Epilepsy is a common neurological disorder. However, current antiseizure drugs are limited in terms of their low efficacy and severe side effects. TCM has attracted attention owing to its widespread use in traditional and folk medicine for the treatment and prevention of epilepsy (Lin and Hsieh, 2021; Wu et al., 2023). The main finding of the present study was that pretreatment with NRICM101, a TCM formula, has anticonvulsant effects on KA-induced seizures in rats that are accompanied by significant attenuation of KA-induced neuronal loss, astrocytosis, increased glutamate, inflammatory molecules (HMGB1, NF-kB, IL-1β, IL-6, and TNF-α) and necroptotic markers (RIPK-3 and MLKL) in the cortex and hippocampus. This article is the first to report on the effects of NRICM101 in a KA-induced seizure model.
KA is a glutamate analog that stimulates excessive glutamate release and glutamate receptor activation. Systemic injection of KA in rodents induces seizures, epileptiform discharges and neuronal death in specific regions of the brain, including the cortex and hippocampus; these findings are similar to those observed in human epilepsy (Levesque and Avoli, 2013). Therefore, KA is the most commonly used compound to induce seizures in preliminary tests for the screening of potential anticonvulsant drugs. In the present study, rats that were i.p administered 15 mg/kg KA had a significantly shorter latency to seizure onset, fewer EEG ictal spikes, and more evident cortical and hippocampal neuronal damage. These results are consistent with those of previous studies (Friedman et al., 1994; Chang et al., 2022; Jean et al., 2022). Oral pretreatment with NRICM101 at 300 mg/kg significantly reduced seizure severity, prolonged the latency to seizure onset, suppressed EEG ictal spikes, and prevented cortical and hippocampal neuronal damage in KA-treated rats. Furthermore, 100 mg/kg NRICM101 + 50 mg/kg CBZ was also able to prevent the seizure generation induced by KA. The same effects were also observed with the reference drug CBZ, a well-known antiseizure drug used to treat generalized seizures. Our results are preliminary and the first to demonstrate the antiepileptogenic and neuroprotective actions of NRICM101 in an experimental seizure model. However, the combination of NRICM101 and CBZ may offer a new way to prevent epilepsy.
Excess glutamate-mediated neuronal excitation is generally considered a critical factor in the pathological process of epilepsy (During and Spencer, 1993; Soukupova et al., 2015). Elevated glutamate in the brain is likely due either to increased glutamate release and/or impaired reuptake. Astrocytic GLT-1 and GLAST take up synaptic glutamate, GS converts glutamate to glutamine within astrocytes, GDH metabolizes glutamate to α-ketoglutarate, and GAD67 converts glutamate into GABA (Danbolt, 2001; Rose et al., 2017). These proteins are critical for maintaining glutamate concentrations at normal levels, and their deficiency contributes to glutamate elevation, thus increasing synaptic excitability and seizure susceptibility (Swamy et al., 2011; Eid et al., 2012; Green et al., 2021; Hotz et al., 2022. In the present study, a significant increase in glutamate and a marked decrease in the protein levels of GLT-1, GLAST, GS, GDH, and GAD67 were found in the cortex and hippocampus of KA-treated rats. These results coincide with previous studies (Friedman et al., 1994; van der Hel, 2014; Lin et al., 2016) and suggest that decreased uptake and metabolism of glutamate leads to an increase in glutamate in KA-treated rats. Additionally, NRICM101 pretreatment decreased the level of glutamate and increased the protein levels of GLT-1, GLAST, GS, GDH, and GAD67 in the cortex and hippocampus of KA-treated rats. These findings suggested that NRICM101 preserves the normal metabolism and clearance of glutamate, a likely explanation for the decreased glutamate level in KA-treated rats. Since seizures induced by KA are due to the enhancement of glutamatergic neurotransmission, the findings in this study suggest that a decrease in glutamate levels in the brain might be a mechanism for the neuroprotective effects of NRICM101 against KA-induced epilepsy.
Neuroinflammation has been associated with glutamatergic hyperactivity in epilepsy (Vezzani and Granata, 2005; Vezzani and Viviani, 2015; Vezzani et al., 2019). In particular, astrogliosis and subsequent increases in inflammatory cytokines (IL-1β, IL-6, and TNF-α) are assumed to enhance glutamate levels and glutamate receptor activation, resulting in neuronal loss, which plays a crucial role in the development of seizures (Viviani et al., 2003; Devinsky et al., 2013). For example, in reactive astrocytes, IL-1β and TNF-α decrease GLT-1 and GS expression, and IL-6 triggers the release of glutamate from reactive astrocytes, which increases the glutamate concentration in the brain and decreases the threshold for inducing seizures (Kang et al., 2005; Tilleux and Hermans, 2007; Perez et al., 2012; Terrone et al., 2020). In addition, IL-1β/IL-1R1 pathway activation can enhance glutamatergic NMDA receptor activation, resulting in excitotoxicity and seizures (Viviani et al., 2003). IL-6 promotes the production of GFAP, IL-1β, IL-6, and TNF-α by activating the JAK2/STAT3 pathway through binding to the IL-6 receptor, which exacerbates the inflammation that contributes to the generation of seizures and the activation of neuronal death (Xu et al., 2011; Han et al., 2018; Abd EI-Aal et al., 2022). TNF-α can bind to TNFR1 to activate NF-κB by phosphorylating IκB, resulting in the upregulation of the expression of inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α, etc.) and, on the other hand, the phosphorylation of RIPK3 and MLKL, leading to necroptosis (Liu et al., 2017; Dhuriya and Sharma, 2018; Abd EI-Aal et al., 2022). Furthermore, TNF-α/TNFR1-mediated inflammation promotion and necroptosis are enhanced in epilepsy (Soltani et al., 2022). In the present study, KA administration increased astrocyte proliferation and GFAP expression in the cortex and hippocampus, indicating reactive astrogliosis. Moreover, the levels of IL-1β, IL-1R1, IL-6, p-JAK2, p-STAT3, TNF-α, TNFR1, p-IκBα, p-RIPK3, and p-MLKL were significantly increased in the cortex and hippocampus of KA-treated rats. These findings are consistent with previous results (Hoda et al., 2017; Abd EI-Aal et al., 2022; Soltani et al., 2022). On the other hand, NRICM101 pretreatment decreased the number of reactive astrocytes and downregulated the expression of GFAP, IL-1β, IL-1R1, IL-6, p-JAK2, p-STAT3, TNF-α, TNFR1, p-IκB, p-RIPK3, and p-MLKL in the cortex and hippocampus of KA-treated rats. In addition, a decrease in cytosolic p65-NFκB expression was observed in the cortex and hippocampus from the KA group, and this phenomenon was also prevented by NRICM101 pretreatment. These results suggested that the anticonvulsant and neuroprotective effects of NRICM101 might be achieved by suppressing astrocytosis and reducing the levels of inflammatory cytokines. Consistent with our findings, Lin et al. demonstrated that NRICM101 attenuates the inflammatory process in acid intratracheal instillation-induced acute lung injury in mice mainly via a reduction in TNF-α and IL-6; this phenomenon is related to the downregulation of the STAT3 pathway (Lin et al., 2023). Additionally, previous studies demonstrated that NRICM101 attenuates lipopolysaccharide (LPS)-induced production of inflammatory cytokines, e.g., TNF-α, IL-1β, and IL-6, in murine alveolar macrophages (Wei et al., 2013; Tsai et al., 2021). Our study is the first to show the anti-inflammatory effects of NRICM101 in brain tissue, and these effects might be related to its ability to prevent glutamate elevation and neuronal death in KA-treated rats.
The HMGB1/TLR4 cascade has been shown to aggravate astrogliosis and augment epileptogenic inflammatory signaling (van Vliet et al., 2018; Zhang et al., 2022). HMGB1 is a ubiquitous nuclear protein that is released from damaged neurons and glial cells and binds to TLR4 to activate NF-κB and, consequently, the inflammatory response (Paudel et al., 2019). HMGB1 and TLR4 levels are increased in the serum and brain tissue of both epileptic patients and animal models (Luo et al., 2013; Kaya et al., 2021; Yue et al., 2023). Inhibiting the HMGB1/TLR4 pathway can increase the seizure threshold, alleviate the inflammatory response, and lessen nerve damage after epileptic seizures (Li et al., 2013; Zhao et al., 2017). Consistent with these studies, the present study revealed an increase in the serum HMGB1 concentration, as well as in the HMGB1 and TLR4 expression in the cortex and hippocampus, in the KA group. On the other hand, NRICM101 pretreatment decreased the serum HMGB1 concentration and the cortical and hippocampal expression of both HMGB1 and TLR4 in KA-treated rats, suggesting that the suppression of HMGB1/TLR4 pathway activation is involved. Based on the present data, we infer that NRICM101 prevents HMGB1/TLR4/NF-κB pathway activation resulting in the suppression of downstream signaling events (IL-6/p-JAK2/p-STAT3, IL-1β/IL-1R1, TNF-α/TNFR1/NF-κB, and necroptosis signaling RIPK3/MLKL), leading to the inhibition of inflammatory cytokines (IL-1β, IL-6, and TNF-α) and astrogliosis; these effects consequently increase the levels of glutamate metabolism-associated enzymes (GS, GDH, and GAD67) and glutamate reuptake-associated proteins (GLAST and GLT-1) resulting in a decrease in the glutamate concentration in the brain. This difference might be related to the attenuation of neuronal hyperexcitability, seizure generation, and neuronal damage in KA-treated rats (Fig. 11).
In the present study, the chemical fingerprint of NRICM101 revealed that flavonoids were the major components, particularly baicalin, which is consistent with the findings of a previous study (Tsai et al., 2021). Flavonoids have been reported to interact with glutamatergic neurotransmission (Lin et al., 2015; Jean et al., 2022; Pai et al., 2023). In addition, baicalin has been shown to produce antiepileptic effects by inhibiting oxidative stress and the inflammatory response (Yang et al., 2021; Li et al., 2022). Thus, the anticonvulsant effect observed in the present study may be explained by the presence of flavonoids in NRICM101. On the other hand, NRICM 101 at 300 mg/kg for 7 days had no effect on rat body weight, a sign of animal health status. In addition, no changes in the serum ALT or AST concentration or in the histology of the liver or kidney were observed. The results of our study suggest that NRICM101 at 300 mg/kg does not induce significant toxic effects.