Epilepsy is a chronic brain disease caused by the abnormal discharge of brain neurons. At present, the pathogenesis of epilepsy is not clear. The existing antiepileptic drugs primarily suppress neuronal excitability by regulating neurotransmitter receptors (such as glutamate, GABA and acetylcholine) or ion channels [29, 30, 31]. These are in fact anticonvulsant medications rather than antiepileptic treatments, as they do not target the implicit underlying etiology. Epilepsy can cause the activation and proliferation of microglia and astrocytes, which are involved in various pathological processes such as inflammation and apoptosis in central system diseases [32, 33]. At the same time, epileptic seizures will increase oxidative stress and promote the production of reactive oxygen species, leading to excessive neuronal excitement, oxidative damage, cells apoptosis and long-term intracranial biochemical changes [34, 35]. Therefore, it is believed that the inflammatory and oxidative stress responses are the underlying mechanisms of epilepsy neuropathology. In this experiment, we chose the PTZ-induced epilepsy model as it is similar to the human epilepsy model , and PTZ is related to nerve excitotoxicity and the expression of reactive oxygen species .
JAK2/STAT3 is an important inflammatory response-related pathway. The activation of this pathway can initiate gene transcription in the nucleus, and regulate the inflammatory response and the expression of apoptotic factors . JAK2 is a kind of non-receptor tyrosine protein kinase family. STAT3 is a denucleotide binding protein, which is the substrate and downstream factor of JAK2 . JAK2 and STAT3 are widely distributed throughout the central nervous system, and can be activated by many cytokines and growth factors (such as interleukins, colony stimulating factors, growth hormones, etc.) . Activated JAK2 can activate STAT3, and transmit extra-membrane stimulus signals into cells through the tyrosine residues of various target proteins, leading to the phosphorylation of JAK2 and STAT3. p-STAT3 dimerizes and transfers to the nucleus, which can bind specific promoter sequence and regulate gene transcription . In this experiment, the results of Western blot and qRT-PCR showed that the expression of the JAK2/STAT3 signaling pathway as well as apoptotic proteins caspase3 and Bax, increased significantly after epilepsy. This implicates the JAK2/STAT3 signaling pathway in the pathological process of epilepsy. In combination with experimental results, we considered the following possible mechanisms: ①Epilepsy can induce the activation of JAK2/STAT3 signaling. The activated JAK2/STAT3 signal promotes the expression of downstream inflammatory factors TNF-α and IL-1β . At the same time, the high expression of TNF-α and IL-1β can bind to their receptors, and further activate JAK2 and STAT3, leading to their phosphorylation . The combination of p-JAK2 and p-STAT3 with DNA increases the expression of cytokine genes and produces more interleukins and cytokines. This vicious cycle can lead to persistent inflammation that is difficult to control. ②The downstream signals regulated by JAK2/STAT3 also include the apoptotic targets caspase3, Bax, and Bcl-2 family proteins. Therefore, the activation of JAK2/STAT3 can lead to neuronal apoptosis and aggravate the pathological process of brain injury . ③JAK2/STAT3 signaling can mediate the activation of microglia and astrocytes, which can in turn, secrete TNF-α and IL-1β, leading to neuroinflammation and brain damage after epilepsy. Yang's research shows that JAK2/STAT3 signaling can cause brain damage and behavioral abnormalities through increasing the expression of IL-1β in neonatal rats with hypoxic-ischemic encephalopathy  while Liu's research shows that inhibiting the JAK2/STAT3 signaling pathway can reduce seizure-induced brain injury . These research results are consistent with our findings and support our proposed mechanisms of epileptic-pathology.
Keap1 is a multi-domain repressor protein of the Kelch family, which binds to Nrf2 through the Cul3 ubiquitin ligase containing E3. Nrf2 is an important transcription factor that regulates cellular anti-oxidative stress and can promote the expression of a series of antioxidant protective proteins . At the same time, it can coordinate many protective detoxification and anti-inflammatory genes, and synergistically improve the efficiency of the cellular defense system . Under physiological conditions, Keap1 combines with and inhibits Nrf2. Under the influence of external oxidative stressors, Nrf2 is decoupled with Keap1 and combined with the antioxidant response element ARE to activate the Nrf2 signaling pathway, thereby increasing the expression of antioxidant proteins HO-1, SOD, NQO1, etc., and reducing oxidative damage and accumulation of toxicity metabolites [49,50]. In a study by Wang et al. when the amygdala was rapidly ignited, the protein and gene levels of Nrf2, HO-1 and NQO1 increased significantly in the hippocampus, which confirmed that the Nrf2 signaling pathway plays an important role in the early stages of epileptic seizures . Wu et al. found that activating the Nrf2-ARE signaling pathway had anticonvulsant effects and improved the cognitive function of epileptic rats, while Nrf2 knockout mice had more severe seizures and obvious cognitive impairment . In our experiment, the results of Western blot and qPCR showed that the expression of Nrf2, HO-1, NQO1 decreased and Keap1 increased significantly after epilepsy, suggesting inhibition of the Nrf2 antioxidant pathway, and activation of oxidative stress pathways, which is consistent with previous studies. At the same time, the Keap1/Nrf2 signaling pathway was activated after Genistein treatment, and the expression of IL-6 inflammatory mediators was reduced. This indicates that the overexpression of Nrf2 can inhibit the inflammatory response against post-epileptic brain injury, and this effect may be related to the inhibition of JAK3/STAT2 and TLR4/NF-kB inflammation signal pathway .
Microglia are the key regulators of immune response in the central nervous system. Under normal circumstances, microglia are in a static state but can be activated when subjected to abnormal stimulation, leading to rapid proliferation and migration to the injured site, and mediating neuroinflammation and neuronal apoptosis . Therefore, inhibiting the activation and proliferation of microglia may be an effective measure to reduce epilepsy-induced brain injury. Astrocytes are macroglial cells in the central nervous system. They play an important role in maintaining the stability of the internal environment, forming the blood-brain barrier, and transmitting nerve excitation . Astrocytes are the place where glutamate (Glu) and γ-aminobutyric acid (GABA) are metabolized. They have a spatial buffering effect on extracellular potassium ions and can maintain ion balance around neurons [56, 57]. Astrocytes can participate in the immune response of the central nervous system, and can cause neuroinflammation under the stimulation of central nervous system damage, infection, toxins or autoimmunity . In the pathological process of epilepsy, K+ channels are involved in regulating cell membrane resting potentials and the repolarization process of action potentials, and are closely related to the excitability of neurons . At the same time, during epileptic seizures, the excitatory and inhibitory amino acids are unbalanced within and outside the cell, and as mentioned, the inflammatory pathway is activated . Thus we propose that astrocytes are closely related to the pathological process of epilepsy. In this experiment, the immunofluorescence results showed that Iba-1 and GFAP positive cells increased significantly after epilepsy, which confirmed that the activation and proliferation of microglia and astrocytes were involved in the pathological process of PTZ induced-epilepsy. Activated microglia and astrocytes can cause neuronal damage and apoptosis through pro-inflammatory mediators, cytokines and ROS . We found that NeuN positive cells decreased significantly after epilepsy, indicating that the number of surviving neurons decreased. Meanwhile, the results of Western blot and qPCR showed that the apoptotic proteins caspase3 and Bax, and the inflammatory factor, IL-6 increased significantly, confirming that neuronal apoptosis and inflammation are involved in the pathological process of epilepsy. The specific mechanism considers that activated astrocytes and microglia secrete a variety of immune effector molecules, such as interleukins IL-1, IL-6, IL-8, NF-kB and oxygen free radicals , which can damage neurons, glial cells and the blood-brain barrier, leading to local or extensive damage of the central nervous system. Furthermore, abnormal glial cell function can cause a decrease in potassium buffer capacity or an excessive intake of GABA, which may induce seizures and aggravate brain damage . At the same time, the production of inflammatory factors can further activate microglia and astrocytes and promote the pathological process of epilepsy . These potential mechanisms of action are consistent with the results of our study.
Genistein is a natural isoflavone compound that has anti-tumor, -oxidation, and -inflammatory properties, and offers synaptic protection, a reduction of astrocyte aggregation and improvements in learning and memory . Some studies show that Genistein can reduce the NF-kB-induced inflammatory response by reducing ROS levels, blocking mitochondrial-dependent apoptosis and thereby reducing the area of cerebral infarction . Genistein can also directly act on vascular endothelial cells, alleviating oxidative stress, inflammation and vascular damage by increasing the protein expression of eNOS . At present, the research on Genistein mainly focuses on ischemic stroke, brain injury and Alzheimer's disease [68, 69], with few reports on epilepsy. In this experiment, we observed that activated microglia and astrocytes, expression of JAK2/STAT3 inflammation pathway, and apoptotic caspase3 and Bax increased significantly after epilepsy, while the number of neurons, expression of Nrf2 anti-oxidative stress pathway and anti-apoptotic protein Bcl-2 were reduced. Genistein was found to reverse these pathological processes, thereby playing a protective effect in epilepsy-induced brain damage. We consider the following as potential underlying mechanisms for this effect: ①Genistein reduces the expression of inflammatory factors by inhibiting the JAK2/STAT3 signaling pathway; Masakatsu demonstrated that Genistein can block JAK2 phosphorylation and EPO-induced glutamate release in a cerebral ischemia model, indicating Genistein has a neuroprotective effect consistent with our findings ; ②Genistein inhibits mitochondrial damage and the initiation of apoptosis programs by increasing the synthesis of anti-apoptotic proteins, thereby increasing the number of surviving neurons; ③ Genistein increases the expression of antioxidant proteins HO-1 and NQO1 and inhibits the generation of reactive oxygen species, thereby reducing the oxidative damage process induced by epilepsy; in Miao's research, Genistein increased the expression of Nrf2 and reduced brain damage caused by oxidation stress , which is consistent with our study; ④Genistein reduces the expression of inflammatory factors by inhibiting the activation of astrocytes and microglia; ⑤ Studies have also shown that Genistein inhibits the Ca2+ influx and glutamate release of hippocampal synaptosomes[74,75], thereby reducing seizures. This experiment proved that Genistein can reduce the seizure intensity and seizure duration, and prolong seizure latency, which is consistent with results in the literature. At the same time, it was found that Genistein has a greater therapeutic effect at a dose of 15 mg/kg compared to 5 mg/kg, indicating that the protective effect of Genistein on brain damage after epilepsy may be dose-dependent.