ambient temperature of 22 ± 3 °C and in a two cycles of 12 h of light and 12 h of darkness. Ad libitum access was provided to animals for water and food. It was ensured that animals included in the experiment were acclimatized to laboratory conditions before starting the tests. All experiments were carried out in a blinded setting and were conducted in the hours from 09:00 to 17:00. Each experimental group consisted of six animals. The Animal Ethics Committee of Sivas Cumhuriyet University approved the study protocol (Approval no: 65202830-050.04.04-357).

Drug Administration

Pantoprazole and pentylenetetrazole (PTZ) (Sigma-Aldrich Co., St Louis, MO, USA) were dissolved in physiological saline. Each drug solution was prepared freshly on the days of the experiments.

Experimental Protocols

The animals were assigned to three experimental groups randomly (n = 6 for each group). Group 1 was defined as the control group. The rats in group 2 received saline intraperitoneally (i.p.) at a dose of 1 mL /kg for 7 days. Group 3 was the pantoprazole group and the rats in this group received 10 mg/kg dose of pantoprazole for 7 days (i.p). Pentylenetetrazole (PTZ) was given to the rats to induce epileptic seizures at doses of 45 mg/kg (i.p). After 30 min, the administration of the last dose of the medicine was made. The seizure severity was scored based on Modifies Racine's Convulsion Scale (RCS) where seizure stages are defined by the RCS as follows: 0 = no convulsion; 1 = twitching of vibrissae and pinnae; 2 = motor arrest with more pronounced twitching; 3 = myoclonic jerks; 4 = tonic–clonic seizure while the animal remained on its feet; 5 = tonic–clonic seizure with loss of the righting reflex; 6 = tonic–clonic seizure with wild climbing and jumping; and 7 = lethal seizure (Erdogan et al. 2019). The rats were observed for 30 min following PTZ injection, both for behavioral scoring according to the RCS and for determining the time of the first myoclonic jerk (FMJ). Twenty-four hours after PTZ injection, a passive avoidance test trial was performed and then the animals were sacrificed by decapitation. The brain tissue obtained from the animals underwent assessments.

Passive Avoidance Test

The passive avoidance (PA) learning test based on negative reinforcement was carried out. The apparatus had a grid floor and comprised two compartments: one dark and the other one lighted, with a small gate connecting these two parts. This test was performed with the knowledge that rats have a native preference for a dark environment. Before beginning the training session, the animals were familiarized with the apparatus for two successive days (300 s per day). On the ensuing day, they were placed in the lighted compartment and the time latency for entering the dark compartment was noted down. During the training phase, the animals were located in the lighted compartment while facing towards the walls away from the gate, and received an electric shock (1 mA, 5 s duration) when they entered the dark part. The animals were then returned to their cages. In the retention test phase that was carried out 1 h after the training sessions, the rats were placed in the light compartment, and time latency to enter the dark compartment was recorded.

Preparation of Brain Tissue Homogenates

After mixing the brain tissue samples (both cortex and hippocampus area of the brain by separating) of the animals with a cold phosphate-buffered saline (PBS, pH 7.4) solution, the tissue samples were homogenized using a mechanical homogenizer (Analytic Jena Speed Mill Plus, Jena, Germany). The homogenates were centrifuged at 4000 rpm for 10 min at a temperature of 4 °C. Then, the supernatants were obtained and stored in ice until biochemical analysis was performed. A Bradford protein assay kit (Merck Millipore, Darmstadt, Germany) was used to determine the total protein levels in the samples (Ernst and Zor 2010).

Measurement of 8-Hydroxy-2′-Deoxyguanosine (8-Ohdg), Caspase-3, and Brain-Derived Neurotrophic Factor (BDNF)

The levels of 8-OHdG, caspase-3, and BDNF from brain supernatants were measured using rat ELISA commercial kits (Shanghai YL Biotech Co., Shanghai, China). The operation protocols were according to the manufacturer’s instructions. In brief, standard and tissue samples were added in the plate and incubated for 60 min at 37° C. After the washing step, staining solutions were added and incubated for 15 min at 37° C. Stop solution was added and read at 450 nm by the ELISA reader (Thermo Fisher Scientific, Altrincham, UK). Standard curves were used for calculations for all of these kits. The coefficients of variation within and between the plates were less than 10%.

In Vitro Studies

Cell Culture

SHSY-5Y (CRL-2266) cell lines were obtained from American Type Culture Collection and cultured in DMEM (Thermo Fisher Scientific, Altrincham, UK) containing 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich Co., St Louis, MO, USA), 1% l-glutamine (Sigma-Aldrich Co., St Louis, MO, USA), and 1% penicillin/streptomycin (Sigma-Aldrich Co., St Louis, MO, USA). The cells were maintained at 37 °C within a 5% CO2 humidified atmosphere. Pantoprazole and pentylenetetrazole (PTZ) (Sigma-Aldrich Co., St Louis, MO, USA) were dissolved in DMEM and stock solutions were prepared before treatment.

Cell Viability Assay

Cell viability was assessed using the XTT assay (Roche Diagnostic, MA, USA). SHSY-5Y cells were seeded in 96-well plates at a density of 1 × 104 cells per well in 100-μL DMEM and grown overnight before treatment. The following day, four cell groups were prepared to evaluate the neuroprotective effect of pantoprazole. The control group did not undergo any treatment. Cells in the PTZ group were treated with 30 mM PTZ for 24 h. Cells in the pantoprazole group were treated with various concentrations (10, 25, 50, 100, 200, 400 µM) of pantoprazole for 24 h. Cells in the PTZ + pantoprazole group were pre-treated with various concentrations (12.5, 25, 50, 100, 200, 400 µM) of pantoprazole for 1 h and then exposed to 30 mM PTZ for 24 h. After incubation, the medium was removed and wells were washed two times with phosphate-buffered saline. In the last step, 100 μL DMEM without phenol red, and a mixture of 50 μL XTT labeling solution was added to all the wells, and then the plates were maintained at 37 °C for 4 h. The plates were shaken and the absorbance was detected using an ELISA micro-plate reader (Thermo Fisher Scientific, Altrincham, UK) at 450 nm. All the experiments were performed three times and the cell viability was measured as viable cell amount percentages compared to untreated cells (control).

Annexin V Binding Assay

Apoptosis was evaluated using the Muse Annexin V/Dead Cell (Merck Millipore, Darmstadt, Germany) assay. Shortly after, the cells were seeded into six-well plates and were allowed to attach overnight before treatment. The cells were then treated with pantoprazole (10 µM), PTZ (30 mM) or their combinations and incubated for 24 h. After incubation, an Annexin V assay was performed according to the report in our previous study (Ergul and Bakar-Ates 2019).

Preparation of Cell Homogenates

The cells were collected by sterile tubes and were centrifuged at 2000 RPM for approximately 10 min. The supernatants were removed and the components of the cells, which were at the bottom of the tubes, were suspended using PBS (pH 7.4) to dilute cell suspension of the cell concentration to approximately 1 million/mL. The cells were damaged through repeated freeze–thaw cycles to let out the internal components and were then centrifuged at 4000 rpm for 10 min at a temperature of 4 °C. Then, the supernatants were collected for biochemical analysis of total antioxidant status (TAS) and total oxidant status (TOS) by using TAS and TOS commercial kits (Rel Assay Kit Diagnostics, Antep, Turkey). The Bradford protein assay kit (Merck Millipore, Darmstadt, Germany) was used to determine the total protein levels in the samples.

Measurement of TAS

The TAS concentrations at the cell supernatants were determined with an automated assay method that was previously developed by Erel (Erel 2005). The method is based on monitoring the reaction rate of free radicals by measuring the absorbance of colored dianisidyl radicals during free radical reactions starting with the production of hydroxyl radicals in Fenton reaction. Antioxidants in the tissue samples should suppress coloring proportionally to their concentrations (Erel 2005). Accordingly, the results were expressed in micromolar Trolox equivalents per milligram tissue protein (μmol Trolox Eq/mg protein).

Measurement of TOS

Tissue TOS concentrations at the cell supernatants were quantified with the automated assay method of Erel (Erel 2004). As ferrous ions are oxidized into ferric ions when adequate quantities of oxidants are available in the medium, the method allows for quantifying TOS levels by measuring tissue levels of ferric ions with the use of xylenol orange. Hydrogen peroxide was used for the calibration of the assay (Erel 2004). The results of the assay were expressed in micromolar hydrogen peroxide equivalents per milligram tissue protein (μmol H2O2 Eq/mg protein).

Statistical Analysis

The results were expressed as a mean ± standard error of the mean (SEM). The data analyses were performed with SPSS Version 23.0 for Windows. The data were evaluated using a one-way analysis of variance (ANOVA). A post hoc Tukey test was utilized to identify the differences between the experimental groups, and a value of P < 0.05 was accepted as statistically significant.

Results

Effects of Pantoprazole on the Seizure Stage and First Myoclonic Jerk After PTZ-Induced Seizure in the Rats

In the present study, epileptic parameters were determined using the video recordings after PTZ injection to rats. As shown in Fig. 2, the epileptic seizure scores according to the Racine scale were statistically significant different between the saline + PTZ and the pantoprazole + PTZ groups (P < 0.05). Moreover, the FMJ time in pantoprazole + PTZ group was significantly higher than the saline + PTZ group (P < 0.01).

Fig. 2
figure 2

Effects of pantoprazole on seizure stage (RCS) and first myoclonic jerk (FMJ) after PTZ-induced seizure in rats (n = 6). Values are presented as mean ± SEM; *P < 0.05 and **P < 0.01 compared to the saline + PTZ group

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Effects of Pantoprazole on the Passive Avoidance Test following PTZ-induced Seizure in the Rats

In order to evaluate memory impairment after PTZ-induced seizures, the passive avoidance test was used. There were no statistically significant differences between the groups in terms of training trails (P > 0.05; Fig. 3a). However, there was statistically significant difference between the saline + PTZ and the control group in terms of the test trials (P < 0.001; Fig. 3b). On the other hand, the test trial time in the pantoprazole + PTZ group was significantly higher than the saline + PTZ group (P < 0.001; Fig. 3b).

Fig. 3
figure 3

Effects of pantoprazole on passive avoidance test after PTZ-induced seizure in rats (n = 6). Values are presented as mean ± SEM; ###P < 0.001 compared to control group; ***P < 0.001 compared to the saline + PTZ group

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Effects of Pantoprazole on 8-Ohdg Levels in the Cortex and Hippocampus After PTZ-Induced Seizure in the Rats

The 8-OHdG levels in the cortex and hippocampus were evaluated using the ELISA kit and 8-OHdG levels were found higher in the saline + PTZ group than in the control group in the cortex and hippocampus (P < 0.001; Fig. 4a and b). Nevertheless, pantoprazole significantly reduced 8-OHdG levels in both the cortex and hippocampus compared to the saline + PTZ group (P < 0.001; Fig. 4a and b).

Fig. 4
figure 4

Effects of pantoprazole on 8-OHdG levels in the cortex (a) and hippocampus (b) after PTZ-induced seizure in rats (n = 6). Values are presented as mean ± SEM; ###P < 0.001 compared to the control group; ***P < 0.001 compared to the saline + PTZ group

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Effects of Pantoprazole on Caspase-3 Levels in the Cortex and Hippocampus After PTZ-Induced Seizure in the Rats

The ELISA measurements were also performed to assess the effects of pantoprazole in the cortex and hippocampus on caspase-3 levels in the control, saline + PTZ, and pantoprazole + PTZ groups. Caspase-3 levels were found higher in the saline + PTZ group than in the control group in both the cortex and hippocampus (P < 0.01; Fig. 5a and b). However, pantoprazole significantly decreased caspase-3 levels only in the hippocampus when compared to the saline + PTZ group (P < 0.05; Fig. 5b).

Fig. 5
figure 5

Effects of pantoprazole on caspase-3 levels in the cortex (a) and hippocampus (b) after PTZ-induced seizure in rats (n = 6). Values are presented as mean ± SEM; #P < 0.05 and ##P < 0.01 compared to the control group; *P < 0.05 compared to the saline + PTZ group

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Effects of Pantoprazole on BDNF Levels in the Cortex and Hippocampus After PTZ-Induced Seizure in the Rats

BDNF levels in the cortex and hippocampus were determined using an ELISA kit. There were no statistically significant differences between the groups in the cortex levels of BDNF (P > 0.05; Fig. 6a). However, BDNF levels were found be higher in the hippocampus in the saline + PTZ, and pantoprazole + PTZ groups than in the control group (P < 0.05 to P < 0.001; Fig. 6b). Furthermore, pantoprazole significantly increased BDNF levels in the hippocampus compared to the saline + PTZ group (P < 0.001; Fig. 6b).

Fig. 6
figure 6

Effects of pantoprazole on BDNF levels in the cortex (a) and hippocampus (b) after PTZ-induced seizure in rats (n = 6). Values are presented as mean ± SEM; #P < 0.05 and ###P < 0.001 compared to control group; *P < 0.05 compared to PTZ group

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Effects of Pantoprazole on Cell Viability After PTZ-Induced Neurotoxicity in SH-SY5Y Cells

An XTT cell proliferation assay was performed to evaluate the neuroprotective effects of pantoprazole on PTZ-induced SH-SY5Y cell toxicity. In our previous study, the IC50 value of PTZ in the SH-SY5Y cell was found as 30 mM and this value was used in this study (Kalantaripour et al. 2017). As presented in Fig. 7, 30 mM constant concentration of PTZ treatment significantly reduced cell viability compared to the control (P < 0.001). Moreover, when applied together, pantoprazole exhibited significant neuroprotective effects at 12.5–100 mg/mL concentrations (P < 0.01). In addition, treatment of pantoprazole at the dose of 200 mg/mL showed toxicity in SH-SY5Y cells (P < 0.05; Fig. 7), but not at the concentrations of 12.5–100 mg/mL (P > 0.05; Fig. 7).

Fig. 7
figure 7

Effects of pantoprazole on cell viability after PTZ-induced neurotoxicity in SH-SY5Y cells. Values are presented as mean ± SEM; ###P < 0.001 compared to the control group; **P < 0.01 compared to the saline + PTZ group

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Effects of Pantoprazole on Apoptosis After PTZ-Induced Neurotoxicity in SH-SY5Y Cells

Flow cytometry was performed to evaluate the anti-apoptotic effects of pantoprazole on PTZ-induced SH-SY5Y cells. The results of flow cytometry analysis are given in Fig. 8 and it is exhibited that PTZ treatment remarkably increased the proportion of apoptotic cells at IC50 concentration for 24 h when compared to the control (P < 0.01 to P < 0.001). Furthermore, pantoprazole treatment at a dose of 50 mg/mL significantly reduced the apoptotic percentage of SH-SY5Y cells after PTZ-induced neurotoxicity (P < 0.01 to P < 0.001). However, treatment of pantoprazole alone did not demonstrate significant apoptotic effects in SH-SY5Y cells (P > 0.05; Fig. 7).

Fig. 8
figure 8

Effects of pantoprazole on apoptosis after PTZ-induced neurotoxicity in SH-SY5Y cells. Values are presented as mean ± SEM; ##P < 0.01 and ###P < 0.001 compared to the control group; **P < 0.01 and **P < 0.001 compared to the saline + PTZ group

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Effects of Pantoprazole on TAS and TOS Levels After PTZ-Induced Neurotoxicity in SH-SY5Y Cells

The TAS and TOS levels in SH-SY5Y cells were measured using commercial kits. TAS levels in SH-SY5Y cells were significantly decreased in only PTZ-treated cells compared to the control (P < 0.01; Fig. 9a). Pantoprazole treatment at a dose of 50 mg/mL significantly improved TAS levels in SH-SY5Y cells after PTZ-induced neurotoxicity (P < 0.05; Fig. 9a). Moreover, pantoprazole treatment at a dose of 50 mg/mL significantly reduced the TOS level in PTZ-induced SH-SY5Y cells (P < 0.01; Fig. 9b). However, pantoprazole alone had no significant effect on the TOS level in SH-SY5Y cells (P > 0.05; Fig. 9b).

Fig. 9
figure 9

Effects of pantoprazole on TAS (a) and TOS (b) levels after PTZ-induced neurotoxicity in SH-SY5Y cells. Values are presented as mean ± SEM; ##P < 0.01 and ###P < 0.001 compared to control group; *P < 0.05 and ***P < 0.001 compared to the saline + PTZ group

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Discussion

In this study, the effect of pantoprazole, a proton pump inhibitor, on both PTZ-induced seizures in rats and on PTZ-induced neurotoxicity in the SH-SY5Y neuroblastoma cell line was evaluated. With respect to in vivo, it was found that pantoprazole significantly decreased the seizure stages and increased first myoclonic jerk time in rats. Furthermore, pantoprazole alleviated the memory impairment that occurred after epileptic seizures. Moreover, pantoprazole decreased 8-OHdG, caspase-3 levels, and also increased BDNF levels after PTZ-induced epileptic seizures in rats. Regarding the in vitro trial, pantoprazole pretreatment in SH-SY5Y cells decreased neuronal death after PTZ-induced neurotoxicity. On the other hand, pantoprazole pretreatment increased TAS levels while reducing TOS levels in SH-SY5Y cells after PTZ-induced neurotoxicity.

Oxidative stress, because of an oxidant and antioxidant defense imbalance, has an important role in the onset of epileptic seizures and the progression of epileptogenesis. This has already been demonstrated in clinical and experimental studies (Aguiar et al. 2012). One clinical study has shown that lipid peroxidation was significantly higher in epileptic patients than in healthy persons (Hamed et al. 2004). Another study has demonstrated the levels of antioxidant systems markers such as glutathione reductase, vitamins C, E, and A were significantly lower in epileptic patients than in healthy persons (Waldbaum and Patel 2010). Moreover, experimental studies have shown that PTZ-induced epileptic seizures give rise to the production of free radicals and oxidative damage to proteins, lipids, and cell DNA. The high levels of mitochondrial superoxide, the inactivation of iron- and sulfur-dependent enzymes, and also iron-induced toxicity may contribute to oxidative damage to neurons following epileptic seizures (Sudha et al. 2001). In this study, oxidative damage marker (8-OHdG) levels in PTZ-induced rats increased in both the cortex and hippocampus. However, pantoprazole decreased 8-OHdG levels in the cortex and hippocampus following the rats being induced with PTZ. In addition, TAS levels, which are related to the antioxidant system, decreased and TOS levels, which are related to oxidative species production, increased after PTZ-induced neurotoxicity in SH-SY5Y cells. On the other hand, pantoprazole raised TAS levels while decreasing TOS levels after PTZ-induced neurotoxicity in SH-SY5Y cells. Previous studies have shown that proton pump inhibitors had antioxidant properties in different tissues, and they also reduced oxidative stress (Numico et al. 2017; Onda et al. 2017). These studies are line with our study. For this reason, regulating the effect of pantoprazole on antioxidant and oxidant systems may be one of the possible positive mechanisms of PTZ-induced epileptic seizures in rats and PTZ-induced neurotoxicity.

Apoptosis is defined as programmed cell death. It occurs through two main ways as intrinsic and extrinsic pathways caused by aging in the cell, oxidative stress, and extracellular stimulation (Elmore 2007). The common result of both is the activation of caspase-3 starting the cell's death cascade (Hengartner 2000). Epileptic seizures induce apoptosis in neurons by activating caspase-3 in different brain regions, including the cortex, thalamus, amygdala, and hippocampus (Méndez-Armenta et al. 2014). On the other hand, several studies have demonstrated that PTZ-induced seizures caused neuronal damage and apoptosis in the cortex and hippocampus (Branco et al. 2013). Moreover, damage to the neurons in the hippocampus causes memory and learning impairment. The role of the hippocampus as a system involved in memory processes has been well explained, and damage to this region of the brain can cause severe forgetfulness (Whitlock 2006). It has been reported that PTZ-induced epilepsy in rats caused impairment of passive avoidance memory and significantly reduced latency to enter the dark chamber in the shuttle box test, which is consistent with our results. In addition, studies have shown that PTZ-induced epileptic seizures cause spatial memory impairment, learning disorders, and passive avoidance memory in laboratory animals (Mehla et al. 2010). It has been shown that one of the proton pump inhibitors, omeprazole, protected neuronal damage against traumatic brain injury by decreasing caspase-3 and oxidative stress formation (Ozay et al. 2017). In the present study, the apoptosis marker levels, caspase-3, increased in both the cortex and hippocampus following PTZ-inducement in the rats. However, pantoprazole decreased caspase-3 levels in the hippocampus after PTZ-inducement in the rats. Moreover, pantoprazole significantly improved passive avoidance memory in the rats due to PTZ-induced epileptic seizure memory impairment. In addition, pantoprazole decreased the percentage of late apoptotic and dead cells, while increasing the percentage of live cells after PTZ-induced neurotoxicity in SH-SY5Y cells.

BDNF plays an important role in synaptic plasticity that is associated with the cellular model of learning and memory (Whitlock 2006). In addition to BDNF’s protective effects on the nervous system, exposure of adult hippocampal slices to BDNF causes increased excitatory transmission (Ji et al. 2010). In epileptogenesis-related brain areas, such as the cortex and hippocampus, BDNF is up-regulated during the occurrence of epileptic seizures (Isgor et al. 2015). Moreover, it also has been found that serum BDNF levels increased in epileptic patients, and it may be used as a biomarker for epilepsy (Jefferys 2010). Consistent with these findings, in the present study, BDNF levels increased in the hippocampus but not in the cortex after PTZ-induced epileptic seizures in the rats. In addition, pantoprazole raised BDNF levels in the hippocampus after PTZ-induced epileptic seizures. Pantoprazole can promote BDNF expression in the hippocampus, which may be a potential underlying pathway for its neuroprotective effects against PTZ-induced comorbidities involving learning and memory as well-matching passive avoidance test results.

It has been shown that the cholinergic system involves in the pathophysiology of epileptic seizures (Ventura et al. 2010). Moreover, it has been noted that PTZ-induced seizures increase acetylcholinesterase (AChE) activity and catalyze the breakdown of acetylcholine, which is one of the essential neurotransmitters in the brain, in different brain regions such as the cortex and hippocampus (Mishra and Goel 2015). Moreover, one of the more recent studies has demonstrated that PPIs inhibit acetylcholine biosynthesis (Kumar et al. 2020). Therefore, inhibiting acetylcholine levels in the brain may explain the antiepileptic effect of pantoprazole. However, this is needed to be established by further investigations.

This study has potential limitations. Although it has been shown that pantoprazole pretreatment before PTZ-induced seizures increases BDNF and protects memory, the effect of pantoprazole without PTZ-induced seizures on biochemical markers, such as BDNF, and also memory is not clear in vivo. On the other hand, the PTZ-induced neurotoxicity model has been used to imitate acute seizure in vitro and oxidative stress interaction. However, the PTZ exploration time (24 h) of the cells is too long compared to acute in vivo models.

Conclusion

The findings of this study showed that pantoprazole has antiepileptic activity in PTZ-induced epileptic seizures in rats and prevents neuroblastoma cells against PTZ-induced toxicity as well as memory impairment after epileptic seizures in rats. These effects may occur, possibly through inhibition of oxidative stress and activation of BDNF release. Thus, pantoprazole and PPIs might be potential therapeutic and supportive agents in epilepsy treatment in the clinic. Further studies are required to answer the questions raised about the probable mechanisms involved.