Zingerone Targets Status Epilepticus by Blocking Hippocampal Neurodegeneration via Regulation of Redox Imbalance, Inflammation and Apoptosis

Epilepsy is an intricate neurological disease where the neurons are severely affected, leading to the mortality of millions worldwide. Status epilepticus (SE), induced by lithium chloride (LiCl) and pilocarpine, is the most accepted model for epilepsy. The current work aims to unravel the mechanisms underlying the anti-epileptic efficacy of zingerone (an active ingredient of ginger), which has beneficial pharmacological activities on seizure-induced behavioral, histological, neurochemical, and molecular patterns in mice. Zingerone restored cognitive function by diminishing seizure activity, escape latency, and subsequent hippocampal damage manifested in histology. Seizures are associated with local inflammation, redox imbalance, and neural loss, confirmed by the present study of SE, and was attenuated by zingerone treatment. Nuclear factor-kappa B and its downstream signaling molecules (TNF-α, IL-1β, IL-6, NO, MPO) were activated in the LiCl-and-pilocarpine-induced group leading to inflammatory signaling, which was substantially ameliorated by zingerone treatment. The intrinsic apoptotic process was triggered subsequent to SE, as demonstrated by augmentation of cleaved caspase-3, downregulation of Bcl-2. However, zingerone treatment downregulated caspase-3 and upregulated Bcl-2, increasing cell survival and decreasing hippocampal neural death, deciphering involvement of apoptosis in SE. Therefore, zingerone plays an essential role in neuroprotection, probably by precluding oxidative stress, inflammation, and obstructing the mitochondrial pathway of apoptosis.


Introduction
Epilepsy is the fourth most widespread, chronic, non-communicable illness of the brain affecting people of all ages. Roughly 50 million individuals are afflicted by this disease worldwide; the rate of active epilepsy population at a given time is between 4 to 10 persons per 1000 people [1]. The major symptoms are recurrent seizures, which could involve a part of the body or the whole body, sometimes together with the loss of Table 1. Zingerone and sodium valproate effects on seizure severity and latency to onset of SE in LiCl-and-pilocarpineinduced epilepsy in mice. Results are representative of mean ± SE of fourteen mice per group. The results that we obtained are significantly different from the LiCl and pilocarpine group as the main comparison is with LiCl and pilocarpine group only (** p < 0.01 and *** p < 0.001). Group I: Normal saline (

Effect of Zingerone and Sodium Valproate on Escape Latency and Probe Trial of Morris Water Maze
Escape latency diminished with progress in days in all groups. Group II mice demonstrated substantially (*** p < 0.001) greater latency than group I mice. Pretreatment with zingerone at 25 mg/kg exhibited a noteworthy ( ## p < 0.05) decline in the escape latency to find a hidden platform as compared to the group that received LiCl and pilocarpine only (group II). The decrease in escape latency for group III and group V was more substantial ( ### p < 0.001) in comparison with positive control mice ( Figure 1A). When the platform was removed, group II devoted less time pointedly (*** p < 0.001), owing to forgetfulness in the target quadrant in comparison with group I mice. The percentage of time given in the target quadrant was significantly increased by the pretreatment of zingerone of a lower dose ( ## p < 0.01) and a higher dose ( ### p < 0.001) when contrasted with group II mice. Sodium valproate, the standard antiepileptic drug administered to mice of group III, displayed substantial ( ### p < 0.001) growth in the time being in the target quadrant in comparison to the positive control group ( Figure 1B). Results are representative of mean ± SE of fourteen mice per group. In group-II, the escape latency was increased significantly (*** p < 0.001) as compared to control group (group I). Treatment with sodium valproate and zingerone (25 and 50 mg/kg b.w.) significantly attenuated escape latency level in group III ( ### p < 0.001), group IV ( ## p < 0.01), and group V ( ### p < 0.001) as compared to group II. Group I: Normal saline ( Results are representative of mean ± SE of fourteen mice per group. In group-II, the escape latency was increased significantly (*** p < 0.001) as compared to control group (group I). Treatment with sodium valproate and zingerone ( We observed a substantial (*** p < 0.001) rise in MDA in mice of group II in comparison with negative control mice. A noteworthy ( # p < 0.05) diminution was observed in group IV mice that had received zingerone (25 mg/kg) while an extremely substantial ( ### p < 0.001) fall in levels of MDA was seen in mice administered zingerone (50 mg/kg) compared to levels in the toxic group that had received LiCl and pilocarpine only (group II). Sodium valproate, the standard antiepileptic drug administered to mice of group III exhibited a substantial ( ### p < 0.001) fall in MDA when compared to group II (Table 2). Substantial (*** p < 0.001) fall in SOD was detected in mice of positive control when compared with negative control mice. Increase in SOD ( # p < 0.05) was detected substantially in group IV mice that had received zingerone (25 mg/kg) while more significant ( ### p < 0.001) rise was seen in mice administered zingerone (50 mg/kg) compared to LiCl and pilocarpine only treated group (group II). Sodium valproate, the standard antiepileptic drug administered to mice of group III, displayed a substantial ( ### p < 0.001) rise in SOD in comparison to group II (Table 2).

Catalase Activity (CAT)
A substantial reduction occurred in CAT in mice of group II in comparison to activity in control mice. A marked rise ( # p < 0.05) in CAT was detected in group IV mice that had received zingerone (25 mg/kg) while a significant ( ### p < 0.001) rise was seen in mice with administered zingerone (50 mg/kg) compared to levels in the group that had received LiCl and pilocarpine only (group II). Sodium valproate, the standard antiepileptic drug administered to mice of group III, showed an extremely substantial ( ### p < 0.001) rise in CAT activity as contrasted to group II mice ( Table 2).

Glutathione Reductase Levels (GR)
An extremely significant (*** p < 0.001) fall occurred in GR in mice of group II in comparison to levels in control mice (Group I). A non-significant (ns) rise was detected in mice of group IV that had received zingerone (25 mg/kg) while a highly significant ( ## p < 0.01) rise was detected in mice treated with zingerone (50 mg/kg) compared to levels in LiCl and pilocarpine mice (group II). Sodium valproate, the standard antiepileptic drug administered to mice of group III revealed a noteworthy ( ### p < 0.001) rise in GR (Table 2).

Inflammatory Markers
Group  From the above results, we conclude that zingerone (50 mg/kg b.w.) demonstrates better protection in LiCl and pilocarpine administration. So, for further histopathological and immunohistochemistry experiments, we have selected only the higher dose of zingerone that is 50 mg/kg b.w.

Histopathological Analysis of Zingerone and Sodium Valproate Treatment in LiCl-and-Pilocarpine-Induced Epilepsy
Histological examination of hippocampal sections revealed normal morphology of neurons of group I animals. No deformity in CA1 sections of normal mice was observed and healthy neurons are marked by black arrow (Figure 3). Neural condensation, necrosis, and nuclear degeneration in the CA1 region were observed in the hippocampus of mice of group II animals that had received LiCl and pilocarpine only as indicated by yellow arrows (*** p < 0.001). Mild condensation and less degeneration of neurons were observed in the hippocampus of mice of group V that had been administered 50 mg/kg b.w. of zingerone in comparison with the positive control ( ### p < 0.001) ( Figure 3).

Effect of Zingerone on Choline Acetyl Transferase (ChAT), Bcl-2, and Caspase Expression
The deficiency in cholinergic activity is reported by the decrease in the ChAT expression in the hippocampal region of the brain in LiCl and pilocarpine-administered mice (group II). The black arrows represent the neural loss and decreased ChAT activity ( Figure 4). Zingerone at the 50 mg/kg treatment enhanced the expression of ChAT activity in the hippocampus of mice in comparison with the positive control (group II) (Figure 4). Bcl-2 expression was upregulated in the hippocampus of LiCl and pilocarpine-administered mice in comparison with the control group ( Figure 5). Zingerone as 50 mg/kg successfully downregulated the Bcl-2 activities in group V treated with the higher dose of zingerone ( Figure 5) as compared to the positive control (group II) demonstrating the survival of neurons. Hence, there was an increase in the survival of neurons.    Negligible activated caspase-3-positive neurons were visible in the control group (group I) ( Figure 6). In group II, with the treatment of LiCl and pilocarpine in mice, activated caspase-3 expression is upregulated in the hippocampal region of the brain ( Figure 6). Black arrows show degenerated neurons with high expression of activated caspase-3. Zingerone at the 50 mg/kg attenuated caspase-3 expression in the group treated with a higher dose of zingerone as juxtaposed to a positive control (group II) indicating survival of neurons ( Figure 6).

Discussion
Initial pathology of SE involves unwarranted ROS production, strong neuroinflammatory retorts, discerning neural deterioration, and interruption of BBB, following consequent impairment in cognition [23]. The purpose of SE treatment is to terminate seizures as quickly as possible with either first-line or second-line drug therapy such as antidepressants and anti-epileptic drugs which include phenobarbital or valproic acid, respectively. Apparently, these drugs terminate seizures immediately. However, they do not efficiently recover the pathophysiological variations which occur following epileptic seizure episodes leading to neural injury and cognitive deficits [24]. Consequently, new treatment approaches are highly needed to mitigate the pathophysiological damage in the brain after SE. We hypothesized that zingerone may provide beneficial outcomes in SE centered on previous studies [25].
Seizure intensity is assessed usually in seizure models of animals to do behavioral analysis. One of the most frequently used seizure intensity measurements in epilepsy is done by Racine's scale. This scale has become efficient over the years to better define the development of seizures initiated by various chemicals [26]. Zingerone treatment demonstrated a reduction in the score of seizures and a boost in latency of seizures. The results were more significant and comparable with sodium valproate when higher doses of zingerone were administered. The function of sodium valproate is to enhance GABAergic activity besides diminishing excitatory neurotransmission [27]. The effect of zingerone in altering epileptogenesis could be attributed to its role in decreasing stimulatory neural transmission, elevating GABAergic activity, and perturbing ion-channel activity, as reported previously [28][29][30][31][32]. In the case of epileptogenesis, there are reports of decreased GABAergic activity of neurons leading to seizures [33]. Zingerone has been previously reported in the seizure model of mice to enhance the GABAergic activity by an increase in the Ca ++ influx through ionophores of presynaptic 5HT3 receptors causing an anticonvulsant effect [34]. Similarly, the ion channels are also reported to be dysfunctional in epilepsy-inducing seizures [35]. Zingerone has prevented the ion channel disruptions, as previously reported by Hosseini and Mirazi [33]. However, the exact mentioned mechanism by which zingerone acts in the present model of epilepsy is not clear and needs further study.
One of the clinical manifestations in SE-induced brain damage is cognitive impairment. Long-term seizures which occur repeatedly over a long period of time in SE inescapably have an impact on patients and affect their quality of life. Unfortunately, AEDs do not alleviate memory deficit caused by seizures and their long-term usage elevates oxidative stress and prompts cognitive imbalance. Moreover, epilepsy itself contributes to oxidative stress, which is further heightened by AEDs, indicating that antioxidants, anti-inflammatory, and anti-apoptotic agents may have the potential to impede seizures and the pathophysiology associated with SE deciphering antiepileptic therapy [36]. The Morris water maze (MWM) is the interpretation of memory and learning. LiCl-and-pilocarpine-induced SE lowers the behavioral pattern of learning as evidenced by higher values of escape latency in the positive control group, which, however, was counteracted by zingerone in groups IV and V, suggesting the protective role of zingerone in the improvement of neural damage. Results of the MWM reveal that positive control animals either stayed for a smaller amount of time in the target quadrant or took a longer amount of time to grasp the escape stand or entirely could not grasp the stand, indicating poor cognition and visual-spatial memory, as reported previously [7,37], when compared with control animals. Moreover, the explicit basis of poor cognition following SE is not known. Nevertheless, imbalances in neurochemical signaling and progressive structural damage to neurons induced by recurrent transitory seizures in SE may be one of the reasons for neurobehavioral manifestations. Our results are further validated by previous findings.
One of the hallmarks and fundamental cytotoxic mechanism of recurrent epileptic seizure is the induction and activation of oxidative stress which further exacerbate neural toxicity, dysfunction, and death. It is well evidenced from augmented oxidized proteins and lipid levels found clinically in epilepsy patients. However, oxidation of biomolecules that contribute to excitatory neural damage and collapse was also found in epileptic brain tissue surgically resected, indicating that oxidative stress is an important phenomenon in SE [38]. Pilocarpine results in surging ROS, NO content, and lipid peroxidation formation, as reported previously. It decreases antioxidants like nitric oxide synthases (NOS), CAT, SOD, GR, GSH amounts in different areas of the brain, which include the hippocampus, cortex, or other areas or entire brain. We also obtained similar results in the present study as ones reported previously. However, antioxidant therapy is found to exhibit beneficial effects on various epilepsy models by ameliorating oxidative stress in the brain [39]. Similar results were obtained in our current study. The brain consumes the largest quantity of oxygen in comparison to the rest of the areas of the body which makes it prone to redox imbalance. It also comprises a large amount of polyunsaturated fatty acids, therefore, it is predisposed to peroxidative lipids [8]. SOD, CAT, GPx, GR are endogenous antioxidant machinery enzymes implicated in the cleansing of ROS where superoxide anion radicals are dismutated by SOD, H 2 O 2 is decomposed by CAT decreasing oxidative stress, effectively protecting itself from redox damage. The hydroxyl ion (OH 0 ), which is the most damaging free radical, attacks molecules by hydrogen abstraction, subsequent repeated attacks trigger injury to genetic material, proteins, lipids, and cellular membranes of mitochondria and nucleus. H 2 O 2 indeed is a non-free radical; nevertheless, it is exceptionally detrimental and has the ability to traverse lipid layers, reacting with transition metals. Besides, it can stimulate chromosomal modifications and oxidize sulfhydryl compounds (in the absence of catalase). In the present study, we show increased H 2 O 2 formation in pilocarpineinduced group in comparison with negative control, as stated before [40]. However, treatment with zingerone scavenged H 2 O 2 production, thereby, a decrease in content and similar results were obtained in the sodium valproate group. GPx shields cells against oxidative damage and meanwhile involves GSH as its cofactor. GSH is a highly recognized non-enzymic antioxidant that defends against peroxidative damage of cell membranes endogenously [41]. In the present study, we found a decrease in SOD, CAT, GR, and GSH in LiCl-and-pilocarpine-induced group as reported previously [42]. Administration of zingerone notably in high doses or sodium valproate diminishes oxidative damage with the restoration of the antioxidant armory. These results are in agreement with earlier reports [24,37,43,44].
The oxidative stress and generation of free radicals in epilepsy has been established. Pearson-Smith et al., Diniz et al., and Clayton et al. studied oxidative stress in epilepsy models [38][39][40], and there has been increasing evidence supporting the use of several natural compounds in combating the ROS generated by unstable radicals as described by Rehman [11,36,[41][42][43]. However, our study is novel in establishing the neuroprotective and antioxidant role of zingerone in the SE model of mice targeting inflammation, neural cell death, and redox imbalance.
The role of the cholinergic system is well documented in pilocarpine-induced SE. AChE is critically involved in cholinergic neural communication and breakdown of acetylcholine to dismiss the propagation of neural signals. When acetylcholine gets accumulated, there is an activation of muscarinic and nicotinic receptors excessively. Therefore, an elevated cholinergic signal in the brain is associated with SE [45]. The increase in the activity of AChE in epilepsy is well documented. Studies have confirmed the upregulation of AChE allied with elevated immune response, facilitating the epileptogenic process [46]. The anti-inflammatory role of zingerone in the present work is presumed to be associated with the downregulation of AChE, elevating the Ach levels which in turn inhibit the innate immune response a1nd proinflammatory cytokines. We hypothesize the role of zingerone in improving cholinergic dysfunction, possibly by downregulation of AChE; this needs further research to uphold.
Inflammation in the neurons plays a vital role in epileptogenesis, which has been well evidenced by clinical signs and neuropathology events in epilepsy in the last decade. Proinflammatory cytokines have been shown remarkably to be the most upregulated molecules during epileptogenesis. Neural inflammatory molecules elevate neural excitability via augmenting synaptic transmission by mediating glutamate receptors leading to downregulation of seizure threshold [47]. Henceforward, one of the objectives of the current work was to elucidate the functional importance of NF-κB and inflammatory mediators in LiCl-and-pilocarpine-induced epilepsy. NF-κB, a redox-sensitive molecule that is directly proportional to pro-inflammatory mediators' expression in neural excitability and gliosis in the hippocampus. Therefore, it becomes a practically medicinal target in the management of epilepsy [48]. In the current work, we examined NF-κB in the hippocampus region. Our results decipher that NF-κB is substantially elevated in LiCl-and-pilocarpine-induced epilepsy in comparison with the negative control. Though, prophylactic treatment with zingerone downregulated NF-κB in the hippocampi regions of mice as reported previously [9]. The elevated inflammatory marker levels like TNF-α, IL-1β, and IL-6 in toxic control group are attributed to activation of the innate immune response. Any ischemic, epileptic, or excitotoxic damage causes initiation of proinflammatory cytokine storm including TNF-α, IL-6, IL-1β as reported previously [47,49]. Our study confirmed it further that there was an increase in cytokine levels in the LiCl and pilocarpine group, indicative of inflammatory reactions leading to seizures. However, treatment with zingerone alleviated cytokine storm as reported previously by Rehman et al. and Wali et al. [50,51] and therefore regulating neuroinflammation and, hence, controlling seizures.
Neuroinflammation induced by epileptic seizures results in neural death in several brain regions, particularly the hippocampus. If inflammation persists excessively it results in cellular damage and neurotoxicity, as reported in the literature. However, the limitation of the present study is that we have not studied specific marker proteins of microglia and glial cells, which makes it hard to be certain about a particular cell's involvement. It has been reported that microglial activation facilitates the release of cytokines and chemokines like NF-κB, TNF-α, and IL-1β which is found in the current study, as reported previously [52].
Instigation of inflammatory pathways like the NO pathway in various experimental and clinical studies has been found to have a critical position in the pathology of epileptic seizures and neural lesions. NO is a neurotransmitter in the brain found in the gaseous state and NOS regulates the biosynthesis of NO. NO is a free radical which has low reactivity with most of the biomolecules. Nonetheless, it may react with other free radicals, generating peroxynitrite. The latter is a potent inducer of cell death [53,54]. Our results reveal that NO content in the hippocampus is augmented following seizures, representative of epileptic seizures. However, zingerone treatment reduces NO content, alleviating inflammation and toxicity of NO on brain tissue, as reported previously by Mir et al. [55].
Myeloperoxidase is an inflammatory marker found in leucocytes and is the only peroxidase that uses H 2 O 2 to oxidize halides forming hypohalous acids. So, it involves the production of RNS and ROS. It also inhibits inhibitors of matrix metalloproteinases, leading to leakage in BBB, which is associated with the advancement and initiation of seizures. We show an increase in MPO activity in positive control animals, as reported previously [56]. However, treatment with zingerone alleviated MPO content, indicating the anti-inflammatory role of zingerone, as reported [57].
Apoptosis related to redox signaling is associated with epilepsy. Induction of apoptotic signaling pathway is reported to worsen seizure-induced brain damage resulting in extended epileptic seizures. In reaction to seizure-stimulated brain injury, permeabilization of mitochondria happens and proapoptotic proteins are secreted from mitochondria, leading to downstream executioner caspase (caspase-3) activation causing brain lesions [58]. Evidence suggests that there is cross-talk between oxidative stress and mitochondrial dysfunction followed by apoptosis in seizure-induced neural damage, making both changes in mitochondria and oxidative stress elements for epileptogenesis [59]. Furthermore, mitochondria are vulnerable to oxidant damage as they are predominant sites of ROS generation. Current data suggest that targeting oxidative stress, inflammation, and mitochondrial disruption may be a new approach for precluding neural death and inhibiting epilepsy, and controlling seizures [60,61]. The outcomes of zingerone on neural lesions following epileptic seizures by LiCl and pilocarpine administration, and on mitochondrialapoptosis-pathway-related regulator Bcl-2 and caspase-3 were studied, which is an imperative procedure of neuroprotective potential of zingerone to lessen cell death and endorse survival as reported [19,20]. The results indicate that zingerone intervened and limited brain damage, increased Bcl-2 protein expression in hippocampal neurons, downregulated apoptotic protein activated caspase-3 expression, suggesting that zingerone regulates the expressions of these mitochondrial intrinsic apoptotic-pathway-associated molecules to diminish lesions and death of hippocampal cells and play a beneficial role in protection of brain [19,20].
Histology shows neural thickness was considerably diminished in the positive control group in the CA1 region of the hippocampus which may be attributed to the unnecessary increase of intracellular Ca 2+ levels due to overtly activated glutamate receptors, as reported earlier [62]. However, treatment with zingerone in group V markedly increased neural density, indicating growth and neural survival. Apoptotic cell death, necrosis, and pyknotic nuclei are seen in positive control in comparison to the negative control. Additionally, to validate the hippocampal lesions in the brain, we also explored intrinsic-pathwayapoptosis-related factors like Bcl-2 and caspase-3 which showed that zingerone treatment augmented Bcl-2 and alleviated caspase-3 expression in group V [63].

Animal Study
Male swiss albino mice weighing 25-35 g, of 4-6 weeks old, were housed in the institutional animal facility, having easy access to food and drinking water with a relative humidity of 45-55% in a temperature of 23-25 • C of 12 h dark/12 h light period. Institutional Animal Care and Ethics Committee (No: RAKMHSU-REC-08-2019-F-P) permitted all the experimental procedures described in the manuscript. Animals were randomly divided into five groups of 14 mice in each group.

Preparation of Drugs and Treatment Protocol
Pilocarpine was used to induce epilepsy and was freshly prepared in water and given intraperitoneally (i.p.) at the dose of 30 mg/kg body weight to mice. Lithium chloride (3 mEq/kg, i.p.) was administered 15-20 h prior to pilocarpine injection. Lithium potentiates pilocarpine effect by activating T-lymphocytes and mononuclear cells indirectly, resulting in higher serum IL-1β levels, thus varying the BBB permeability and augmenting pilocarpine uptake. Researchers have also found pre-treatment with LiCl to increase acetylcholine release leading to more acetylcholine crossing the synaptic cleft and reaching the postsynaptic membrane, activating muscarinic receptors and decreasing time to SE onset. The LiCl pilocarpine model has been shown to generate more consistent and prolonged seizures with reproducible results with a low mortality rate, thus making it a promising model for studying SE [64]. Sodium valproate was used as a standard antiepileptic drug, which was prepared in a similar way as pilocarpine and was given at the same dose to one of the groups to compare the results with our natural compound group. Zingerone was freshly prepared in normal saline and given as 25 and 50 mg/kg body weight based on previous reports [56,[65][66][67] For the treatment schedule, refer to Figure 7. Control animals were treated with an equivalent volume of water (10 mL/kg b.w.) for 15 days preceding pilocarpine [68].
promising model for studying SE [64]. Sodium valproate was used as a standard antiepileptic drug, which was prepared in a similar way as pilocarpine and was given at the same dose to one of the groups to compare the results with our natural compound group. Zingerone was freshly prepared in normal saline and given as 25 and 50 mg/kg body weight based on previous reports [56,[65][66][67] For the treatment schedule, refer to Figure 7. Control animals were treated with an equivalent volume of water (10 mL/kg b.w.) for 15 days preceding pilocarpine [68].

Experimental Regimen
Before the start of the experiment, all animals were trained in the Morris water maze. The animals were randomly selected into five groups of fourteen animals each.
Group I: Negative control. Mice were provided with the standard diet with free access to oral drinking water.
Group II: Positive control/pilocarpine group. In this, mice were provided with a standard diet and drinking water. Pilocarpine was injected 30 mg/kg b.w i.p. on the 15th day. LiCl (3 mEq/kg b.w.) was given intraperitoneally 15-20 h before pilocarpine injection.
Group III: Standard drug group. In this group, mice were provided with a standard diet and drinking water. LiCl (3 mEq/kg b.w.) was given intraperitoneally 15-20 h before pilocarpine injection. Pilocarpine (30 mg/kg b.w.) i.p. was given on day 15, and Sodium Valproate (300 mg/kg b.w.), which is a standard anti-epileptic drug, was given prophylactically orally for 15 days prior to pilocarpine injection.
Group IV: (Prophylactic Group I). Mice were given a standard diet and drinking water. LiCl (3 mEq/kg b.w.) was given intraperitoneally 15-20 h before pilocarpine injection. Pilocarpine (30 mg/kg b.w.) i.p. was given on day 15, and zingeron as 25 mg/kg b.w. was administered for 15 days orally prior to pilocarpine injection from the start of the study.
Group V: (Prophylactic Group II). Mice were given a standard diet and drinking water. LiCl (3 mEq/kg b.w.) was given intraperitoneally 15-20 h before pilocarpine injection. Pilocarpine (30 mg/kg b.w.) i.p., given on day 15 and zingerone as 50 mg/kg b.w, administered for 15 days orally prior to pilocarpine injection from the start of the study.
After administering pilocarpine, the alterations in seizure activity in different groups were recorded for a period of 1 h as per the following Racine classification [68]. SE was terminated by diazepam (10 mg/kg, i.p.) Eight animals from each group were used for biochemical estimations, and six animals were used for histopathology and immunohistochemistry studies. Furthermore, for histology and immunohistochemistry studies, we proceeded with only three groups on the basis of the best results that we got from our biochemical and behavioral studies, and we found the higher dose of zingerone to be the best for mitigating the process of SE.
Prophylactic activity of zingerone was measured for modifying SE by checking cognitive impairment by MWM, ROS measurement, antioxidant armory, seizure severity, latency to onset of seizures, NFκ-B, inflammatory mediators like TNF-α, IL-1β, and apoptosis pathway proteins; histological alterations were also studied.

Morris Water Maze (MWM)
The already trained mice were assessed for spatial learning and memory abilities. The test was performed for 5 days. MWM (TSE Systems, Chesterfield, MO, USA) is comprised of a round pool with water (25 ± 2 • C) in which mice are trained to swim to a concealed stand and flee from water, as described previously [69]. Before the start of experiments, animals were trained to find the hidden platform by giving four trials each day for each animal for five consecutive days. The platform was fixed at one place permanently during the complete procedure. When animals climbed at the platform, it remained there for 15 s prior to the beginning of the subsequent trial. If in the maximum allowed time of 60 s, mice failed to grasp the escape stand, it was quietly put on the platform and allowed to stay there for 15 s prior to its removal from the pool. Escape latency is the total amount of time required by mice here to grasp the concealed stand, which was evaluated in seconds.
Spatial probe test: The probe test was performed by eliminating the hidden platform from the pool, and mice were permitted for 60 s to freely swim in the pool. The amount of time devoted by the mice in the target quadrant was estimated, and this was the measure of the intensity of memory reinforcement following learning.

Tissue Homogenization
Once behavioral studies were done, animals were sacrificed, the hippocampus blotted dry, weighed (5% w/v), and homogenized in 0.01 mM phosphate-buffered saline having pH 7.4 with 10 µL/mL protease inhibitor cocktail in Potter-Elvehjem homogenizer (Thomas Scientific, Swedesboro, NJ, USA) and centrifuged (TSE Systems, Chesterfield, MO, USA) at 825× g for 5 min at 4 • C to separate debris. The supernatant-1 (S1) was used for the assay of lipid peroxidation, myeloperoxidase, and AChE activity, and the rest was again centrifuged in REMI cooling centrifuge to acquire post mitochondrial fluid (PMF/PMS) of the hippocampus at 10,500× g for 15 min at 4 • C, which was used for the estimation of the antioxidant armory, NO, and inflammatory markers. The absorbance was calculated by UV-1601 (Shimadzu, Japan) for the antioxidant armory and for inflammatory markers by ELISA Plate Reader (benchmark plus, Biorad, Hercules, CA, USA) [20] 20 µL ferric chloride of 100 mM, making 1 mL as total volume. This reaction was incubated at 37 • C kept in a shaking water bath for 1 h. By adding 10% of 1 mL, the trichloroacetic acid reaction was stopped, followed by the addition of 0.67% of 10 mL thiobarbituric acid (TBA), and all the reactions in tubes/vials were placed in a boiling water bath for a period of 20 min. The tubes/vials of the above-mentioned reaction mixture were transferred to an ice bath and then centrifuged at 2500× g for 10 min, and the absorbance of each sample was taken. GSH was calculated as done by Rashid et al. [73]. PMS was mixed with 4.0% sulfosalisylic acid in a 1:1 ratio (v/v). The reaction mixture was incubated for 60 min at 4 • C for and then centrifuged at 1200× g for 15 min at 4 • C. The reaction mixture consisted of 400 µL filtered aliquot, 2200 µL phosphate buffer of 0.1 M with physiological pH and 400 µL DTNB of 10 mM, making up 3 mL as total and then absorbance is taken for each sample in each group.

Assay for Activity of AChE
AChE was estimated as defined by Ellman et al. [74]. Acetylcholine (ATC), is an artificial substrate which is broken down in the presence of AChE to release thiocholine. Thionitrobenzoic acid is formed by reaction of thiocholine with DTNB. The reaction mixture contained 2.6 mL of 0.1 M sodium phosphate buffer having pH 7.4, 100 µL of 10 mM DTNB, 20 µL of ATC, and 400 µL S1 making 3.12 mL volume in total. Absorbance is taken for each sample in each group.

Assay for Myeloperoxidase Activity
The neutrophil quantification is measured as a level of myloperoxidase (MPO) activity and was carried using the Bradley et al. method [75]. Activity was measured by mixing 0.1 mL of the supernatant with 2.9 mL of 50 mM phosphate buffer having pH 6.0, containing 0.167 mg/mL 0-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. Absorbance is taken for each sample in each group.

Reactive Oxygen Species (ROS) Assessment
ROS measurement was observed by the oxidation of 2 7 -dichlorodihydrofluorescein diacetate (DCF-DA) to 2 7 -dichloro-fluorescein (DCFH2) as described by Chan-Min Liua et al. [76]. Equal amounts of supernatants from various groups and Griess reagent (0.1% N-(1naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide, and 2.5% H 3 PO 4 ) were mixed together. Total nitrites were spectrophotometrically measured at 540 nm after incubation of 10 min at room temperature in the dark. The nitrite concentration was calculated by NaNO standard curve [77] The protein was measured by Lowry et al. [78] with BSA reagent as a standard.

Histopathology and Counting of Neuronal Loss
After the experiment, the mice were anesthetized and perfused transcardially with chilled PBS (10 mM, pH 7.4) and then 4% paraformaldehyde. The fixed brains were removed and embedded in paraffin. The paraffin sections at the level of hippocampus (thickness 5 µm) were dewaxed and rehydrated by using different gradient of alcohol for hematoxylin-eosin (H&E) staining. The morphology of the pyramidal neurons in CA1 region of hippocampus was examined under light microscopy (Olympus BX50, Tokyo, Japan) [20].
Neuronal loss was estimated as described by Vaibhav et al. The pyknotic nuclei were defined as darkly stained punctate nuclei and those nuclei that were fragmented were counted as single nucleus. The cells with pyknotic nuclei were regarded as dead cells and not counted in viable cell counting under the microscopic field in each group. In each group, the number of live cells and dead cells were counted in five areas of CA1 region of hippocampus and the average was calculated. The percentage of dead cells to that of viable cells was regarded as neuronal loss. This process was repeated five times [20].

Immunohistochemical Staining of ChAT, Bcl-2, and Caspase-3
Immunohistochemistry was done to identify ChAT, Bcl-2, and caspase-3 protein expressions. Immunohistochemical staining protocol was done as explained by Rashid et al. [79]. Animals were given anesthesia and perfused transcardially for 20 min with 4% paraformaldehyde in normal saline (9%). Brains extracted and fixed in 4% paraformaldehyde in normal saline (9%) for another 24 h. Coronal section of the hippocampus was processed for immunohistochemical staining. We used the following antibodies as antimouse ChAT rabbit antibody (1:200) (Sigma-Aldrich, Taufkirchen, Germany), anti-mouse Bcl-2 polyclonal antibody (1:150) (Thermo Fisher Scientific, Waltham, MA, USA), antimouse caspase-3 polyclonal antibody (activated caspase-3) (1:200) (Thermo Fisher Scientific, Waltham, MA, USA) overnight incubation at 4 • C following the protocol by Rashid et al. Next day, the slides were washed three times in Tris buffers (pH-6.0) and were incubated with a biotinylated Goat Anti-Polyvalent Plus (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. This step was followed by further washing in Tris buffer and incubation of slides at room temperature with Streptavidin Peroxidase Plus (Thermo Fisher Scientific, Waltham, MA, USA) that binds to the biotin present on the secondary antibody. After washing in Tris buffer, the immunostaining reaction product was developed using 3,30-diaminobenzidine (DAB Plus substrate, Thermo Fisher Scientific, Waltham, MA, USA). After immunoreactivity, slides were dipped in distilled water, counterstained with Harris hematoxylin and dried, and finally, the sections were mounted with DPX and covered with cover slips. The slides were ready to be observed under microscope. (Olympus BX50, Tokyo, Japan). The positive neural cells were counted at five different areas of CA1 region of hippocampus by microscope (magnification X 200) and the process was repeated five times. The cells expressing the protein were plotted as mean percentage of total cells counted [20,79,80].

Statistical Analysis
The data are displayed as the mean ± standard error of the mean (SEM) from all the groups individually. Analysis of variance (ANOVA) was utilized to determine the differences between groups followed by Tukey-Kramer multiple comparisons test. Criterion for statistical significance minimally is set at p < 0.05 for all comparisons unless stated otherwise.

Conclusions
Zingerone alleviated LiCl-and-pilocarpine-induced epilepsy pattern and showed neural survival and protection in the hippocampus of mice after SE by reducing redox imbalance, inhibition of expression of inflammatory mediators, and increasing survival of neurons by regulating apoptosis, which are closely associated with onset of seizures. Henceforth, zingerone deciphered promising therapeutic efficacy in SE mice, offering a novel beneficial window for mitigation of epilepsy in vivo.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.