Semen Strychni, the dried seeds of Strychnos nux-vomica L. (Loganiaceae) with severe toxicity (Lu et al., 2020), has a long history of clinical applications in improving blood circulation, treating cancer and relieving rheumatic pain. It plays an essential role in the treatment of rheumatoid arthritis (Li et al., 2015). Studies indicated that strychnine and brucine, which account for about 70% of Strychnos alkaloids (SAs), are the main biologically active components of Semen Strychni (Lin et al., 2016). Semen Strychni is characterized by a narrow therapeutic window and individual differences, it has a prominent toxic effect, especially its neurotoxicity, as it can cross the blood-brain barrier (BBB). The blood-brain AUC ratios of brucine and strychnine were dose-related, limiting its clinical applications (Wu et al., 2012; Ren et al., 2018). Semen Strychni-induced neurotoxicity makes the central nervous system reflex center more sensitive to afferent stimuli and blocks postsynaptic inhibitory Glycine (Gly) receptors in the spinal cord and brain stem (Guo et al., 2018; Li et al., 2018).

Processing and compatibility are the most common methods to reduce the toxicity of Semen Strychni (Guo et al., 2018). Licorice (Glycyrrhizae Radix et Rhizoma) is the root of Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L. or Glycyrrhiza inflata Bat., it is frequently used in traditional Chinese medicine. Licorice can reduce the toxicity of Semen Strychni without characterization of their active constituents (Gu et al., 2014; Zhang et al., 2018a; Ramalingam et al., 2018). Isoliquiritigenin (ISL), a flavonoid found in Glycyrrhiza glabrate, exerts neuroprotective effects, mainly through antioxidant, anti-inflammatory, anti-epilepsy, increasing energy metabolism (Zhan and Yang, 2006; Zhu et al., 2019a; Gao et al., 2020; Shi et al., 2020; Wang et al., 2020). However, little is known about the mechanism of action between ISL and Semen Strychni.

Neurotransmitters can be regarded as biomarkers of SAs-induced neurotoxicity (Sun et al., 2018; Vogt, 2019). It mainly including monoamines, amino acids, and choline. Glutamate (Glu) and γ-aminobutyric acid (GABA) are the primary excitatory and inhibitory amino acid neurotransmitters in the brain. An imbalance between Glu and GABA neurotransmission can cause epileptic seizures and irreversible neuronal damage (Yang et al., 2020; Akyuz et al., 2021). Gly is an inhibitory amino acid neurotransmitter that inhibits neuromotor (Fernando et al., 2015; Spiering, 2018). Due to this, an overdose of Semen Strychni developed widespread muscle spasms and convulsions (Naik and Chakrapani, 2009), severe cases due to clonic seizures resulting in tonic epilepsy and died of respiratory arrest (Malone et al., 1992). Acetylcholine (ACh) can act as a neurotransmitter or neuromodulator (Vogt, 2018), and it is involved in controlling breathing (Mutolo et al., 2011). As the study indicated, the level of ACh in rat serum of the Semen Strychni group showed a significant elevation, and Acetylcholinesterase (AchE) activity decreased (Li et al., 2018; Sun et al., 2018).

In neurotransmitter metabolism, Dopamine (DA) and Tryptophan (Trp) pathways are two critical metabolic pathways. In the DA pathway, the synthesis of DA, epinephrine (E), and norepinephrine (NE) are from Tyrosine (Tyr) by the enzyme Tyrosine Hydroxylase (TH), and further metabolized to 3,4-dihydroxyphenylacetic acid (DOPAC) and Homovanillic acid (HVA) via Catechol-O-methyl transferase (COMT) and Monoamine oxidase (MAO). They play an essential role in mood, behavior, and motor control (Matsubara et al., 2011; Dimic et al., 2017; Yao et al., 2020). In the Trp pathway, serotonin (5-hydroxytryptamine, 5-HT) is synthesized from Trp and biotransformed to 5-hydroxyindole acetic acid (5-HIAA) by MAO. Neurotoxicity can alter neurodevelopment and neurotransmitter metabolism-related genes expression patterns (Tian et al., 2021). Metabolic disorders in the 5-HT and DA pathways are associated with central nervous system diseases, such as sudden death in epilepsy (Wu et al., 2012; Comai et al., 2020). The above results are similar to the symptoms of Semen Strychni poisoning. Maybe Semen Strychni neurotoxicity is associated with these neurotransmitters.

In the present study, we evaluated the degree of neuronal damage in Semen Strychni extract (SSE) and the alleviating effect of ISL by examining the degeneration of hippocampal neurons and the synthesis, release, transport, and metabolism of neurotransmitters. Our research aimed to provide a reference for improving the clinical application safety of Semen Strychni.

In this study, the rats were observed for symptoms of SSE-induced neurotoxicity. By intraperitoneal injection of SSE (125 mg/kg) for seven consecutive days, severely neurons degenerated in the CA1 region. Neuronal injury impairs cognitive and motor function. Over time, damaged neurons impair hippocampal function, gradually leading to memory deficits (Lazarov and Hollands, 2016). Strychnine exposure altered neuronal synapses during embryonic development and impaired motor ability in adulthood (Roy et al., 2012). In our current study, neuronal degeneration was detected by FJ-B staining, which proved that Semen Strychni-induced neurotoxicity could affect the development of neurons and potentially cause cognitive impairment. Furthermore, ISL has a detoxification effect on SSE.

When severe Semen Strychni poisoning occurs, rats die of tonic seizures following clonic convulsions. With oral administration, it is rapidly absorbed in the gastrointestinal tract, and the first pass effect will reduce the amount of Semen Strychni entering the brain. At the same time, intraperitoneal injection induces clonic convulsions without subsequent tonic extension seizures and high Mortality Rates (Malone et al., 1992). Although studies have shown that rats orally administrated with SSE (552 mg/kg) for 15 d were found neuronal necrosis, intracellular lipid accumulation, and cell apoptosis in the cerebral cortex (Li et al., 2018; Sun et al., 2018). Our results indicated that compared with the oral administration for 15 days, the intraperitoneal administration for 7 days can evoke the symptoms of Semen Strychni neurotoxicity more quickly.

In addition to neuron damage, we also found changes in the concentration of neurotransmitters in different brain regions. According to our results, the number of neurotransmitters with significant differences in serum is higher than plasma. Therefore, detection of serum is sufficient. In addition, a comparison of significant changes in the number of neurotransmitters in the blood and the brain showed that changes in the serum were not representative of the brain because of the BBB (Gupta et al., 2019). The neurotoxicity may be more accurately studied by directly examining the relevant brain tissues (Ge et al., 2013).

In terms of amino acid neurotransmitters, in the SSE group, the concentrations of GABA and Glu in the cerebellum and hippocampus were significantly increased, which is consistent with the reports of Shi et al. (2017) and Sun et al. (2018). Glu is an excitatory neurotransmitter, and GABA is an inhibitory neurotransmitter. The comparative balance keeps the brain in a controlled environment. Once the balance is destroyed, the abnormal discharge of neurons will appear. The pathogenesis of convulsions is the relative imbalance of excitatory and inhibitory neurotransmitters (Akyuz et al., 2021). The symptoms of Semen Strychni poisoning showed that the excitatory effect of Glu was more significant than the inhibitory effect of GABA. In that case, the content of Glu in the synaptic cleft increases, its circulation is unbalanced, and a large amount of Ca²⁺ flows into neurons, which can cause Glu-induced neurotoxicity (Yang et al., 2012; Hu et al., 2018; Yang et al., 2020). Thus, we also examined the Glu receptors. Glutamatergic N-methyl-D-aspartic acid receptors (NMDARs, which are special ionic Glu receptors with Ca²⁺-gated channels) are essential receptors of Glu. It composes of NR1 (which binds the co-agonist glycine), NR2 (which binds glutamate), and NR3 subunits (Reis et al., 2009). NMDARs can mediate distinct cellular responses because of the regionalized receptor activities. Activation of synaptic NMDARs initiates plasticity and stimulates cell survival while activating extra-synaptic NMDARs promotes cell death (Wang and Reddy, 2017). In this study, the neurotoxicity of SSE inhibited the mRNA level of NR1, NR2A, NR2B, which probably resulted in degeneration of neurons and inhibition of glycine excitation.

GABA exists in the cerebellum, hippocampus (Yoon and Lee, 2014). There are two types of GABA receptors: GABA_A and GABA_B receptors (Brohan and Goudra, 2017). GABA mediates its effects through the ionotropic GABA_A receptors (GABRs), which belong to the Cys-loop superfamily of ligand-gated ion channels. The GABRs are pentameric, membrane-bound proteins surrounding an anion-selective pore (Giraudo et al., 2019). When GABA binds to receptors, it suppresses neuronal activity in the adult brain by opening a transmembrane channel permeable to chloride (Sigel and Steinmann, 2012; Zhu et al., 2018; Masiulis et al., 2019). However, in our results, the mRNA level of GABA receptors was decreased and could not bind with the increased GABA, thus the inhibitory effect of GABA on the neurotoxicity of Strychnos was weakened.

Besides, the concentrations of Tyr in the cerebellum and hypothalamus; Trp in the striatum and hypothalamus decreased, while Trp cerebellum increased in our results. Overall, the concentration of Trp went up (Sun et al., 2018), which indicates that the crucial role of Trp is in the cerebellum, and it tends to aggregate in the cerebellum (Sarna et al., 1991). Studies have described that when rats are given SSE, their exercise ability decreases (Shi et al., 2017). The surface area of the cerebellar cortex is 80% that of the cerebral cortex. The cerebellum has traditionally been thought to coordinate movement and maintain a sense of balance, but now research has shown that it also has a cognitive function (Koziol et al., 2014; Sereno et al., 2020; Van Overwalle et al., 2020). Once it’s damaged, there’s a lot of neurotransmitters that are affected. According to our results, the number of changes in neurotransmitters was highest in the cerebellum, followed by the hippocampus. The hippocampus is an important structure for learning and memory and is the site of a great deal of neurogenesis, it is also susceptible to biochemical and neurochemical alterations (Kang et al., 2017; Fernandes et al., 2020). In general, the structure and function of cerebellum and hippocampus were severely damaged when SSE was poisoned.

Trp and Tyr are involved in the two most important metabolic pathways of neurotransmitters--DA and Trp pathways. DA mainly exists in the striatum, providing learning signals by regulating synaptic plasticity (Berke, 2018; Walters et al., 2020). The striatum is associated with motor function—damage to the striatum results in aberrant sequencing of spontaneous movements and sensory-guided movements (Markowitz and Datta, 2020). Trp metabolism could occur both peripherally and centrally, and the 5-HT pathway is one of the main pathways (Li et al., 2020). Our study showed no significant changes in 5-HT and DA, but the concentration of NE, E, DOPAC, HVA, and 5-HIAA was significantly changed by neurotoxicity of SSE. The striatum and cerebellar endothelium may have the ability to convert Trp to 5-HIAA (Sarna et al., 1991), which is why the concentration of 5-HT was not affected. In addition, the ratio of 5-HIAA/5-HT and DOPAC/DA in the cerebellum and the activity of MAO were decreased, indicating that the inhibition of MAO inhibited 5-HT and DA metabolism. MAO is one of the main metabolic enzymes for DA metabolism. The downregulation of MAO leads to the decrease of DA metabolism (Graves et al., 2020). MAO is the essential metabolic enzyme to DA metabolism and catalyzes 5-HT to 5-HIAA (Wu et al., 2015). Previous reports demonstrated that Ca²⁺ selectively enhanced MAO activity in mice, rats, monkeys, and human Brains. But an antagonist of Ca²⁺ permeable NMDARs could potentially reduce MAO activity (Robinson et al., 2016). Therefore, inhibition of NMDARs mRNA expression by SSE neurotoxicity may be one of the reasons for MAO reduction.

Curiously, in our research, there was no significant change in the level of ACh, and the concentration of Gly was elevated in the prefrontal cortex in the SSE group. Ach is a central stimulant of the autonomic nervous system and mediated by cholinergic and nicotinic receptors. Increasing evidence suggests that the dysfunction of nicotinic acetylcholine receptor (nAChR), which is widely expressed in hippocampal and cortical neurons, may be related to the pathogenesis of epilepsy (Akyuz et al., 2021). The neurotoxicity of SSE may be due to its influence on nAChR, which is involved in epilepsy and affects Ach expression. Gly is a major inhibitory neurotransmitter in the brain stem and spinal cord. In the hippocampus, glycine synaptic plasticity is mainly controlled by the GlyT1 transporter. GlyT1 overexpression in the epileptic brain leads to dysregulation of Gly signaling (Shen et al., 2015). Neurotransmitters are unstable and quickly degraded (Hyman, 2005). Perhaps we can study the effect of SSE on glycine by examining the receptor GlyT1.

In the present study, oral administration of ISL (50 mg/kg) immediately after intraperitoneal injection of SSE attenuated the symptoms of severe twitches, myotonia, and breathing difficulty in rats. Previous studies found that oral administration of ISL (>25 mg/kg) has sedative-hypnotic effects by positive allosteric modulation of GABA_A-BZD receptors (Cho et al., 2011). That is likely to be an important mechanism in the alleviation of ISL on Semen Strychni neurotoxicity. Additionally, ISL improved neuronal degeneration, restored the abnormal levels of neurotransmitters such as Gly, Trp, DOPAC, Glu, returned the mRNA expression of Glu, GABA receptors and the level of metabolic enzymes MAO, COMT to normal, which suggests that ISL has a detoxification effect.

ISL has memory enhancing and anti-Alzheimer’s activities, it can improve age-related neurodegenerative disorders (Ramalingam et al., 2018). ISL improved the degenerative neurons probably by inhibiting the mitochondrial protein mitoNEET (Chen et al., 2019) and reduced Trp expression by activating the extracellular signal-regulated protein kinase pathway (Lv et al., 2020). Moreover, excessively released Glu in the system can lead to the overstimulation of postsynaptic Glu receptors that leads to excitotoxic neuronal injury in vitro (Im et al., 2016). ISL can prevent Glu-induced toxicity by mitigating mitochondrial dysfunction (Ramalingam et al., 2018). The most important point is that ISL can act on receptors. ISL is a novel NMDA receptor antagonist that is bound to NMDA receptors to inhibit the Glu-induced increase in Ca²⁺ influx (Kawakami et al., 2011) and inhibits cocaine-induced striatal dopamine release by regulating the GABA_B receptor (Jang et al., 2008). Besides, Pan et al. found ISL inhibited the activity of MAO-A and MAO-B in brain mitochondria of normal rats (Pan et al., 2000). However, our results indicate the opposite. When damage occurs in the body, blood vessels, cells and other tissues will change (Wang and Li, 2018). Due to this, under normal circumstances, ISL reducing MAO activities, the opposite was observed in the case of Semen Strychni poisoning. We speculated that when ISL interacts with SSE, ISL has another neuroprotective mechanism of action to antagonize SSE-induced neurotoxicity. Further studies are required to confirm our speculation.

In summary, ISL has the function of regulating neurotransmitters and preventing damage to the BBB and neurons (Wang et al., 2008; Zhang et al., 2018b; Zhu et al., 2019b), which may be why ISL can detoxify the neurotoxicity of SSE. Therefore, the potential function and mechanism of the compatibility of ISL and Semen Strychni to alleviate the neurotoxicity of Semen Strychni deserve further study.

We investigated the mechanism of Semen Strychni neurotoxicity and the detoxification mechanism of ISL from the perspective of neurotransmitter metabolic pathways. ISL has detoxifying effects when taken orally immediately after intraperitoneal injection of SSE by reducing the degeneration of neurons, restoring the metabolic pathways of Trp and DA. What’s innovative in our studies is that Semen Strychni-induced neurotoxicity suppressed the mRNA relative expression of GABRs and NMDARs, which not only reduced the activity of MAO and affected the metabolism of DA and 5-HT, but also may be one of the reasons for the neuronal degeneration and the inhibition of Gly excitation. In addition, we suggested that detecting the receptor GlyT1 can further investigate the effect of SSE on Gly. Taken together, from our studies, we speculated that Semen Strychni-induced neurotoxicity might risk neurological diseases. Intimately monitoring and timely rescue measures should be taken in clinical practice.