The current study assessed the impact of possible effects of SUG on PTZ-induced seizures in mice. As far as we know from the literature, this is the first study to evaluate SUG’s effect on seizures and potential mechanisms, including oxidative stress, apoptosis, and autophagy pathways. The present study showed that SUG significantly decreased the RCS and dGTCS time and increased FMJ and oGTCS time in mice. Moreover, TOS levels were decreased, and SUG increased TAS levels after PTZ-induced epileptic seizures in mice. On the one hand, SUG reduced 4-HNE and 3,3 Dityrosine immunohistochemically scores but did not change 8-OhDG immunohistochemically scores in the hippocampal CA1 region after PTZ-induced seizures in mice. On the other hand, SUG alleviated Caspase-3 and LC3B immunohistochemically scores, while it did not change AIFM1 immunohistochemically scores in the hippocampal CA1 region after PTZ-induced seizures in mice.
Oxidative stress is critical at the beginning of epileptic seizures and the progression of epileptogenesis because of an oxidant and antioxidant defense imbalance. Both clinical and experimental studies showed the imbalance between Oxidant-antioxidant systems (Aguiar et al. 2012). One clinical study has demonstrated that peroxidation was significantly higher in epileptic patients than in healthy persons (Hamed et al. 2004). Sudha et al. showed that epileptic patients have lower levels of antioxidant systems markers such as glutathione reductase and vitamins C, E, and A than healthy people (Sudha et al. 2001). Furthermore, it has been shown that free radicals and oxidative damage to proteins, lipids, and cell DNA were produced by PTZ-induced epileptic seizures (Taskiran et al. 2021; Taskiran and Ergul 2021). High mitochondrial superoxide levels, the inactivation of iron and sulfur dependents, and iron-induced toxicity may increase oxidative neuron injury following epileptic episodes (Waldbaum and Patel 2010). In the present study, biochemical oxidative injury marker (TOS) levels in PTZ-induced mice increased in the brain sites, including the cortex and hippocampus. However, SUG decreased the oxidative stress in the cortex and hippocampus, followed by seizures. In addition, TAS levels, a biochemical antioxidant marker, decreased after PTZ-induced seizures in mice. On the other hand, SUG improved TAS levels after seizures. Some immunochemical features indicate oxidative damage in different cell components, such as 8-OhDG, related to oxidative damage to DNA, 4-HNE, lipid peroxidation products following oxidative damage, and 3,3 Dityrosine, associated with oxidative damage to protein (Balasubramanian and Kanwar 2002; Omari Shekaftik and Nasirzadeh 2021; Li et al. 2022b). Moreover, previous studies have demonstrated that these markers increase in the brain after PTZ-induced seizures (Mao et al. 2019b; Taskiran and Ergul 2021). In the current study, the immunochemical score of 8-OhDG did not change, but an immunochemical score of 4-HNE and 3,3 Dityrosine elevated in the hippocampal CA1 region after PTZ-induced seizures in mice. However, SUG reversed the immunochemical score of 4-HNE and 3,3 Dityrosine in the hippocampal CA1 region after PTZ-induced seizures in mice. Ozbilgin et al. have demonstrated that SUG has antioxidant properties in the brain after cerebral ischemia/reperfusion injury by increasing total antioxidant capacity and decreasing lipid peroxidation marker MDA levels (Ozbilgin et al. 2016). Like Ozbilgin, Uludag has reported that SUG raises the antioxidant marker, GSH, and reduces MDA levels in the brain after cranial neurotoxicity in rats (Uludağ 2019). However, it has been claimed that SUG does not affect antioxidant and oxidant systems in the muscle after ischemia/reperfusion (Alagöz et al. 2020).
Apoptosis is defined as programmed cell death. It occurs through intrinsic and extrinsic pathways caused by aging in the cell, oxidative stress, and extracellular stimulation (Elmore 2007). The typical 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 cause neuronal damage and apoptosis in the brain (Branco et al. 2013; Sevki Taskıran et al. 2018). The present study showed that caspase-3 levels increased in mice's hippocampal CA1 region after PTZ-induced seizures. However, SUG decreased caspase-3 levels in the hippocampal CA1 region after PTZ-induced seizures in mice. Moreover, similar to our study, SUG reduced space-3 levels in the brain after experimental trauma in rats (Mucuoglu et al. 2022).
Apoptosis-inducing factor (AIF) is a mitochondrion-localized flavoprotein with NADH oxidase activity encoded by a nuclear gene. AIF has been shown to translocate from mitochondria to the cytosol and nucleus when apoptosis is induced. Mitochondrion-localized (eutopic) AIF is considered inert far as apoptosis modulation is concerned. In contrast, extra-mitochondrial (ectopic) AIF causes cell death. AIF is believed to mediate caspase-independent end because of inhibition of caspase activation (e.g., by knock-out of Apaf-1) or caspase activity (by addition of synthetic pseudosubstrates) (Candé et al. 2004). Previous studies have indicated that AIF expression increases in the brain following neurological disorders (Ghavami et al. 2014; Rodriguez et al. 2020). AIF gene expression and protein levels rise in mice's brains after the PTZ-kindling model (Zheng et al. 2020). However, in the present study, it has been observed that there is no change in the immunohistochemical ore AIF in the hippocampal CA1 region after PTZ-induced seizures in mice; this may be related to the different epileptic seizure models used in these two studies.
Autophagy, a process of self-eating of cellular materials, is exquisite to reprocessing the damaged cell organelles to procure new building blocks for maintaining cellular homeostasis. Autophagy is obligatory for cell survival during stress conditions (nutrient deprivation) by regulating cellular bioenergetics and eliminating toxic protein agglomerates. Autophagy is ordinally accompanied by cell death, possibly via remodeling autophagic constituents for cell death or by inducing apoptotic cell death (Kumariya et al. 2021). In addition, autophagy is crucial for neuronal function, and losing vital autophagic components leads to progressive neurodegeneration and structural defects in neuronal synapses (Tavernarakis 2020). It also has been reported that autophagy plays an essential role in neurological disorders (Fleming et al. 2022). Besides, autophagy has been used to develop seizures and epilepsy (Grishchuk et al. 2011; Guo et al. 2022). LC3B is considered one of the markers of autophagy (Hwang and Kim 2022). Further, studies have demonstrated that LC3B levels elevate the hippocampus after PTZ-induced seizures (Wang et al. 2020; Li et al. 2022a). In the current study, LC3B levels increased in the hippocampal CA1 region after PTZ-induced seizures in mice, like in previous studies. Moreover, SUG alleviated LC3B levels in the hippocampus after PTZ-induced seizures in mice.