Genistein Protects Epilepsy-Induced Brain Injury Through Regulating The JAK2/STAT3 and Keap1/Nrf2 Signaling Pathways in The Developing Rats

Qing-peng Hu The Second Hospital,University of South China Xiang-yi Huang (  hqpeng26986@126.com ) The Second Hospital, University of South China Wei Feng The Second Hospital,University of South China Fen-fang Chen The Second Hospital,University of South China Hong-xia Yan The Second Hospital,University of South China Xin Zhang The Second Hospital,University of South China Yang -wan Zhou The Second Hospital,University of South China


Background
Epilepsy is a chronic neurological disease that impacts all demographics and places a heavy socioeconomic burden on both patients and the healthcare system.It is estimated that 10.5 million children worldwide suffer from epilepsy, and 80% of epilepsy patients live in low-and middle-income countries [1] .The causes of epilepsy are complex and diverse, including genetic and metabolic factors, immune abnormalities, trauma, infection, and systemic diseases [2,3,4] .Antiepileptic drugs can inhibit epilepsy or improve symptoms without affecting the pathological process of the disease, and about 30% of patients have refractory epilepsy [5] .It is therefore of great importance to explore the pathogenesis of epilepsy and nd new, targeted anti-epileptic drugs.
The JAK-STAT signal pathway is a cytokine-stimulated signal transduction pathway discovered in recent years.It participates in many important biological processes such as cell proliferation, differentiation, in ammation, apoptosis and immune regulation [6,7] .Janus kinase (JAK) can sense extracellular signals by binding to receptors such as interferon, interleukin, growth factor, etc. [8] , and transmit information to signal transducer and activator of transcription (STAT) [9] .Phosphorylated STATs can dimerize and translocate into nucleus where they bind to the promoter DNA sequence, causing changes in DNA transcription and activity levels, which in turn affects basic cell functions such as cell growth, differentiation and death [10] .JAK2 and STAT3 are mainly expressed in brain tissue.Studies have found that the activated JAK2/STAT3 pathway after brain injury promotes the activation and proliferation of microglia, and participates in various pathological processes such as in ammation and apoptosis in central system diseases [11,12] .The current research on JAK2/STAT3 signaling pathway is mainly focused on hypoxic-ischemic encephalopathy, hematological tumors, immune dysfunction and cardiovascular diseases [13,14] , with few reports on developmental epilepsy.
The oxidative stress pathway plays an important role in the occurrence and development of many diseases [15,16] .Kelch-like epichlorohydrin-related protein-1 (Keap1) is a multi-regional repressor protein of the Kelch family and a cytoplasmic inhibitor of Nrf2 [17] .Nuclear factor E2-related factor 2 (Nrf2) is a key factor in the oxidative stress response which is regulated by Keap1 and regulates the expression of antioxidant protein and phase II detoxi cation enzymes by interacting with the antioxidant response element ARE [18] .Under the stimulation of hypoxia, shock, infection, cytokines, etc., the Nrf2 signaling pathway can be activated to initiate the expression of multiple downstream target proteins such as hemeoxygenase 1 (HO-1) and dependent reduced coenzyme/ Quinone oxidoreductase 1 (NQO1) [19] .HO-1 and NQO1 can reduce the toxic effects of oxygen free radicals and endogenous toxins on neurons, thus having anti-oxidation, anti-apoptosis and anti-infection properties [20] .The current research on the Keap1/Nrf2 signaling pathway mainly focuses on cerebral hemorrhage, cerebral infarction and neurodegenerative diseases [21,22] , with again, few reports on the pathogenesis of epilepsy.Genistein (Gen) is an iso avone from soy extract.It is a protein tyrosinase inhibitor with strong effects of anti-angiogenesis, anti-free radical, anti-oxidation, anti-in ammatory factor release and anti-tumor [23,24] .
Studies have shown that Genistein can inhibit the excessive proliferation and activation of astrocytes and microglia induced by cerebral ischemic damage [25,26] , thereby exerting protective effects on nerve function.Genistein can also affect the expression of transcription factors [27] , thereby regulating the in ammatory and immune response.However, the mechanism of Genistein in the pathogenesis of epilepsy is still unclear.The purpose of this study is to establish a developing rat epilepsy model in order to observe the regulation of the JAK2/STAT3 and Keap1/Nrf2 signaling pathways, as well as the protective mechanism of Genistein in treating epilepsy-induced brain injury.Methods 1.1 Animals 80 healthy male Sprague-Dawley rats (20 days old, weighing 60-80g) were provided by the Experimental Animal Center, University of South China.All rats were randomly divided into 4 groups, and each group consisted of 20 animals: (1) Control group; (2) PTZ group; (3) PTZ+5mg/kg Gen group (4) PTZ +10mg/kg Gen group.The feeding conditions of all experimental animals were alternated between day and night for 12 hours.The ambient temperature was 22°C-26°C, and the humidity was approximately 55%.Rats were allowed free access to food and water.This experiment was approved by the Ethics Committee of the Second Hospital, University of South China and meets the requirements of the National Health Center.

Establish rat epilepsy model
Intraperitoneal injection of 35 mg/kg PTZ for 5 days to induce a developmental epilepsy model [28] .The PTZ+Gen groups were given intraperitoneal injection of Genistein (5 or 10mg/kg) 30 min before each PTZ administration.The Control group was only injected with saline.The latency, duration and intensity of each seizure were recorded, and the average values were calculated for statistical analysis.Seizure intensity scores were graded on the Racine scale [73] : Grade : generalized tonic-clonic seizures and falls; Grade : standing with forelimb clonus; Grade : forelimb clonus; Grade : rhythmic nodding; Grade : tremor, facial muscle twitching, chewing; Grade 0: no seizures.A -grade indicates successful seizure modeling.A total of 60 rats were injected with KA.There were 3 rats that died due to status epilepticus and 2 rats that did not exhibit seizures.The success rate of modeling was 91.6%.Finally, 16 rats in each group were selected for subsequent experiments.The rats were sacri ced with an anesthetic (1% barbital sodium, 500 mg/kg) 24h after epilepsy.The rats were then perfused intracardially with saline and hippocampal tissue were immediately collected.The samples were stored at -80°C for subsequent experiments.

Immuno uorescence staining
The rats were anesthetized with iso urane and transcardially perfused with normal saline followed by 4% paraformaldehyde until the liver turned white.The brain tissues were removed and xed in 4% paraformaldehyde for 24 hours, and then soaked in 30% sucrose solution for dehydration.After OCT embedding, the brain tissues were cut into 5μm thick slices on a cryostat.The tissue sections were permeabilized with 0.5% TritonX-100 at room temperature for 20 min.After blocking with 10% goat serum at room temperature for 1 h to eliminate non-speci c staining, the tissue sections were incubated with primary antibodies: NeuN (rabbit anti-rat 1:500), Iba-1 (rabbit anti-rat 1:250) and GFAP (rabbit anti-rat 1:500) at 4°C overnight.After washing 3 times with PBS, the tissue sections were incubated with uorescently labeled secondary antibody (1:1000) at 37°C for 2 h.After washing 3 times with PBS, the tissue sections were incubated with DAPI (1:1000) for 30 min to stain the nucleus.Finally, sections were mounted onto gelatin-coated glass slides and air-dried.The number of neurons, activated microglia and astrocytes were observed with a uorescence microscope.Five high-power elds (×400) with the same area were randomly selected for each slice, and the number of NeuN, Iba1 and GFAP stained positive cells in each eld were visually counted, and the average value was used for statistical analysis.

Western blot to detect the protein levels
The rats were decapitated and hippocampal tissue was separated.After adding RIPA lysate and PMSF protease inhibitor, tissue was homogenized with a glass rod until fully broken.The dissolved tissue was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was the total protein.The supernatant was collected and the protein concentration was determined by BCA method.Stacking and separation gel were prepared according to the instructions and placed in the electrophoresis tank.After adding the protein samples and the protein marker to the sample wells, the electrophoresis tank was set to a constant voltage of 80V.Once the protein sample and the marker were electrophoresed to the separation gel position, voltage was set to 100V until the end of electrophoresis.After adding the transfer buffer to the transfer tank, the current was set to 200 mA and the protein was transferred to the PVDF membrane.The transferred PVDF membrane was put into 5% skimmed milk for blocking non-speci c binding at 37°C for 2 h.The membranes were incubated at 4°C overnight with primary antibodies: p-JAK2 (1:1000), p-STAT3 (1:2000), TNF-α (1:1000), IL-1β (1:1000), Keap1 (1:1000), Nrf2 (1:1000), HO-1 (1:1000), NQO1 (1:1000), caspase3 (1:1000), Bax (1:1000), Bcl-2 (1:1000) and β-actin (1:1000).After washing 3 times with PBST, the membranes were incubated with secondary antibody (1:2000) at 37°C for 2 h.ECL luminescent solutions A and B were mixed in equal volumes, and the PVDF membrane was immersed in the luminescent solution for 5 min.After three PBST rinses, the protein bands were scanned with the Image Quant ™ LAS 4000 imaging system, and the scan results were analyzed with ImageJ software.

Table 1 Nucleotide sequences of qRT-PCR primers 1.7 Statistical analysis
All the data analyses were performed using SPSS 20.0 statistical software.The data were expressed as Mean±SD.Means comparison between two groups were analyzed by t-test and P<0.05 was considered statistically signi cant.Means comparison between multiple groups were analyzed by one-way analysis of variance (ANOVA), and the Bonferroni post-test correction was used to reduce the likelihood of false positives.There were 5 comparisons for Western blot, qRT-PCR, TUNEL and Immuno uorescence staining, and the signi cance level was adjusted accordingly to P<0.01.
Compared with the PTZ group, the Genistein treatment groups reduced seizure intensity and duration, and prolonged seizure latency (Figures 1-3).Further comparison found that the Genistein dose of 15mg/kg had a greater therapeutic effect than 5mg/kg.These results suggest that Genistein can inhibit seizure activity.

Genistein increases mature neuronal cell count and reduces activated microglia and astrocytes
Immuno uorescence staining was used to observe the number of mature neurons, activated microglia and astrocytes in the hippocampal CA1 region 24 h after epilepsy (Figure 4, 5, 6).The results are as follows: in the Control group, the number of mature neurons (NeuN positive cells) was normal and the distribution was neat and orderly (Figure 4A1), while activated microglia (Iba-1 positive cells) and astrocytes (GFAP positive cells) were almost invisible (Figure 5A1,6A1); In the PTZ group, the number of mature neurons was signi cantly reduced (Figure 4B1) and the distribution was disordered; activated microglia and astrocytes were signi cantly increased (p<0.01), with the cell volume of microglia becoming larger with an amoeba or round appearance, and shorter, thickened or fused cell protrusions (Figure 5B1).Compared with the PTZ group, the number of neurons signi cantly increased in the Genistein (5 or 15mg/kg) treatment groups (Figure 4C1,4D1) (p<0.01);activated microglia were signi cantly reduced in the PTZ+15mg/kg Gen group (Figure 5D1) (p<0.01) and the cell volume became smaller and the protrusions more slender; activated astrocyte were also signi cantly reduced in the Genistein (5 or 15mg/kg) treatment groups (Figure 6C1,6D1) (p<0.05).Further comparison found that 15mg/kg Genistein was more effective than the 5mg/kg dose.
Immuno uorescence staining suggests that Genistein can increase mature neuronal cells and reduce activated microglia and astrocytes, thus supporting its protective effect on brain damage post epilepsy.

Genistein inhibits the expression of JAK2/STAT3 signaling pathway
To explore the effect of Genistein on the JAK2/STAT3 signaling pathway, Western blot and qRT-PCR were used to determine the protein and mRNA levels of p-JAK2, p-STAT3, TNF-α and IL-1β in the hippocampus after PTZ-induced epilepsy (Figures 7 and 8).The optical density of the band was used to further analyze the relative expression of the protein (Figure 7B).Our ndings indicate that the protein and mRNA levels of p-JAK2, p-STAT3, TNF-α and IL-1β increased signi cantly in the PTZ group and these increases were attenuated by Genistein treatment (Fig. 7B and 8, p<0.01).Further comparison revealed that the protein levels of p-JAK2 and IL-1β, and the mRNA levels of p-JAK2, TNF-α and IL-1β in the PTZ+15mg/kg Gen group were signi cantly lower than that in the PTZ+5mg/kg Gen group (p<0.01).
Western blot and qRT-PCR results thus indicate that Genistein can inhibit the expression of the JAK2/STAT3 signaling pathway on PTZ-induced epilepsy in developing rats.

Genistein activates the Keap1/Nrf2 signaling pathways
To further investigate the effect of Genistein on the Keap1/Nrf2 antioxidant stress pathway, Western blot and qRT-PCR were used to detect the protein and mRNA levels of Keap1, Nrf2, HO-1 and NQO1 in the hippocampus after epilepsy (Figures 9 and 10).The results were as follows: The protein and mRNA levels of Keap1 increased signi cantly in the PTZ group compared to the Control group (p<0.01), and this increase was inhibited by Genistein treatment (p<0.01) (Fig. 9B and 10); The protein and mRNA levels of Nrf2, HO-1 and NQO1 in the PTZ group were signi cantly lower than the Control group levels (p<0.01), and these expression changes were reversed by Genistein treatment; (Fig. 9B and 10); Further comparison revealed that the protein and mRNA levels of Keap1 and NQO1 were signi cantly different in PTZ+5mg/kg Gen group and PTZ+15mg/kg Gen group (p<0.01); the protein level of HO-1 and the mRNA level of Nrf2 in the PTZ+15mg/kg Gen group was signi cantly higher than that in the PTZ+5mg/kg Gen group (p<0.01).
These result suggests that Genistein can activate the Keap1/Nrf2 antioxidant stress pathway after PTZinduced epilepsy.Furthermore the inhibitory effect is dose-dependent, with 15mg/kg Genistein having a greater inhibitory effect.

Genistein inhibits the expression of apoptotic markers
In order to explore the effects of Genistein on apoptosis-related proteins, Western blot and qPCR were used to measure the protein and mRNA levels, respectively, of caspase3, Bax and Bcl2 in the hippocampus after epilepsy (Figure 11, 12).The results were as follows: The protein and mRNA levels of caspase3 and Bax in the PTZ group increased signi cantly after epilepsy (p<0.01), while Genistein treatment was shown to reduce the expression of both; The protein and mRNA levels of Bcl2 in the PTZ group decreased signi cantly compared to the Control group (p<0.01), and these expression changes were reversed by Genistein treatment (Fig. 11 and 12); Further comparison found that Bax protein and caspase3 mRNA were signi cantly lower, and Bcl2 mRNA expression was signi cantly higher in the PTZ+15mg/kg Gen group than in the PTZ+5mg/kg Gen group.
These results demonstrate that Genistein can reduce the ratio of apoptotic proteins caspase3 and Bax to anti-apoptotic protein Bcl2, thereby exerting a protective effect on cell apoptosis.At the same time, a heightened treatment effect is correlated with a heightened Genistein dose.

Discussion
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 in ammation 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 in ammatory 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 [36] , and PTZ is related to nerve excitotoxicity and the expression of reactive oxygen species [37] .JAK2/STAT3 is an important in ammatory response-related pathway.The activation of this pathway can initiate gene transcription in the nucleus, and regulate the in ammatory response and the expression of apoptotic factors [38] .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 [39] .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.) [40]   .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 speci c promoter sequence and regulate gene transcription [41] .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 signi cantly 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: Epilepsy can induce the activation of JAK2/STAT3 signaling.The activated JAK2/STAT3 signal promotes the expression of downstream in ammatory factors TNF-α and IL-1β [42] .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 [43] .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 in ammation that is di cult to control.The 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 [44] .JAK2/STAT3 signaling can mediate the activation of microglia and astrocytes, which can in turn, secrete TNF-α and IL-1β, leading to neuroin ammation 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 [45] while Liu's research shows that inhibiting the JAK2/STAT3 signaling pathway can reduce seizure-induced brain injury [46] .These research results are consistent with our ndings 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 antioxidative stress and can promote the expression of a series of antioxidant protective proteins [47] .At the same time, it can coordinate many protective detoxi cation and anti-in ammatory genes, and synergistically improve the e ciency of the cellular defense system [48] .Under physiological conditions, Keap1 combines with and inhibits Nrf2.Under the in uence 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 signi cantly in the hippocampus, which con rmed that the Nrf2 signaling pathway plays an important role in the early stages of epileptic seizures [51] .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 [52] .In our experiment, the results of Western blot and qPCR showed that the expression of Nrf2, HO-1, NQO1 decreased and Keap1 increased signi cantly 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 in ammatory mediators was reduced.This indicates that the overexpression of Nrf2 can inhibit the in ammatory response against post-epileptic brain injury, and this effect may be related to the inhibition of JAK3/STAT2 and TLR4/NF-kB in ammation signal pathway [53] .
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 neuroin ammation and neuronal apoptosis [54] .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 [55] .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 neuroin ammation under the stimulation of central nervous system damage, infection, toxins or autoimmunity [58] .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 [59] .At the same time, during epileptic seizures, the excitatory and inhibitory amino acids are unbalanced within and outside the cell, and as mentioned, the in ammatory pathway is activated [60] .Thus we propose that astrocytes are closely related to the pathological process of epilepsy.In this experiment, the immuno uorescence results showed that Iba-1 and GFAP positive cells increased signi cantly after epilepsy, which con rmed 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-in ammatory mediators, cytokines and ROS [61] .We found that NeuN positive cells decreased signi cantly 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 in ammatory factor, IL-6 increased signi cantly, con rming that neuronal apoptosis and in ammation are involved in the pathological process of epilepsy.The speci c 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 [62] , 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 [63] .At the same time, the production of in ammatory factors can further activate microglia and astrocytes and promote the pathological process of epilepsy [64] .These potential mechanisms of action are consistent with the results of our study.
Genistein is a natural iso avone compound that has anti-tumor, -oxidation, and -in ammatory properties, and offers synaptic protection, a reduction of astrocyte aggregation and improvements in learning and memory [65] .Some studies show that Genistein can reduce the NF-kB-induced in ammatory response by reducing ROS levels, blocking mitochondrial-dependent apoptosis and thereby reducing the area of cerebral infarction [66] .Genistein can also directly act on vascular endothelial cells, alleviating oxidative stress, in ammation and vascular damage by increasing the protein expression of eNOS [67] .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 in ammation pathway, and apoptotic caspase3 and Bax increased signi cantly 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: Genistein reduces the expression of in ammatory 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 ndings [70] ; Genistein inhibits mitochondrial damage and the initiation of apoptosis programs by increasing the synthesis of anti-apoptotic proteins, thereby increasing the number of surviving neurons [71] ; 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 [72] , which is consistent with our study; Genistein reduces the expression of in ammatory factors by inhibiting the activation of astrocytes and microglia; Studies have also shown that Genistein inhibits the Ca 2+ in ux 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.

Conclusions
In conclusion, Genistein can reduce the intensity and duration of seizures, inhibit the activation of microglia and astrocytes as well as the expression of JAK2/STAT3 in ammatory pathways, while activating the Keap1/Nrf2 oxidative stress pathway, and increasing anti-apoptotic proteins and neuronal survival.This experiment thereby reveals the potential protective effects and underlying mechanisms of Genistein on brain damage after developmental epilepsy, and also provides a theoretical basis for the development of new anti-epileptic drugs.

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