Taiwan Chingguan Yihau (NRICM101) prevents kainic acid-induced seizures in rats by modulating neuroinflammation and the glutamatergic system

Taiwan Chingguan Yihau (NRICM101) prevents kainic acid-induced seizures in rats by modulating neuroinflammation and the glutamatergic system | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Taiwan Chingguan Yihau (NRICM101) prevents kainic acid-induced seizures in rats by modulating neuroinflammation and the glutamatergic system Chi-Feng Hung, Wei-Che Chiu, Jia-Cih Chen, Wu-Chang Chuang, Su-Jane Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3932956/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Taiwan Chingguan Yihau (NRICM101) is a Traditional Chinese medicine (TCM) formula used to treat coronavirus disease 2019; however, its impact on epilepsy has not been revealed. Therefore, the present study evaluated the anti-epileptogenic effect of orally administered NRICM101 on kainic acid (KA)-induced seizures in rats and investigated its possible mechanisms of action. Sprague‒Dawley rats were administered NRICM101 (300 mg/kg) by oral gavage for 7 consecutive days before receiving an intraperitoneal injection of KA (15 mg/kg). NRICM101 considerably reduced the seizure behavior and electroencephalographic seizures induced by KA in rats. NRICM101 also significantly decreased the neuronal loss and glutamate increase and increased GLAST, GLT-1, GAD67, GDH and GS levels in the cortex and hippocampus of KA-treated rats. In addition, NRICM101 significantly suppressed astrogliosis (as determined by decreased GFAP expression); neuroinflammatory signaling (as determined by reduced HMGB1, TLR-4, IL-1β, IL-1R, IL-6, p-JAK2, p-STAT3, TNF-α, TNFR1 and p-IκB levels, and increased cytosolic p65-NFκB levels); and necroptosis (as determined by decreased p-RIPK3 and p-MLKL levels) in the cortex and hippocampus of KA-treated rats. The effects of NRICM101 were similar to those of carbamazepine, a well-recognized antiseizure drug. Furthermore, no toxic effects of NRICM101 on the liver and kidney were observed in NRICM101-treated rats. The results indicate that NRICM101 has antiepileptogenic and neuroprotective effects through the suppression of the inflammatory cues (HMGB1/TLR4, Il-1β/IL-1R1, IL-6/p-JAK2/p-STAT3, and TNF-α/TNFR1/NF-κB) and necroptosis signaling pathways (TNF-α/TNFR1/RIP3/MLKL) associated with glutamate level regulation in the brain and is innocuous. Our findings highlight the promising role of NRICM101 in the management of epilepsy. NRICM101 antiseizure neuroprotection anti-neuroinflammation glutamate kainic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Epilepsy is a common neurological disorder that can occur at any age. Approximately 70 million people worldwide suffer from epilepsy (Ngugi et al., 2011 ). This disease is characterized by spontaneous and recurrent seizures caused by excessive discharge of cerebral neurons due to an imbalance between the inhibitory γ-aminobutyric acid (GABA) and excitatory glutamate systems in the brain (Lason et al., 2013 ). Current antiseizure drugs act mainly to maintain the stability of neuronal cells by modulating ion channels and neurotransmitters in the brain (Sills and Rogawski, 2020 ). Despite the availability of many antiseizure drugs, approximately one-third of patients do not respond to current antiseizure medications, and approximately 25% of patients discontinue treatment due to significant adverse effects from the medications (Perucca and Gilliam, 2012 ; Loscher et al., 2020). Moreover, current antiseizure drugs are used mainly to suppress already diagnosed seizures but are not used to prevent the development of seizures in patients who are at risk of epilepsy (Billakota et al., 2020 ). Therefore, there is a need to develop new medications that enable successful treatment not only of patients with epilepsy but also of those who are at risk for disease development. Traditional Chinese medicine (TCM) has attracted attention due to its widespread use in traditional and folk medicine for the prevention and treatment of epilepsy (Lin and Hsieh, 2021 ; Wu et al., 2923). Today, many herbs have been confirmed to produce antiepileptic effects and are suggested as alternative medicines for antiepileptic purposes (Zhu et al., 2014 ; He et al., 2021 ; Lu et al., 2023 ). Taiwan Chingguan Yihau (NRICM101) is a well-known traditional Chinese formula prescribed for the treatment of coronavirus disease 2019 (COVID-19) that has antiviral, immunomodulatory, and anti-inflammatory effects (Tsai et al., 2021 ; Cheng et al., 2022; Singh and Yang, 2022; Wei et al., 2023 ). NRICM101 contains Scutellaria Root, Heartleaf Houttuynia, Mulberry Leaf, Saposhnikovia Root, Mongolian Snakegourd Fruit, Indigowoad Root, baked Liquorice Root, Magnolia Bark, Peppermint Herb, and Fineleaf Nepeta. Additionally, various bioactive ingredients, such as baicalin, epigoitrin, liquiritin, quercetin 3-galactoside, quercetin 3-rhamnoside, scutellarin, rutin, wogonoside, and caffeoylquinic acids, have been identified in NRICM101 and might be responsible for its pharmacological properties (Tsai et al., 2021 ). However, there are no data on the antiseizure efficacy of NRICM101. Therefore, the aims of present work were to evaluate the effect of NRICM101 on kainic acid (KA)-induced seizures in rats, which is a reliable and widely used model that mimics the clinical and neuropathological features of human epilepsy (Levesque and Avoli, 2013); to compare the effect of NRICM101 with that of the conventional antiseizure drug carbamazepine (CBZ); and to investigate the possible involvement of neuroinflammation and glutamate in epilepsy (Green et al., 2021 ; Hotz et al., 2022 ; Soltani et al., 2022). Methods Animals Adult Sprague‒Dawley male rats (weighing 170–190 g; BioLASCO, Taipei, Taiwan) were housed in standard cages maintained at 22 ± 2°C and with a 12/12-h light/dark cycle. The rats were fed and provided water ad libitum. The rats were left undisturbed for a minimum of 3 days to allow adaptation to the new environment. The rats were weighed daily in the morning, after which body weights were recorded. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) and following the regulations of the Committee for Animal Use of the Fu Jen Catholic University (project number A11206). Drugs and Chemicals NRICM101 was obtained from Sun Ten Pharmaceutical Co., Ltd. (Taipei, Taiwan). KA, CBZ, and all other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Seizure model establishment and drug treatment Seizures were induced by intraperitoneal (i.p.) administration of 15 mg/kg KA to rats. Rats were randomly subdivided into 4 groups (each containing 10 rats) as follows: (a) Control group: rats received a 0.3 mL normal saline oral gavage for 7 days; (b) KA group: rats received a 0.3 mL normal saline oral gavage for 7 days and a KA injection (15 mg/kg in 0.3 mL saline, i.p.) on the 8th day; (c) NRICM101 + KA group: rats received an NRICM101 oral gavage (300 mg/kg in 0.3 mL saline) for 7 days and a KA injection (i.p.) on the 8th day; and (d) CBZ + KA group: rats received a CBZ oral gavage (100 mg/kg in 0.3 mL saline) for 7 days and a KA injection (i.p.) on the 8th day. The doses and schedules for drug administration were selected based on previous studies and pilot experiments (Friedman et al., 1994 ; Spigolon et al., 2010 ; Chang et al., 2022 ). Before KA injection, the body weights of the rats in the control and NRICM101 groups were measured, and liver, kidney and blood samples were collected from the control and NRICM101 rats after they were sacrificed for hematoxylin-eosin (H&E) staining and serum alanine transaminase (ALT) and aspartate transaminase (AST) measurement, respectively. After each KA injection, each rat was placed in a cage to record its seizure behavior for 3 h. The latency to seizure onset (min) and seizure score were recorded based on Racine’s scale: 0, normal; 1, mouth and facial movements, hyperactivity, grooming, sniffing, scratching, and wet dog shakes; 2, head nodding, staring, and tremors; 3, forelimb clonus; 4, forelimb clonus with rearing; and 5, rearing, jumping, and falling (Racine, 1972 ). During the behavioral assessments, interictal epileptiform spikes were also monitored via electroencephalography (EEG). On the 9th and 11th days, the rats were sacrificed for subsequent experiments (Fig. 1 A). EEG recording EEG recordings were conducted using a three-channel EEG system (Pinnacle Technology Inc., Lawrence, KS, USA) in accordance with the previously stated method (Hsieh et al., 2022 ; Jean et al., 2022 ). After anesthesia with 3% sevoflurane (RWD Life Science, Shenzhen, China), the rats were secured in a stereotaxic frame (RWD, Life. Science, Dover, USA) and surgically implanted with four electrodes. Two electrodes were placed bilaterally over the frontal cortex relative to bregma (anteroposterior (AP), + 3.9; mediolateral M), ± 2.0), and two electrodes were placed over the parietal cortex (AP − 6.4, ML ± 4.0). The surgeries were conducted under sevoflurane anesthesia. Three days after surgery, the rats were placed in transparent cages to connect to the EEG recording amplifier (mode #8213-SE3), and the EEG signals were recorded for 3 h using a data acquisition system (Pinnacle Technology, Inc., Lawrence, KS, USA). The criteria for determining whether a recorded event was a seizure were high-amplitude, rhythmic discharges, including repetitive spikes and spike-and-wave discharges, which had a duration of at least 10 s. The number and duration of EEG ictal spikes were analyzed using PAL-8200 EEG software (Pinnacle Technologies). Histological and immunohistochemical analyses The rats were euthanized by administering Zoletil 50 (40 mg/kg i.p.; Virbac, France) under deep anesthesia. Subsequently, the rats were transcardially perfused with 100 mL of saline (0.9%). Thereafter, the rats were perfused with 50 mL of paraformaldehyde prepared in 0.05 M sodium phosphate (pH 7.4, containing 0.8% saline). The brains were isolated, fixed in 4% paraformaldehyde at 4°C for 24 h and transferred to 30% sucrose solution for 7 days at 4°C. The frozen brains were postfixed in the same perfusion fixative and then washed and embedded in paraffin to prepare paraffin blocks which were sectioned into 30-µm-thick coronal sections using a frozen microtome. The brain sections were mounted onto gelatin-coated slides for Nissl and Fluoro-Jade B (FJB) staining and glial fibrillary acidic protein (GFAP) immunohistochemical staining. In addition, liver and kidney samples were collected and fixed in 4% paraformaldehyde at 4°C for H&E staining. Nissl staining was performed to detect neuronal damage; this is a classic nucleic acid staining method for nervous system tissue. Briefly, slide-mounted brain sections were rehydrated in distilled water and stained in 0.5% cresyl violet solution (Abcam, Cambridge, UK) for 6 min. After rinsing with distilled water, the slides were dehydrated in graded ethanol solutions (70%, 95% and 100%), cleared in xylene and finally covered with dibutyl phthalate in xylene medium (DPX, Sigma‒Aldrich). In addition, FJB staining was used to analyze degenerated neurons in brain sections from rats as described previously (Lin et al., 2016 ; Pai et al., 2023 ). The slide-mounted brain sections were rinsed in 100% ethanol and distilled water. Afterward, the slides were rinsed in 0.06% potassium permanganate for 15 min and then washed 3 times in distilled water for 1 min each. Sections were subsequently stained in 0.001% FJB (Biosensis, Thebarton, South Australia) in 0.1% acetic acid for 20 min in darkness. The slides were subsequently washed 3 times in distilled water for 1 min each and then dried overnight at room temperature. Dried slides were cleared in xylene and mounted with DPX. GFAP immunohistochemical staining was performed to analyze the activation of astrocytes (Chang et al., 2022 ). Briefly, the brain sections were washed in phosphate buffer saline (PBS), blocked with 5% normal horse serum for 30 min, and then incubated overnight with a GFAP monoclonal antibody (1:500; Cell Signaling, Beverly, MA, USA) at 4°C. The sections were washed and then incubated with a biotinylated secondary antibody (1:200; Gentex, Zeeland, MI, USA) for 2 h. The sections were then incubated for 1 h at room temperature in ExtrAvidin peroxidase (1:1000) and 3,3’-diaminobenzidine in H 2 O 2 (DAB kit, Vector, CA) to visualize the reaction as a brown. The sections were then air-dried, dehydrated, cleared with xylene, and coverslipped with DPX. The entorhinal cortex and hippocampal CA1 and CA3 regions of brain sections were photographed and analyzed under 100× magnification using an upright fluorescence microscope (Zeiss Axioskop 40, Goettingen, Germany). The number of positively stained cells per mm 2 was calculated in 4 consecutive coronal sections for each animal by an examiner blinded to the experimental conditions. The data were averaged for each animal using ImageJ image analysis software (NIH Image, National Institutes of Health, Bethesda, MD, USA). H&E staining was performed as previously reported to evaluate the histological morphology of the liver and kidney (Yeh et al., 2022 ). Formalin-fixed liver and kidney tissues were processed into paraffin-embedded tissues and sectioned into 20 µm thick sections using a microtome. The sections were dewaxed with xylene and dehydrated with gradient ethanol (100–75%). After being rinsed with double distilled water, the slices were placed in a solution of hematoxylin for 3 min and then stained with a solution of eosin for 5 min. Finally, neutral gum was used to mount the sections. The stained sections were photographed by an upright fluorescence microscope (Zeiss Axioskop 40, Goettingen, Germany) at 200× magnification and examined blindly by expert pathologists. Brain tissue and blood sampling for biochemical analysis Rats from each group were sacrificed by decapitation, and 1 mL blood was collected and centrifuged at 2000 × g for 10 min, after which the supernatant was collected and stored at − 80°C for use in an enzyme-linked immunosorbent assay (ELISA). Moreover, the brains of the mice were quickly removed and dissected on ice, and the cortex and hippocampus were stored at − 80°C for high-performance liquid chromatography (HPLC), quantitative real-time polymerase chain reaction (qRT–PCR) and western blot analysis. HPLC The glutamate levels in the cortex and hippocampus tissues were measured using HPLC as described previously (Chang et al., 2022 ; Jean et al., 2022 ). The brain tissue (50 mg) was homogenized in HEPES buffer medium and centrifuged at 15,000 × g for 10 min at 4°C. The supernatant (10 µL) was filtered through a 0.22 µm membrane filter and injected into an HPLC instrument (HTEC-500, Eicom, Kyoto, Japan). The glutamate concentration was determined using peak areas with an external standard method and is expressed herein as ng/mg protein. qRT‒PCR The mRNA expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cortical and hippocampal tissues was measured by qRT‒PCR (Lin et al., 2015 ). The frozen cortex and hippocampus were homogenized with a TissueRuptor homogenizer (Qiagen, CA, USA), and total RNA was extracted using a Total RNA Isolation Kit (GeneDireX®, Vegas, NV, USA; #NA021-0100) and subsequently quantified spectrophotometrically. cDNA was synthesized from 1 µg of total RNA using an iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and was subsequently buffered in a volume of 20 µL. Then, 20 µL of cDNA was diluted to a total volume of 200 µL. RT-PCR was carried out with 0.2 µL of cDNA as a template in a total 2 µL volume of reaction mixture containing 10 µL of Power SYBR Green PCR Master Mix (Thermo Scientific, MA, USA) and 2.5 µM of each primer in a Step One Real-time PCR system (Applied Biosystems, USA). Amplification and detection were performed using a thermal cycler. The cycling parameters were as follows: initial denaturation at 95°C for 10 min and 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s. The expression of each gene was normalized to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the 2 −ΔΔct method was used to analyze the relative quantity values (Livak and Schmittgen, 2021 ). The sequences of the primers used are as follows: IL-1β, 5′−GCA ATG GTC GGG ACA TAG TT−3′ (forward) and 5′−AGA CCT GAC TTG GCA GAGGA−3′ (reverse); IL-6, 5′−ACC ACC CAC AAC AGA CCA GT−3′ (forward) and 5′−CGG AAC TCC AGA AGA CCAGA−3′ (reverse); TNF-α, 5′−TGA CCC CCA TTA CTC TGA CC−3′ (forward) and 5′−GCC CAC TAC TTC AGC GTC TC−3′ (reverse); and GAPDH, 5′−GTG GAC CTC ATG GCC TAC AT−3′ (forward) and 5′−GGA TGG AAT TGT GAG GGA GA−3′ (reverse). Western blot The frozen cortex and hippocampus were homogenized in 2% SDS containing 1 mM PMSF, 1 mM Na2VO4, 20 mM NaF, and a mixture of phosphatase-proteinase inhibitors (Sigma Aldrich) using ultrasonic homogenizers. After denaturation at 95°C for 10 min, insoluble debris was removed by centrifugation at 10,000 × g for 10 min at 4°C. The protein concentration in the supernatants was determined using a Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal aliquots of protein (20 µg/lane) from each sample were separated via SDS‒PAGE (10% gel) and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific, MA, USA). The membranes were treated with 5% bovine serum albumin in Tris-buffered saline (TBS) supplemented with 0.1% Tween - 20 (TBST) for 40 min at room temperature and then probed with specific primary antibodies overnight at 4°C. The primary antibodies were used as follows: GLAST (1:10000; Abcam, Cambridge, UK), GLT-1 (1:100000; Abcam, Cambridge, UK), GS (1:50000; Cell Signaling, Beverly, MA, USA), GDH (1: 30000; Invitrogen, Waltham, MA, USA), GAD67 (1: 2000; Invitrogen, Waltham, MA, USA), GFAP (1:10000, Cell Signaling, Beverly, MA, USA), IL-1β (1: 2000; Abcam, Cambridge, UK), IL-6 (1: 800; Abcam, Cambridge, UK), IL-1R1 (1: 10000; Abcam, Cambridge, UK), p-JAK2 (1:800, Cell Signaling, Beverly, MA, USA), p-STAT3 (1:1000, Cell Signaling, Beverly, MA, USA), TNF-α (1:4000; Abcam, Cambridge, UK), TNFR1 (1:800, Abcam, Cambridge, UK), p-RIPK3 (1:2000; Abcam, Cambridge, UK), pMLKL (1:1000, Abcam, Cambridge, UK), p-IκBα (1:1000, Cell Signaling, Beverly, MA, USA), p65-NFκB (1:5000, Cell Signaling, Beverly, MA, USA), HMGB1 (1:10000; Abcam, Cambridge, UK), TLR4 (1:5000; Abcam, Cambridge, UK) and β-actin (1:10000, Cell Signaling, Beverly, MA, USA). After washing with TBST 5 times for 6 min, the membranes were incubated for 2 h at 25°C with horseradish peroxidase-conjugated secondary antibodies (1:2000, Gentex, Zeeland, MI, USA). Expressed proteins of interest were visualized by enhanced chemiluminescence solution (Amersham Biosciences Corp., Amersham, Buckinghamshire, UK). The films were scanned using a scanner and quantified with ImageJ software (NIH Image, National Institutes of Health, Bethesda, MD, USA), after which the protein/β-actin ratio was calculated (Chang et al., 2022 ; Yeh et al., 2022 ). ELISA The serum levels of HMGB1, AST and ALT in rats were detected using ELISA kits (HMGB1, Novus, #NBP3-06661; ALT, Abcam, #ab234579; AST, Abcam, #ab263883) with a microplate ELISA reader. The samples were diluted and analyzed in triplicate following the manufacturer's instructions. The relative concentrations of HMGB1, AST and ALT were calculated via standard curves. HPLC fingerprinting of NRICM101 For HPLC analysis, 0.5 g of NRICM101 was extracted using 20 mL of 70% methanol through ultrasonic oscillation at 25°C for 20 min. The NRICM101 sample was then filtered through a 0.45 µm syringe filter. The Waters HPLC system (Milford, Massachusetts, USA) was composed of a Waters 600 pump system, Waters 2996 photodiode array detector, Waters 717 plus autosampler, and Sugai U-620 column oven (Wakayama City, Japan). A Cosmosil 5C18-MS-II reversed phase column (5 µm, 4.6 mm × 250 mm; Nacalai Tesque, Japan) equipped with a Lichrospher RP-18 end-capped guard column (5 µm, 4.0 mm × 10 mm, Merck; Germany) was used as the stationary phase. The gradient elution mixture was composed of eluents A, B, and C (A: H 2 O/KH 2 PO 4 /10% H 3 PO 4 = 1000 mL/2.72 g/1 mL; B: acetonitrile; C: H 2 O) according to the following profile: 0–30 min, 90%-75% A and 10%-25% B; 30–40 min, 75%-65% A and 25%-35% B; 40–55 min, 65%-0% A, 35%-75% B and 0%-25% C; 55–60 min, 75%-10% B and 25%-90% C; 60–65 min, 0%-90% A, 10%-10% B and 90%-0% C. The gradient elution was used for 3D fingerprint analysis and quantification of liquiritin (25.94 min, 280 nm), rosmarinic acid (29.44 min, 320 nm), baicalin (34.61 min, 280 nm), oroxylin A-7- O -glucuronide (40.39 min. 280 nm), wogonin-7- O -glucuronide (42.53 min, 280 nm), glycyrrhizic acid (51.37 min, 250 nm), and magnolol (61.79 min, 290 nm). The flow rate was 1 mL/min, and the column temperature was maintained at 35°C. Statistical analysis The data are presented as the means ± standard errors of the mean (S.E.M.) and were analyzed using Prism 8.1 software (GraphPad Software, San Diego, California, USA). The data normality was evaluated using the Kolmogorov‒Smirnov test, and the results were considered parametric. Multiple comparisons among groups were performed by one-way analysis of variance (ANOVA) followed by Tukey’s test. The results were considered significant if p > 0.05. Results Effect of NRICM101 on convulsive behavior and EEG signals in KA-treated rats Seven days after the administration of NRICM101 (100, 200, and 300 mg/kg, oral gavage) or CBZ (100 mg/kg, oral gavage) to the rats, they were injected with 15 mg/kg KA (i.p.) and observed for the occurrence of seizures. As shown in Fig. 1 B, NRICM101 at 300 mg/kg had a significant effect on seizure onset time and seizure score ( p 0.05). The percentage of animals that were seizure free was highest in the 300 mg/kg NRICM101 + KA group (77%, p 0.05) and the 100 mg/kg/day treatment group (40%, p > 0.05). Moreover, the effects of 300 mg/kg NRICM101 on seizure onset time and seizure score were similar to those of 100 mg/kg CBZ ( p > 0.05). Additionally, in comparison to the KA group, the 100 mg/kg NRICM101 + 50 mg/kg CBZ + KA group exhibited a significant reduction in the KA-induced seizure score and a significant increase in the latency to seizures ( p 0.05). Since the most effective dose of NRICM101 that prevented seizures induced by KA was 300 mg/kg, this dose was chosen for subsequent experiments. To confirm the behavioral data, we performed an EEG analysis to assess interictal epileptiform spikes in the brains of the rats. As shown in Fig. 1 C and D, compared with those in the control group, the number and duration of EEG ictal spikes in the KA group was greater ( p < 0.001). Additionally, in comparison to those in the KA group, the 300 mg/kg NRICM101 + KA, 100 mg/kg CBZ + KA or 100 mg/kg NRICM101 + 50 mg/kg CBZ + KA groups exhibited significant reductions in KA-induced ictal spikes ( p 0.05). Effects of NRICM101 on the neuronal damage in KA-treated rats We next determined whether NRICM101 prevents the neuronal death that has been implicated in the process of epileptogenesis in the KA model. As shown in Fig. 2 A and B, observation of the Nissl-stained sections clearly showed that the neurons in the entorhinal cortex and hippocampus (CA1 and CA3) of each rat in the control group were clustered close together, and the neuronal shape and number were normal. However, compared with the control group, KA treatment disrupted the regular arrangement of neurons and reduced their number in these regions ( p < 0.001). Conversely, compared with KA treatment, NRICM101 or CBZ pretreatment significantly attenuated these alterations in the structures of the entorhinal cortex and hippocampus, respectively ( p < 0.01). A similar protective effect of NRICM101 was observed through labeling with the neuronal degeneration marker FJB. As shown in Fig. 2 C and D, FJB labeling was never observed in the entorhinal cortex and hippocampus of control rats that did not receive KA. However, a marked increase in the number of FJB-positive neurons in these regions was observed in the KA group compared with that in the control group ( p < 0.001). On the other hand, rats pretreated with NRICM101 or CBZ did not exhibit any FJB-positive neurons. The number of FJB-positive neurons was significantly less in the NRICM101 or CBZ + KA groups than in the KA group (p 0.05). Effects of NIRCM101 on the glutamate, glutamate transporter (GLT-1 and GLAST), and glutamate metabolism-associated enzyme (GS, GDH, and GAD67) levels in the cortex and hippocampus Excess glutamate in the brain is known to contribute to the pathogenesis of epilepsy (During and Spencer, 1993 ). To investigate whether NRICM101 might modulate the glutamatergic system, contributing to its antiseizure and neuroprotective effects, we evaluated the effect of NRICM101 on glutamate levels in the cortex and hippocampus of rats via HPLC. As shown in Fig. 3 A, the concentration of glutamate was significantly greater in the cortex and hippocampus in the KA group than in the control group ( p < 0.001). However, NRICM101 and CBZ pretreatment of KA-treated rats resulted in a significantly lower glutamate concentration than that in the KA group ( p < 0.001), and the glutamate concentration was not different from that in the control group ( p = 0.91). No significant difference was observed in the glutamate concentration between the NRICM101- and CBZ-treated groups ( p > 0.05). Since glutamate elevation in the brain is related to impaired glutamate uptake and metabolism (Eid et al., 2012 ; Green et al., 2021 ; Hotz et al., 2022 ), we analyzed the protein levels of the glutamate transporters GLAST and GLT-1, as well as the glutamate metabolism-associated enzymes glutamine synthetase (GS), glutamate dehydrogenase (GDH), and glutamic acid decarboxylase 67 (GAD67), in the cortex and hippocampus (Fig. 3 B). As shown in Fig. 3 C and D, the protein expression of GLT-1, GLAST, GS, GDH, and GAD67 in the cortex and hippocampus was significantly lower in the KA group than in the control group ( p < 0.0001). In contrast, compared with those in the KA group, the expression of these proteins in the NRICM101 or CBZ groups was significantly increased. No significant differences were observed in the protein levels of GLT-1, GLAST, GS, GDH, or GAD67 of the NRICM101- and CBZ-treated groups ( p > 0.05). Effects of NRICM101 on astrocyte activation in the cortex and hippocampus The inflammatory response, especially astrogliosis, has been shown to contribute to excess glutamate, and enhanced excitability of the neural network and seizure generation (Devinsky et al., 2013 ; Bedner et al., 2015 ). Astrocyte activation was observed in the entorhinal cortex and hippocampus of KA-treated rats, as assessed using an anti-GFAP antibody. Figure 4 A and B show a marked increase in the number of GFAP-labeled cells in the entorhinal cortex and hippocampus (CA1 and CA3) regions in the KA group in contrast to those in the control group ( p < 0.001). However, compared with that in the KA group, the numbers of labeled cells in the NRICM101- and CBZ-treated groups were significantly lower ( p < 0.001). Furthermore, compared with those in the control group, the protein expression of GFAP in the cortex and hippocampus in the KA group was significantly greater (p < 0.001; Fig. 4 C and D). Alternatively, NRICM101 or CBZ significantly decreased the expression of GFAP in the cortex and hippocampus in comparison with that in the KA group ( p 0.05). Effects of NRICM101 on the mRNA levels of IL-1β, IL-6, and TNF-α in the cortex and hippocampus Since elevated levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) have been observed in patients with epilepsy and in several animal models (Vezzani et al., 2008 , 2013 ; Soltani et al., 2022), the expression of IL-1β, IL-6, and TNF-α at the mRNA level was analyzed by qRT-PCR. As shown in Fig. 5 , the mRNA levels of IL-1β, IL-6, and TNF-α in the cortex and hippocampus were significantly greater in the KA group than in the control group ( p < 0.0001). In contrast, the NRICM101 and CBZ groups exhibited significantly lower expression of these genes than did the KA group ( p 0.05). Effects of NRICM101 on the protein levels of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 in the cortex and hippocampus The neuroimmune inflammatory response mediated by the IL-1β/IL-1R1 and IL-6/p-JAK2/p-STAT3 pathways has been shown to regulate the expression of proinflammatory cytokines (Fig. 6 A) (Van Vliet et al., 2018 ; Zhang et al., 2022 ). The protein levels of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 were analyzed via Western blotting. As shown in Fig. 6 B and C, KA treatment substantially increased the protein expression of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 in the cortex and hippocampus in contrast to that in the control group (p < 0.001). In contrast, NRICM101 or CBZ significantly reduced the protein expression of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 compared to that in the KA group ( p 0.05). Effects of NRICM101 on the protein levels of TNF-α, TNFR1, p-IκBα, p65-NFκB, p-RIPK3, and p-MLKL in the cortex and hippocampus TNF-α is known to bind to TNFR1 to activate NF-κB, resulting in the upregulation of inflammatory cytokine expression and the phosphorylation of RIPK3 and MLKL, leading to necroptosis (Fig. 7 A); these phenomena have been linked to the pathogenesis of epilepsy (Abd EI-Aal et al., 2022; Soltani et al., 2022). NF-κB is localized to the cytoplasm and exists primarily as an inactive p50/p65 heterodimer bound to the IκB inhibitory subunit. IκB phosphorylation causes disassociation from p50/p65 heterodimers, leading to NF-κB activation and subsequent translocation into the nucleus, where it induces immune- and inflammation-related gene expression (Liu et al., 2017 ). As shown in Fig. 7 B and C, the protein expression levels of TNF-α, TNFR1, cytosolic p-IκBα, p-RIPK3, and p-MLKL in the cortex and hippocampus in the KA group were significantly increased compared to those in the control group ( p < 0.05), whereas the protein expression levels of TNF-α, TNFR1, p-IκBα, p-RIPK3, and p-MLKL in the cortex and hippocampus were significantly lower in the NRICM101 + KA group than in the KA group ( p < 0.05). In contrast, the cytosolic p65-NFκB protein level was lower in the cortex and hippocampus of KA-treated rats than in those of control rats, indicating that cytosolic p65-NFκB is translocated to the nucleus. Pretreatment with NRICM101 or CBZ significantly increased the protein expression of cytosolic p65-NFκB compared to that in the KA group ( p < 0.001). Effects of NRICM on the protein levels of HMGB1 and TLR4 in the cortex and hippocampus HMGB1 is a ubiquitous protein that has been reported to be increased in the serum and brain tissue of patients with epilepsy and in the brain tissue in of epilepsy model animal; moreover, HMGB1 promotes the synthesis of inflammatory cytokines by activating TLR4, thereby influencing inflammatory responses (Fig. 8 A) (Kaya et al., 2021 ; Chen et al., 2023 ; Yue et al., 2023 ). We observed substantial neuronal damage in the cortex and hippocampus after KA-induced seizures that was concomitant with an increase in the number of activated astrocytes and the expression of inflammatory cytokines. Therefore, we examined whether the neuroprotective effect of NRICM10 is related to HMGB1/TLR4 signaling. As shown in Fig. 8 B and C, compared with those in the control group, the protein levels of HMGB1 and TLR4 in the cortex and hippocampus were significantly greater in the KA-injected groups ( p < 0.001). Notably, the protein levels of HMGB1 and TLR4 were significantly lower in the NRICM101 + KA group than in the KA group ( p < 0.05). In addition, the serum level of HMGB1 was significantly greater in the KA group than in the control group ( p < 0.001). However, rats in the NRICM101 and KA groups had significantly lower levels of HMGB1 than did those in the KA group ( p < 0.001; Fig. 8 D). Phytochemical characterization of NRICM101 Regarding the phytochemical characterization of NRICM101, some standard active compounds were selected on the basis of a review of the literature for identification from the methanol extract. The structures of these compounds were confirmed by 3D fingerprint analysis using the selected standards (approximately 98.99%) (Tsai et al., 2021 ). As given, Fig. 9 shows the chromatogram of NRICM101. The contents of baicalin, oroxylin A-7-O-glucuronide, wogonin-7-O-glucuronide, glycyrrhizic acid, liquiritin, magnolol, and rosmarinic acid in the freeze-dried extract of NRICM101 were 18.34, 1.33, 2.67, 2.42, 1.70, 1.07, and 0.91 mg/g, respectively. Effects of NRICM101 on rat body weight, liver and kidney morphology, and serum ALT and AST levels Figure 10 A shows that the body weight of NRICM101 (300 mg/kg, oral gavage)-treated rats was not significantly different from that of control rats ( p = 0.84). In addition, compared with those of the control rats, the liver and kidney histopathology of the NRICM101 rats showed no obvious signs of pathological changes (Fig. 10 B). Additionally, no significant difference in the serum ALT or AST levels was observed between the NRICM101 rats and the control rats ( p > 0.05; Fig. 10 C). These results imply that NRICM101 administration may be safe. Discussion 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. Conclusion An excessive inflammatory response leads to neuronal damage, resulting in permanent impairment of the structure and function of neural networks, an important underlying cause of the recurrent spontaneous seizures observed in epilepsy. The results obtained from this study revealed that NRICM101 protects model rats against KA-induced seizures and neuronal death and is safe and nontoxic. The suppression of neuroinflammatory trajectories with the consequent maintenance of normal glutamate homeostasis by NRICM101 might explain its antiepileptogenic and neuroprotective effects. Our findings highlight the promising role of NRICM101 in the management of epilepsy. Declarations Author contributions Design of the study: Chi-Feng Hung, Su-Jane Wang. Statistical analysis: Chi-Feng Hung, Wei-Che Chiu, Jia-Cih Chen, Wu-Chang Chuang, Su-Jane Wang. Experiment and data collection: Jia-Cih Chen, Wu-Chang Chuang. Writing the manuscript: Su-Jane Wang. All authors read and approved the final manuscript. Availability of data Data generated during the current study are available from the corresponding author upon reasonable request. Funding This study was supported by the Taiwan Ministry of Science and Technology (112-2320-B-030-010-MY3) and Cathay General Hospital (110-CGH-FJU-02). Acknowledgements We appreciate Sun Ten for providing NRICM101 and their information. Ethics approval The experimental animals were conducted according to the Animal Ethics Committee (IACUC) guidelines issued by the Fu Jen Catholic University (Taipei, Taiwan) (Approval ID: A11206). Consent to Participate Not applicable. Consent for Publication All authors have approved the publication of this paper. Competing interests The authors declare that they have no competing interest. References Abd El-Aal SA, El-Abhar HS, Abulfadl YS (2022) Morin offsets PTZ-induced neuronal degeneration and cognitive decrements in rats: The modulation of TNF. Eur J Pharmacol 931:175213-α/TNFR-1/RIPK1,3/MLKL/PGAM5/Drp-1, IL-6/JAK2/STAT3/GFAP and Keap-1/Nrf-2/HO-1 trajectories Bedner P, Dupper A, Hüttmann K, Müller J, Herde MK, Dublin P, Deshpande T, Schramm J, Häussler U, Haas CA (2015) Astrocyte uncoupling as a cause of human temporal lobe epilepsy. Brain 138:1208–1222 Billakota S, Devinsky O, Kim KW (2020) Why we urgently need improved epilepsy therapies for adult patients. Neuropharmacology 170:107855 Chang A, Chang Y, Wang SJ (2022) Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation. Food Funct 13:10401–10414 Chen Y, Chen X, Liang Y (2023) Meta-analysis of HMGB1 levels in the cerebrospinal fluid and serum of patients with epilepsy. Neurol Sci 44:2329–2337 Cheng YD, Lu CC, Hsu YM, Tsai FJ, Bau DT, Tsai SC, Cheng CC, Lin JJ, Huang YY, Juan YN, Chiu YJ, Kuo SC, Yang JS, Wu LT (2011) In Silico and In Vitro studies of taiwan chingguan yihau (NRICM101) on TNF-α/IL-1β-induced human lung cells. Biomedicine 12:56–71 Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65:1–105 Devinsky O, Vezzani A, Najjar S, De Lanerolle NC, Rogawski MA (2013) Glia and epilepsy: excitability and inflammation. Trends Neurosci 36:174–184 Dhuriya YK, Sharma D (2018) Necroptosis: a regulated infammatory mode of cell death. J Neuroinfammation 15:199 During MJ, Spencer DD (1993) Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain. Lancet 341:1607–1610 Eid T, Behar K, Bumanglag AV, Lee TS (2012) Role of glutamine synthetase inhibition in epilepsy. Neurochem Res 37:2339–2350 Friedman LK, Pellegrini-Giampietro DE, Sperber EF, Bennett MV, Moshe SL, Zukin RS (1994) Kainate-induced status epilepticus alters glutamate and GABAA receptor gene expression in adult rat hippocampus: an in situ hybridization study. J Neurosci 14:1697–1707 Green JL, Dos Santos WF, Fontana ACK (2021) Role of glutamate excitotoxicity and glutamate transporter EAAT2 in epilepsy: Opportunities for novel therapeutics development. Biochem Pharmacol 193:114786 Han CL, Ge M, Liu YP, Zhao XM, Wang KL, Chen N, Meng WJ, Hu W, Zhang JG, Li L, Meng FG (2018) LncRNA H19 contributes to hippocampal glial cell activation via JAK/STAT signaling in a rat model of temporal lobe epilepsy. J Neuroinflammation 15:103 He LY, Hu MB, Li RL, Zhao R, Fan LH, He L, Lu F, Ye X, Huang YL, Wu CJ (2021) Natural medicines for the treatment of epilepsy: Bioactive components, pharmacology and mechanism. Front Pharmacol 12:604–640 Hoda U, Agarwal NB, Vohora D, Parvez S, Raisuddin S (2017) Resveratrol suppressed seizures by attenuating IL-1β, IL1-Ra, IL-6, and TNF-α in the hippocampus and cortex of kindled mice. Nutr Neurosci 20:497–504 Hotz AL, Jamali A, Rieser NN, Niklaus S, Aydin E, Myren-Svelstad S, Lalla L, Jurisch-Yaksi N, Yaksi E, Neuhauss SCF (2022) Loss of glutamate transporter eaat2a leads to aberrant neuronal excitability, recurrent epileptic seizures, and basal hypoactivity. Glia 70:196–214 Hsieh TY, Chang Y, Wang SJ (2022) Piperine provides neuroprotection against kainic acid-induced neurotoxicity via maintaining NGF signalling pathway. Molecules 27:2638 impact, potential mechanisms, and new innovative treatment options. Pharmacol Rev 72:606–638 Jean WH, Huang CT, Hsu JH, Chiu KM, Lee MY, Shieh JS, Lin TY, Wang SJ (2022) Anticonvulsive and neuroprotective effects of eupafolin in rats are associated with the inhibition of glutamate overexcitation and upregulation of the Wnt/β-Catenin signaling pathway. ACS Chem Neurosci 13:1594–1603 Kang N, Xu J, Xu Q, Nedergaard M, Kang J (2005) Astrocytic glutamate release-induced transient depolarization and epileptiform discharges in hippocampal CA1 pyramidal neurons. J Neurophysiol 94:4121–4130 Kaya MA, Erin N, Bozkurt O, Erkek N, Duman O, Haspolat S (2021) Changes of HMGB-1 and sTLR4 levels in cerebrospinal fluid of patients with febrile seizures. Epilepsy Res 169:106516 Lason W, Chlebicka M, Rejdak K (2013) Research advances in basic mechanisms of seizures and antiepileptic drug action. Pharmacol Rep 65:787–801 Lévesque M, Avoli M (2013) The kainic acid model of temporal lobe epilepsy. Neurosci Biobehav Rev 37:2887–2899 Li G, Zhang S, Cheng Y, Lu Y, Jia Z, Yang X, Zhang S, Guo W, Pei L (2022) Baicalin suppresses neuron autophagy and apoptosis by regulating astrocyte polarization in pentylenetetrazol-induced epileptic rats and PC12 cells. Brain Res 1774:147723 Li Z, Li B, Zhu X, Yin P, Liu J, Huang S, Sun R (2013) Neuroprotective effects of anti-high-mobility group box 1 antibody in juvenile rat hippocampus after kainic acid-induced status epilepticus. NeuroReport 24:785–790 Lin CH, Chen YJ, Lin MW, Chang HJ, Yang XR, Lin CS (2023) ACE2 and a Traditional Chinese Medicine Formula NRICM101 Could Alleviate the Inflammation and Pathogenic Process of Acute Lung Injury. Medicina 59:1554 Lin CH, Hsieh CL (2021) Chinese herbal medicine for treating epilepsy. Front Neurosci 15:682821 Lin TY, Lu CW, Wang SJ (2016) Luteolin protects the hippocampus against neuron impairments induced by kainic acid in rats. Neurotoxicology 55:48–57 Lin TY, Lu CW, Wang SJ, Huang SK (2015) Protective effect of hispidulin on kainic acid-induced seizures and neurotoxicity in rats. Eur J Pharmacol 755:6–15 Liu T, Zhang L, Joo D, Sun SC (2017) NF-κB signaling in inflammation. Signal Transduct Target Ther 2:17023 Livak KJ, Schmittgen TD (2021) Analysis of relative gene expression data using real-time quantitative PCR and the 2 – ∆∆CT method. Methods 25:402–408 Löscher W, Potschka H, Sisodiya SM, Vezzani A (2020) Drug resistance in epilepsy. Clinical Lu H, Luo M, Chen R, Luo Y, Xi A, Wang K, Xu Z (2023) Efficacy and safety of traditional Chinese medicine for the treatment of epilepsy: A updated meta-analysis of randomized controlled trials. Epilepsy Res 189:107075 Luo L, Jin Y, Kim ID, Lee JK (2013) Glycyrrhizin attenuates kainic acid-induced neuronal cell death in the mouse hippocampus. Exp Neurobiol 22:107–115 Ngugi AK, Kariuki SM, Bottomley C, Kleinschmidt I, Sander JW, Newton CR (2011) Incidence of epilepsy: a systematic review and meta-analysis. Neurology 77:1005–1012 Pai MS, Wang KC, Yeh KC, Wang SJ (2023) Stabilization of mitochondrial function by chlorogenic acid protects against kainic acid-induced seizures and neuronal cell death in rats. Eur J Pharmacol 961:176197 Paudel YN, Semple BD, Jones NC, Othman I, Shaikh MF (2019) High mobility group box 1 (HMGB1) as a novel frontier in epileptogenesis: from pathogenesis to therapeutic approaches. J Neurochem 151:542–557 Perez EL, Lauritzen F, Wang Y, Lee TS, Kang D, Zaveri HP, Chaudhry FA, Ottersen OP, Bergersen LH, Eid T (2012) Evidence for astrocytes as a potential source of the glutamate excess in temporal lobe epilepsy. Neurobiol Dis 47:331–337 Perucca P, Gilliam FG (2012) Adverse effects of antiepileptic drugs. Lancet Neurol 11:792–802 Racine RJ (1972) Modification of seizure activity by electric stimulation: II. Motor seizure. EEG Clin Neurophysiol 32:281–294 Rose CR, Felix L, Zeug A, Dietrich D, Reiner A, Henneberger C (2017) Astroglial glutamate signaling and uptake in the hippocampus. Front Mol Neurosci 10:451 \ Sills GJ, Rogawski MA (2020) Mechanisms of action of currently used antiseizure drugs. Neuropharmacology 168:107966 Singh S, Yang YF (2011) Pharmacological Mechanism of NRICM101 for COVID-19 Treatments by Combined Network Pharmacology and Pharmacodynamics. Int J Mol Sci 23:15385 Soltani Khaboushan A, Yazdanpanah N, Rezaei N (2022) Neuroinflammation and proinflammatory cytokines in epileptogenesis. Mol Neurobiol 59:1724–1743 Soukupova M, Binaschi A, Falcicchia C, Palma E, Roncon P, Zucchini S, Simonato M (2015) Increased extracellular levels of glutamate in the hippocampus of chronically epileptic rats. Neuroscience 301:246–253 Spigolon G, Veronesi C, Bonny C, Vercelli A (2010) c-Jun n-terminal kinase signaling pathway in excitotoxic cell death following kainic acid-induced status epilepticus. Eur J Neurosci 31:1261–1272 Swamy M, Wan Roslina WY, Sirajudeen KNS, Zulkarnain M, Chandran G (2011) Decreased glutamine synthetase, increased citrulline - nitric oxide cycle activities and oxidative stress in different regions of brain in epilepsy rat model. J Physiol Biochem 67:105–113 Terrone G, Balosso S, Pauletti A, Ravizza T, Vezzani A (2020) Inflammation and reactive oxygen species as disease modifiers in epilepsy. Neuropharmacology 167:107742 Tilleux S, Hermans E (2007) (2007) Neuroinflammation and regulation of glial glutamate uptake in neurological disorders. J Neurosci Res 85: 2059–2070 Tsai KC, Huang YC, Liaw CC, Tsai CI, Chiou CT, Lin CJ, Wei WC, Lin SJ, Tseng YH, Yeh KM, Lin YL, Jan JT, Liang JJ, Liao CC, Chiou WF, Kuo YH, Lee SM, Lee MY, Su YC (2021) A traditional Chinese medicine formula NRICM101 to target COVID-19 through multiple pathways: a bedside-to-bench study. Biomed Pharmacother 133:111037 van der Hel WS, Hessel EV, Bos IW, Mulder SD, Verlinde SA, van Eijsden P, Graan PNEl (2014) Persistent reduction of hippocampal glutamine synthetase expression after status epilepticus in immature rats. Eur J Neurosci 40:3711–3719 van Vliet EA, Aronica E, Vezzani A, Ravizza T (2018) Review: Neuroinflammatory pathways as treatment targets and biomarker candidates in epilepsy: emerging evidence from preclinical and clinical studies. Neuropathol Appl Neurobiol 44:91–111 Vezzani A, Aronica E, Mazarati A, Pittman QJ (2013) Epilepsy and brain inflammation. Exp Neurol 244:11–21 Vezzani A, Balosso S, Ravizza T (2008) The role of cytokines in the pathophysiology of epilepsy. Brain Behav Immun 22:797–803 Vezzani A, Balosso S, Ravizza T (2019) Neuroinflammatory pathways as treatment targets and biomarkers in epilepsy. Nat Rev Neurol 15:459–472 Vezzani A, Granata T (2005) Brain inflammation in epilepsy: experimental and clinical evidence. Epilepsia 46:1724–1743 Vezzani A, Viviani B (2015) Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability. Neuropharmacology 96:70–82 Viviani B, Bartesaghi S, Gardoni F, Vezzani A, Behrens MM, Bartfai T, Binaglia M, Corsini E, Di Luca M, Galli CL, Marinovich M (2003) Interleukin-1beta enhances NMDA receptor-mediated intracellular calcium increase through activation of the Src family of kinases. J Neurosci 23:8692–8700 Wei WC, Tsai KC, Liaw CC, Chiou CT, Tseng YH, Liao GY, Lin YC, Chiou WF, Liou KT, Yu IS, Shen YC, Su YC (2023) NRICM101 ameliorates SARS-CoV-2-S1-induced pulmonary injury in K18-hACE2 mice model. Front Pharmacol 14:1125414 Wu J, Cao M, Peng Y, Dong B, Jiang Y, Hu C, Zhu P, Xing W, Yu L, Xu R, Chen Z (2023) Research progress on the treatment of epilepsy with traditional Chinese medicine. Phytomedicine 120:155022 Xu Z, Xue T, Zhang Z, Wang X, Xu P, Zhang J, Lei X, Li Y, Xie Y, Wang L, Fang M, Chen Y (2011) Role of signal transducer and activator of transcription-3 in up-regulation of GFAP after epilepsy. Neurochem Res 36:2208–2215 Yang J, Jia Z, Xiao Z, Zhao J, Lu Y, Chu L, Shao H, Pei L, Zhang S, Chen Y (2021) Baicalin Rescues Cognitive Dysfunction, Mitigates Neurodegeneration, and Exerts Anti-Epileptic Effects Through Activating TLR4/MYD88/Caspase-3 Pathway in Rats. Drug Des Devel Ther 15:3163–3180 Yeh KC, Hung CF, Lee HL, Hsieh TY, Wang SJ (2022) Soybean meal extract preserves memory ability by increasing presynaptic function and modulating gut microbiota in rats. Mol Neurobiol 59:1649–1664 Yue Z, Tang J, Peng S, Cai X, Rong X, Yang L (2023) Serum concentration of high-mobility group box 1, Toll-like receptor 4 as biomarker in epileptic patients. Epilepsy Res 192:107138 Zhang S, Chen F, Zhai F, Liang S (2022) Role of HMGB1/TLR4 and IL-1β/IL-1R1 signaling pathways in epilepsy. Front Neurol 13:904225 Zhao J, Wang Y, Xu C, Liu K, Wang Y, Chen L, Wu X, Gao F, Guo Y, Zhu J, Wang S, Nishibori M, Chen Z (2017) Therapeutic potential of an anti-high mobility group box-1 monoclonal antibody in epilepsy. Brain Behav Immun 64:308–319 Zhu HL, Wan JB, Wang YT, Li BC, Xiang C, He J, Li P (2014) Medicinal compounds with antiepileptic/anticonvulsant activities. Epilepsia 55:3–16 Additional Declarations No competing interests reported. Supplementary Files graphicabstract.pptx Effect of NRICM101 on rat seizure behavioral and EEG. Schematic representation of the experimental design. Seizure onset time and seizure score during 3 h after KA injection. Typical EEG recorded from the cortex in rats. Number and duration of seizure spikes. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control or KA group < 0.001, ** significant vs KA group < 0.01, # significant vs KA group < 0.05. n = 3−37 rats per group. WBdata.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3932956","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272755272,"identity":"a8f71e62-2f95-4459-b4af-ce5ac400bee9","order_by":0,"name":"Chi-Feng Hung","email":"","orcid":"","institution":"Fu Jen Catholic University","correspondingAuthor":false,"prefix":"","firstName":"Chi-Feng","middleName":"","lastName":"Hung","suffix":""},{"id":272755273,"identity":"466f1914-4c39-4a5c-8345-55512ffd01a0","order_by":1,"name":"Wei-Che Chiu","email":"","orcid":"","institution":"Fu Jen Catholic University","correspondingAuthor":false,"prefix":"","firstName":"Wei-Che","middleName":"","lastName":"Chiu","suffix":""},{"id":272755274,"identity":"a6b679eb-ee24-4f2b-a21e-cf81970ffe1d","order_by":2,"name":"Jia-Cih Chen","email":"","orcid":"","institution":"Fu Jen Catholic University","correspondingAuthor":false,"prefix":"","firstName":"Jia-Cih","middleName":"","lastName":"Chen","suffix":""},{"id":272755276,"identity":"d84855a3-33ce-4a93-b052-fd1e72d79515","order_by":3,"name":"Wu-Chang Chuang","email":"","orcid":"","institution":"Sun Ten Pharmaceutical Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Wu-Chang","middleName":"","lastName":"Chuang","suffix":""},{"id":272755277,"identity":"284c869b-0e73-436e-8683-3c6dbed55195","order_by":4,"name":"Su-Jane Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYHACgwMJBjVyDAyMjQcYbNjAIoS0GD74UHHMGKil4QBDGkxLAl4txoYzzjAnNgBZQC0MhLXotjdvk+ZtY0tf234YaEsCX2IDe/M2CcYfh3FqMTtzrAyoRSZ325lEkBa2xAaeY2USDAl4tNzIMQPZkrvtAFAL4w+gFokcM6CW27i13H8D0sKcbnb+IdQW+TcEtNzgAXs/wewGzGESPAS0nEkrBAWy4bYbQFsSEtiM23jSii0S0v7j1nL88AZQVMqbnU9/+OBDwjHZfvbDG298sEnDqQUVJDAcY2CDMIgHNSSoHQWjYBSMgpECAEvjXVDmtHdgAAAAAElFTkSuQmCC","orcid":"","institution":"Fu Jen Catholic University","correspondingAuthor":true,"prefix":"","firstName":"Su-Jane","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-02-06 05:59:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3932956/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3932956/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51195644,"identity":"7f8ee5f3-77f8-4091-b24d-55d3974b4b95","added_by":"auto","created_at":"2024-02-15 18:38:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149691,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on\u003cstrong\u003e \u003c/strong\u003erat seizure behavioral and EEG. \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the experimental design. \u003cstrong\u003e(B)\u003c/strong\u003e Seizure onset time and seizure score during 3 h after KA injection.\u003cstrong\u003e (C)\u003c/strong\u003e Typical EEG recorded from the cortex in rats. \u003cstrong\u003e(D)\u003c/strong\u003e Number and duration of seizure spikes. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control or KA group \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ** significant vs KA group \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, # significant vs KA group \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. n = 3-37 rats per group.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/a613d5473c78b485fa5f6a42.png"},{"id":51195645,"identity":"e428da9c-917e-4951-9439-0b98b2bf9545","added_by":"auto","created_at":"2024-02-15 18:38:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":425172,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on the morphology and neuronal degeneration of the entorhinal cortex and hippocampus (CA1 and CA3) from different groups. \u003cstrong\u003e(A, C)\u003c/strong\u003e Representative images showing cresyl violet and FJB staining in the entorhinal cortex and hippocampus of rats 72 h after KA injection. \u003cstrong\u003e(B, D)\u003c/strong\u003e Represents the mean of labled cells in the in the entorhinal cortex and hippocampus (CA1and CA3) in different groups. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, # significant vs KA group \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. n = 5 rats per group.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/91862e49ff0be1b9eced5318.png"},{"id":51195648,"identity":"16be8483-0e14-4394-9036-40bcf2d57897","added_by":"auto","created_at":"2024-02-15 18:38:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177607,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on glutamate concentration and protein expression levels of GLAST, GLT-1, GS, GDH, and GAD67 in the cortex and hippocampus from different groups. \u003cstrong\u003e(A) \u003c/strong\u003eGlutamate concentration in cortical and hippocampal brain tissue.\u003cstrong\u003e (B)\u003c/strong\u003e Schematic representation of glutamate uptake and metabolism. \u003cstrong\u003e(C, D)\u003c/strong\u003e Immunoblot results of GLAST, GLT-1, GS, GDH, and GAD67 in the cortex and hippocampus from different groups and the respective bar graphs. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, # significant vs KA group \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. n = 5 rats per group.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/ddb372df6257a60c8083741f.png"},{"id":51195647,"identity":"a5336cc7-f153-4f2f-b004-ee2760eb20f1","added_by":"auto","created_at":"2024-02-15 18:38:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":335743,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on GFAP (marker of astrocytes and astrocytosis) expression in the cortex and hippocampus. \u003cstrong\u003e(A)\u003c/strong\u003e Representative image showing GFAP staining in the entorhinal cortex and hippocampus of rats. Scale bars = 200 μm. \u003cstrong\u003e(B)\u003c/strong\u003e Represents the mean of GFAP-positive cells in the in the entorhinal cortex and hippocampus (CA1and CA3) in different groups. \u003cstrong\u003e(C, D)\u003c/strong\u003e Immunoblot results of GFAP in the cortex and hippocampus from different groups and the respective bar graphs. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, # significant vs KA group \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. n = 5 rats per group.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/92b84b46bd0f54003b385084.png"},{"id":51195655,"identity":"97c957ff-8ca3-4e2f-b6d4-56f7d3bede67","added_by":"auto","created_at":"2024-02-15 18:38:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64384,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on\u003cstrong\u003e \u003c/strong\u003ethe relative expression of IL-1β, IL-6, and TNF-α (at mRNA) in the cortex and hippocampus. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, # significant vs KA group \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. n = 5 rats per group.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/bb319d5fce47c85ef08e2db5.png"},{"id":51195652,"identity":"8285d625-e4a5-41b5-91b6-ca018141b1e1","added_by":"auto","created_at":"2024-02-15 18:38:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":163388,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 proteins in the cortex and hippocampus. \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of IL-1b/IL-1R1 and IL-6/JAK2-STAT3 signaling pathways. \u003cstrong\u003e(B, C) \u003c/strong\u003eImmunoblot results of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 proteins in the cortex and hippocampus from different groups and the respective bar graphs. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, # significant vs KA group \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. n = 5 rats per group.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/3bec852a049d64a75761b013.png"},{"id":51195653,"identity":"cfba9fe8-eda3-440c-90a3-19376cba16e1","added_by":"auto","created_at":"2024-02-15 18:38:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":174222,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on\u003cstrong\u003e \u003c/strong\u003eTNF-α, TNFR1, cytosolic p-IκBa, cytosolic p65-NFkB, p-RIPK3, and p-MLKL proteins in the cortex and hippocampus.\u003cstrong\u003e (A)\u003c/strong\u003e Schematic representation of TNF-dependent inflammatory pathways.\u003cstrong\u003e (B, C)\u003c/strong\u003e Immunoblot results of TNF-α, TNFR1, p-IκBa, p65-NFkB, p-RIPK3, and p-MLKL proteins in the cortex and hippocampus from different groups and the respective bar graphs. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, # significant vs KA group \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. n = 5 rats per group.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/7473570f4f3e9ce14b3dfff0.png"},{"id":51196038,"identity":"afe0f42c-1d7d-4252-8e1a-1af9bcc4003a","added_by":"auto","created_at":"2024-02-15 18:46:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":133161,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on HMGB1 and TLR4 proteins in the cortex and hippocampus. \u003cstrong\u003e(A)\u003c/strong\u003eSchematic representation of HMGB1/TLR4 signaling pathway. \u003cstrong\u003e(B, C)\u003c/strong\u003eImmunoblot results of HMGB1 and TLR4 proteins in the cortex and hippocampus from different groups and the respective bar graphs. \u003cstrong\u003e(D)\u003c/strong\u003e HMGB1 in serum in each group detected with ELISA. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control p \u0026lt; 0.001, # significant vs KA group p \u0026lt; 0.05. n = 5 rats per group.\u003c/p\u003e","description":"","filename":"Slide8.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/a531bba35fe3a1a1c5415d21.png"},{"id":51195656,"identity":"1e660af1-3c31-4d04-a8e0-d206a456504a","added_by":"auto","created_at":"2024-02-15 18:38:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":133072,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC fingerprinting of NRICM101 methanolic extract for 7 standard compounds.\u003c/p\u003e","description":"","filename":"Slide9.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/b2a3cb52c58f34b06d0e2037.png"},{"id":51196039,"identity":"940b45d9-43b3-4d59-9f17-568bdb08b3c6","added_by":"auto","created_at":"2024-02-15 18:46:48","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":267293,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on body weight, liver, kidney, and serum ALT and AST of rats. \u003cstrong\u003e(A)\u003c/strong\u003eGraphical representation of animal body weight changes relative to the initial body weight. \u003cstrong\u003e(B)\u003c/strong\u003e Photomicrographs of rat’s liver and kidneys following H\u0026amp;E staining (200×). Scale bars = 50 μm. \u003cstrong\u003e(C)\u003c/strong\u003e ALT and AST in serum detected with ELISA. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. n = 8-13 rats per group.\u003c/p\u003e","description":"","filename":"Slide10.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/d2fa16eec254cf126c55c5e8.png"},{"id":51195650,"identity":"e744a734-c4e2-4169-a6ec-65296a597719","added_by":"auto","created_at":"2024-02-15 18:38:47","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":26156,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation for the antiseizure and neuroprotective effects of NRICM101 on KA-induced rats.\u003c/p\u003e","description":"","filename":"Slide11.png","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/7189fd536b33493281d83d29.png"},{"id":52304793,"identity":"e5bf38eb-dc51-4e6f-8ef1-0d7540498b67","added_by":"auto","created_at":"2024-03-08 19:12:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2561517,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/89bd2cfb-5892-4976-adab-43a1fddc0588.pdf"},{"id":51195649,"identity":"9a32e819-8b2c-4bf3-a058-4acd25f8f7b7","added_by":"auto","created_at":"2024-02-15 18:38:47","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":244812,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NRICM101 on rat seizure behavioral and EEG. Schematic representation of the experimental design. Seizure onset time and seizure score during 3 h after KA injection. Typical EEG recorded from the cortex in rats. Number and duration of seizure spikes. Data were expressed as mean ± SEM. One-way ANOVA with Tukey posthoc test. *** significant vs control or KA group \u0026lt; 0.001, ** significant vs KA group \u0026lt; 0.01, # significant vs KA group \u0026lt; 0.05. n = 3−37 rats per group.\u003c/p\u003e","description":"","filename":"graphicabstract.pptx","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/029f8eb8d187e99265948721.pptx"},{"id":51196037,"identity":"967468e2-b73c-4aba-8b2f-2521a2f19679","added_by":"auto","created_at":"2024-02-15 18:46:47","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1657199,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"WBdata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3932956/v1/e2ca4336975def45d1e09bd0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Taiwan Chingguan Yihau (NRICM101) prevents kainic acid-induced seizures in rats by modulating neuroinflammation and the glutamatergic system","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy is a common neurological disorder that can occur at any age. Approximately 70\u0026nbsp;million people worldwide suffer from epilepsy (Ngugi et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This disease is characterized by spontaneous and recurrent seizures caused by excessive discharge of cerebral neurons due to an imbalance between the inhibitory γ-aminobutyric acid (GABA) and excitatory glutamate systems in the brain (Lason et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Current antiseizure drugs act mainly to maintain the stability of neuronal cells by modulating ion channels and neurotransmitters in the brain (Sills and Rogawski, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Despite the availability of many antiseizure drugs, approximately one-third of patients do not respond to current antiseizure medications, and approximately 25% of patients discontinue treatment due to significant adverse effects from the medications (Perucca and Gilliam, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Loscher et al., 2020). Moreover, current antiseizure drugs are used mainly to suppress already diagnosed seizures but are not used to prevent the development of seizures in patients who are at risk of epilepsy (Billakota et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, there is a need to develop new medications that enable successful treatment not only of patients with epilepsy but also of those who are at risk for disease development.\u003c/p\u003e \u003cp\u003eTraditional Chinese medicine (TCM) has attracted attention due to its widespread use in traditional and folk medicine for the prevention and treatment of epilepsy (Lin and Hsieh, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al., 2923). Today, many herbs have been confirmed to produce antiepileptic effects and are suggested as alternative medicines for antiepileptic purposes (Zhu et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; He et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Taiwan Chingguan Yihau (NRICM101) is a well-known traditional Chinese formula prescribed for the treatment of coronavirus disease 2019 (COVID-19) that has antiviral, immunomodulatory, and anti-inflammatory effects (Tsai et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cheng et al., 2022; Singh and Yang, 2022; Wei et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). NRICM101 contains Scutellaria Root, Heartleaf Houttuynia, Mulberry Leaf, Saposhnikovia Root, Mongolian Snakegourd Fruit, Indigowoad Root, baked Liquorice Root, Magnolia Bark, Peppermint Herb, and Fineleaf Nepeta. Additionally, various bioactive ingredients, such as baicalin, epigoitrin, liquiritin, quercetin 3-galactoside, quercetin 3-rhamnoside, scutellarin, rutin, wogonoside, and caffeoylquinic acids, have been identified in NRICM101 and might be responsible for its pharmacological properties (Tsai et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, there are no data on the antiseizure efficacy of NRICM101. Therefore, the aims of present work were to evaluate the effect of NRICM101 on kainic acid (KA)-induced seizures in rats, which is a reliable and widely used model that mimics the clinical and neuropathological features of human epilepsy (Levesque and Avoli, 2013); to compare the effect of NRICM101 with that of the conventional antiseizure drug carbamazepine (CBZ); and to investigate the possible involvement of neuroinflammation and glutamate in epilepsy (Green et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hotz et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Soltani et al., 2022).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult Sprague‒Dawley male rats (weighing 170\u0026ndash;190 g; BioLASCO, Taipei, Taiwan) were housed in standard cages maintained at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and with a 12/12-h light/dark cycle. The rats were fed and provided water ad libitum. The rats were left undisturbed for a minimum of 3 days to allow adaptation to the new environment. The rats were weighed daily in the morning, after which body weights were recorded. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80\u0026ndash;23) and following the regulations of the Committee for Animal Use of the Fu Jen Catholic University (project number A11206).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDrugs and Chemicals\u003c/h2\u003e \u003cp\u003eNRICM101 was obtained from Sun Ten Pharmaceutical Co., Ltd. (Taipei, Taiwan). KA, CBZ, and all other reagents were purchased from Sigma\u0026ndash;Aldrich (St. Louis, MO, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSeizure model establishment and drug treatment\u003c/h2\u003e \u003cp\u003eSeizures were induced by intraperitoneal (i.p.) administration of 15 mg/kg KA to rats. Rats were randomly subdivided into 4 groups (each containing 10 rats) as follows: (a) Control group: rats received a 0.3 mL normal saline oral gavage for 7 days; (b) KA group: rats received a 0.3 mL normal saline oral gavage for 7 days and a KA injection (15 mg/kg in 0.3 mL saline, i.p.) on the 8th day; (c) NRICM101\u0026thinsp;+\u0026thinsp;KA group: rats received an NRICM101 oral gavage (300 mg/kg in 0.3 mL saline) for 7 days and a KA injection (i.p.) on the 8th day; and (d) CBZ\u0026thinsp;+\u0026thinsp;KA group: rats received a CBZ oral gavage (100 mg/kg in 0.3 mL saline) for 7 days and a KA injection (i.p.) on the 8th day. The doses and schedules for drug administration were selected based on previous studies and pilot experiments (Friedman et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Spigolon et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Before KA injection, the body weights of the rats in the control and NRICM101 groups were measured, and liver, kidney and blood samples were collected from the control and NRICM101 rats after they were sacrificed for hematoxylin-eosin (H\u0026amp;E) staining and serum alanine transaminase (ALT) and aspartate transaminase (AST) measurement, respectively. After each KA injection, each rat was placed in a cage to record its seizure behavior for 3 h. The latency to seizure onset (min) and seizure score were recorded based on Racine\u0026rsquo;s scale: 0, normal; 1, mouth and facial movements, hyperactivity, grooming, sniffing, scratching, and wet dog shakes; 2, head nodding, staring, and tremors; 3, forelimb clonus; 4, forelimb clonus with rearing; and 5, rearing, jumping, and falling (Racine, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). During the behavioral assessments, interictal epileptiform spikes were also monitored via electroencephalography (EEG). On the 9th and 11th days, the rats were sacrificed for subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEEG recording\u003c/h2\u003e \u003cp\u003eEEG recordings were conducted using a three-channel EEG system (Pinnacle Technology Inc., Lawrence, KS, USA) in accordance with the previously stated method (Hsieh et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jean et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). After anesthesia with 3% sevoflurane (RWD Life Science, Shenzhen, China), the rats were secured in a stereotaxic frame (RWD, Life. Science, Dover, USA) and surgically implanted with four electrodes. Two electrodes were placed bilaterally over the frontal cortex relative to bregma (anteroposterior (AP), + 3.9; mediolateral M), \u0026plusmn; 2.0), and two electrodes were placed over the parietal cortex (AP \u0026minus;\u0026thinsp;6.4, ML\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0). The surgeries were conducted under sevoflurane anesthesia. Three days after surgery, the rats were placed in transparent cages to connect to the EEG recording amplifier (mode #8213-SE3), and the EEG signals were recorded for 3 h using a data acquisition system (Pinnacle Technology, Inc., Lawrence, KS, USA). The criteria for determining whether a recorded event was a seizure were high-amplitude, rhythmic discharges, including repetitive spikes and spike-and-wave discharges, which had a duration of at least 10 s. The number and duration of EEG ictal spikes were analyzed using PAL-8200 EEG software (Pinnacle Technologies).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHistological and immunohistochemical analyses\u003c/h2\u003e \u003cp\u003eThe rats were euthanized by administering Zoletil 50 (40 mg/kg i.p.; Virbac, France) under deep anesthesia. Subsequently, the rats were transcardially perfused with 100 mL of saline (0.9%). Thereafter, the rats were perfused with 50 mL of paraformaldehyde prepared in 0.05 M sodium phosphate (pH 7.4, containing 0.8% saline). The brains were isolated, fixed in 4% paraformaldehyde at 4\u0026deg;C for 24 h and transferred to 30% sucrose solution for 7 days at 4\u0026deg;C. The frozen brains were postfixed in the same perfusion fixative and then washed and embedded in paraffin to prepare paraffin blocks which were sectioned into 30-\u0026micro;m-thick coronal sections using a frozen microtome. The brain sections were mounted onto gelatin-coated slides for Nissl and Fluoro-Jade B (FJB) staining and glial fibrillary acidic protein (GFAP) immunohistochemical staining. In addition, liver and kidney samples were collected and fixed in 4% paraformaldehyde at 4\u0026deg;C for H\u0026amp;E staining.\u003c/p\u003e \u003cp\u003eNissl staining was performed to detect neuronal damage; this is a classic nucleic acid staining method for nervous system tissue. Briefly, slide-mounted brain sections were rehydrated in distilled water and stained in 0.5% cresyl violet solution (Abcam, Cambridge, UK) for 6 min. After rinsing with distilled water, the slides were dehydrated in graded ethanol solutions (70%, 95% and 100%), cleared in xylene and finally covered with dibutyl phthalate in xylene medium (DPX, Sigma‒Aldrich). In addition, FJB staining was used to analyze degenerated neurons in brain sections from rats as described previously (Lin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pai et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The slide-mounted brain sections were rinsed in 100% ethanol and distilled water. Afterward, the slides were rinsed in 0.06% potassium permanganate for 15 min and then washed 3 times in distilled water for 1 min each. Sections were subsequently stained in 0.001% FJB (Biosensis, Thebarton, South Australia) in 0.1% acetic acid for 20 min in darkness. The slides were subsequently washed 3 times in distilled water for 1 min each and then dried overnight at room temperature. Dried slides were cleared in xylene and mounted with DPX. GFAP immunohistochemical staining was performed to analyze the activation of astrocytes (Chang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, the brain sections were washed in phosphate buffer saline (PBS), blocked with 5% normal horse serum for 30 min, and then incubated overnight with a GFAP monoclonal antibody (1:500; Cell Signaling, Beverly, MA, USA) at 4\u0026deg;C. The sections were washed and then incubated with a biotinylated secondary antibody (1:200; Gentex, Zeeland, MI, USA) for 2 h. The sections were then incubated for 1 h at room temperature in ExtrAvidin peroxidase (1:1000) and 3,3\u0026rsquo;-diaminobenzidine in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (DAB kit, Vector, CA) to visualize the reaction as a brown. The sections were then air-dried, dehydrated, cleared with xylene, and coverslipped with DPX. The entorhinal cortex and hippocampal CA1 and CA3 regions of brain sections were photographed and analyzed under 100\u0026times; magnification using an upright fluorescence microscope (Zeiss Axioskop 40, Goettingen, Germany). The number of positively stained cells per mm\u003csup\u003e2\u003c/sup\u003e was calculated in 4 consecutive coronal sections for each animal by an examiner blinded to the experimental conditions. The data were averaged for each animal using ImageJ image analysis software (NIH Image, National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003cp\u003eH\u0026amp;E staining was performed as previously reported to evaluate the histological morphology of the liver and kidney (Yeh et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Formalin-fixed liver and kidney tissues were processed into paraffin-embedded tissues and sectioned into 20 \u0026micro;m thick sections using a microtome. The sections were dewaxed with xylene and dehydrated with gradient ethanol (100\u0026ndash;75%). After being rinsed with double distilled water, the slices were placed in a solution of hematoxylin for 3 min and then stained with a solution of eosin for 5 min. Finally, neutral gum was used to mount the sections. The stained sections were photographed by an upright fluorescence microscope (Zeiss Axioskop 40, Goettingen, Germany) at 200\u0026times; magnification and examined blindly by expert pathologists.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBrain tissue and blood sampling for biochemical analysis\u003c/h2\u003e \u003cp\u003eRats from each group were sacrificed by decapitation, and 1 mL blood was collected and centrifuged at 2000 \u0026times; g for 10 min, after which the supernatant was collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for use in an enzyme-linked immunosorbent assay (ELISA). Moreover, the brains of the mice were quickly removed and dissected on ice, and the cortex and hippocampus were stored at \u0026minus;\u0026thinsp;80\u0026deg;C for high-performance liquid chromatography (HPLC), quantitative real-time polymerase chain reaction (qRT\u0026ndash;PCR) and western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHPLC\u003c/h2\u003e \u003cp\u003eThe glutamate levels in the cortex and hippocampus tissues were measured using HPLC as described previously (Chang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jean et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The brain tissue (50 mg) was homogenized in HEPES buffer medium and centrifuged at 15,000 \u0026times; g for 10 min at 4\u0026deg;C. The supernatant (10 \u0026micro;L) was filtered through a 0.22 \u0026micro;m membrane filter and injected into an HPLC instrument (HTEC-500, Eicom, Kyoto, Japan). The glutamate concentration was determined using peak areas with an external standard method and is expressed herein as ng/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eqRT‒PCR\u003c/h2\u003e \u003cp\u003eThe mRNA expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in cortical and hippocampal tissues was measured by qRT‒PCR (Lin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The frozen cortex and hippocampus were homogenized with a TissueRuptor homogenizer (Qiagen, CA, USA), and total RNA was extracted using a Total RNA Isolation Kit (GeneDireX\u0026reg;, Vegas, NV, USA; #NA021-0100) and subsequently quantified spectrophotometrically. cDNA was synthesized from 1 \u0026micro;g of total RNA using an iScript\u0026trade; cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) and was subsequently buffered in a volume of 20 \u0026micro;L. Then, 20 \u0026micro;L of cDNA was diluted to a total volume of 200 \u0026micro;L. RT-PCR was carried out with 0.2 \u0026micro;L of cDNA as a template in a total 2 \u0026micro;L volume of reaction mixture containing 10 \u0026micro;L of Power SYBR Green PCR Master Mix (Thermo Scientific, MA, USA) and 2.5 \u0026micro;M of each primer in a Step One Real-time PCR system (Applied Biosystems, USA). Amplification and detection were performed using a thermal cycler. The cycling parameters were as follows: initial denaturation at 95\u0026deg;C for 10 min and 40 cycles of denaturation at 95\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 45 s. The expression of each gene was normalized to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the 2\u003csup\u003e\u0026minus;ΔΔct\u003c/sup\u003e method was used to analyze the relative quantity values (Livak and Schmittgen, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The sequences of the primers used are as follows: IL-1β, 5\u0026prime;\u0026minus;GCA ATG GTC GGG ACA TAG TT\u0026minus;3\u0026prime; (forward) and 5\u0026prime;\u0026minus;AGA CCT GAC TTG GCA GAGGA\u0026minus;3\u0026prime; (reverse); IL-6, 5\u0026prime;\u0026minus;ACC ACC CAC AAC AGA CCA GT\u0026minus;3\u0026prime; (forward) and 5\u0026prime;\u0026minus;CGG AAC TCC AGA AGA CCAGA\u0026minus;3\u0026prime; (reverse); TNF-α, 5\u0026prime;\u0026minus;TGA CCC CCA TTA CTC TGA CC\u0026minus;3\u0026prime; (forward) and 5\u0026prime;\u0026minus;GCC CAC TAC TTC AGC GTC TC\u0026minus;3\u0026prime; (reverse); and GAPDH, 5\u0026prime;\u0026minus;GTG GAC CTC ATG GCC TAC AT\u0026minus;3\u0026prime; (forward) and 5\u0026prime;\u0026minus;GGA TGG AAT TGT GAG GGA GA\u0026minus;3\u0026prime; (reverse).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eThe frozen cortex and hippocampus were homogenized in 2% SDS containing 1 mM PMSF, 1 mM Na2VO4, 20 mM NaF, and a mixture of phosphatase-proteinase inhibitors (Sigma Aldrich) using ultrasonic homogenizers. After denaturation at 95\u0026deg;C for 10 min, insoluble debris was removed by centrifugation at 10,000 \u0026times; g for 10 min at 4\u0026deg;C. The protein concentration in the supernatants was determined using a Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal aliquots of protein (20 \u0026micro;g/lane) from each sample were separated via SDS‒PAGE (10% gel) and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Scientific, MA, USA). The membranes were treated with 5% bovine serum albumin in Tris-buffered saline (TBS) supplemented with 0.1% Tween\u003cb\u003e-\u003c/b\u003e20 (TBST) for 40 min at room temperature and then probed with specific primary antibodies overnight at 4\u0026deg;C. The primary antibodies were used as follows: GLAST (1:10000; Abcam, Cambridge, UK), GLT-1 (1:100000; Abcam, Cambridge, UK), GS (1:50000; Cell Signaling, Beverly, MA, USA), GDH (1: 30000; Invitrogen, Waltham, MA, USA), GAD67 (1: 2000; Invitrogen, Waltham, MA, USA), GFAP (1:10000, Cell Signaling, Beverly, MA, USA), IL-1β (1: 2000; Abcam, Cambridge, UK), IL-6 (1: 800; Abcam, Cambridge, UK), IL-1R1 (1: 10000; Abcam, Cambridge, UK), p-JAK2 (1:800, Cell Signaling, Beverly, MA, USA), p-STAT3 (1:1000, Cell Signaling, Beverly, MA, USA), TNF-α (1:4000; Abcam, Cambridge, UK), TNFR1 (1:800, Abcam, Cambridge, UK), p-RIPK3 (1:2000; Abcam, Cambridge, UK), pMLKL (1:1000, Abcam, Cambridge, UK), p-IκBα (1:1000, Cell Signaling, Beverly, MA, USA), p65-NFκB (1:5000, Cell Signaling, Beverly, MA, USA), HMGB1 (1:10000; Abcam, Cambridge, UK), TLR4 (1:5000; Abcam, Cambridge, UK) and β-actin (1:10000, Cell Signaling, Beverly, MA, USA). After washing with TBST 5 times for 6 min, the membranes were incubated for 2 h at 25\u0026deg;C with horseradish peroxidase-conjugated secondary antibodies (1:2000, Gentex, Zeeland, MI, USA). Expressed proteins of interest were visualized by enhanced chemiluminescence solution (Amersham Biosciences Corp., Amersham, Buckinghamshire, UK). The films were scanned using a scanner and quantified with ImageJ software (NIH Image, National Institutes of Health, Bethesda, MD, USA), after which the protein/β-actin ratio was calculated (Chang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yeh et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003eThe serum levels of HMGB1, AST and ALT in rats were detected using ELISA kits (HMGB1, Novus, #NBP3-06661; ALT, Abcam, #ab234579; AST, Abcam, #ab263883) with a microplate ELISA reader. The samples were diluted and analyzed in triplicate following the manufacturer's instructions. The relative concentrations of HMGB1, AST and ALT were calculated via standard curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHPLC fingerprinting of NRICM101\u003c/h2\u003e \u003cp\u003eFor HPLC analysis, 0.5 g of NRICM101 was extracted using 20 mL of 70% methanol through ultrasonic oscillation at 25\u0026deg;C for 20 min. The NRICM101 sample was then filtered through a 0.45 \u0026micro;m syringe filter. The Waters HPLC system (Milford, Massachusetts, USA) was composed of a Waters 600 pump system, Waters 2996 photodiode array detector, Waters 717 plus autosampler, and Sugai U-620 column oven (Wakayama City, Japan). A Cosmosil 5C18-MS-II reversed phase column (5 \u0026micro;m, 4.6 mm \u0026times; 250 mm; Nacalai Tesque, Japan) equipped with a Lichrospher RP-18 end-capped guard column (5 \u0026micro;m, 4.0 mm \u0026times; 10 mm, Merck; Germany) was used as the stationary phase. The gradient elution mixture was composed of eluents A, B, and C (A: H\u003csub\u003e2\u003c/sub\u003eO/KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e/10% H\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1000 mL/2.72 g/1 mL; B: acetonitrile; C: H\u003csub\u003e2\u003c/sub\u003eO) according to the following profile: 0\u0026ndash;30 min, 90%-75% A and 10%-25% B; 30\u0026ndash;40 min, 75%-65% A and 25%-35% B; 40\u0026ndash;55 min, 65%-0% A, 35%-75% B and 0%-25% C; 55\u0026ndash;60 min, 75%-10% B and 25%-90% C; 60\u0026ndash;65 min, 0%-90% A, 10%-10% B and 90%-0% C. The gradient elution was used for 3D fingerprint analysis and quantification of liquiritin (25.94 min, 280 nm), rosmarinic acid (29.44 min, 320 nm), baicalin (34.61 min, 280 nm), oroxylin A-7-\u003cem\u003eO\u003c/em\u003e-glucuronide (40.39 min. 280 nm), wogonin-7-\u003cem\u003eO\u003c/em\u003e-glucuronide (42.53 min, 280 nm), glycyrrhizic acid (51.37 min, 250 nm), and magnolol (61.79 min, 290 nm). The flow rate was 1 mL/min, and the column temperature was maintained at 35\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are presented as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the mean (S.E.M.) and were analyzed using Prism 8.1 software (GraphPad Software, San Diego, California, USA). The data normality was evaluated using the Kolmogorov‒Smirnov test, and the results were considered parametric. Multiple comparisons among groups were performed by one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s test. The results were considered significant if p\u0026thinsp;\u0026gt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of NRICM101 on convulsive behavior and EEG signals in KA-treated rats\u003c/h2\u003e \u003cp\u003eSeven days after the administration of NRICM101 (100, 200, and 300 mg/kg, oral gavage) or CBZ (100 mg/kg, oral gavage) to the rats, they were injected with 15 mg/kg KA (i.p.) and observed for the occurrence of seizures. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, NRICM101 at 300 mg/kg had a significant effect on seizure onset time and seizure score (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but other dosage (100 and 200 mg/kg) had no significant effect on seizure onset time or seizure score compared with those in the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The percentage of animals that were seizure free was highest in the 300 mg/kg NRICM101\u0026thinsp;+\u0026thinsp;KA group (77%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), followed by the 200 mg/kg/day treatment group (33%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and the 100 mg/kg/day treatment group (40%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Moreover, the effects of 300 mg/kg NRICM101 on seizure onset time and seizure score were similar to those of 100 mg/kg CBZ (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Additionally, in comparison to the KA group, the 100 mg/kg NRICM101\u0026thinsp;+\u0026thinsp;50 mg/kg CBZ\u0026thinsp;+\u0026thinsp;KA group exhibited a significant reduction in the KA-induced seizure score and a significant increase in the latency to seizures (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant difference was observed between the NRICM101 group and the CBZ group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Since the most effective dose of NRICM101 that prevented seizures induced by KA was 300 mg/kg, this dose was chosen for subsequent experiments.\u003c/p\u003e \u003cp\u003eTo confirm the behavioral data, we performed an EEG analysis to assess interictal epileptiform spikes in the brains of the rats. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and D, compared with those in the control group, the number and duration of EEG ictal spikes in the KA group was greater (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, in comparison to those in the KA group, the 300 mg/kg NRICM101\u0026thinsp;+\u0026thinsp;KA, 100 mg/kg CBZ\u0026thinsp;+\u0026thinsp;KA or 100 mg/kg NRICM101\u0026thinsp;+\u0026thinsp;50 mg/kg CBZ\u0026thinsp;+\u0026thinsp;KA groups exhibited significant reductions in KA-induced ictal spikes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No significant difference was observed between the NRICM101 group and the CBZ group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffects of NRICM101 on the neuronal damage in KA-treated rats\u003c/h2\u003e \u003cp\u003eWe next determined whether NRICM101 prevents the neuronal death that has been implicated in the process of epileptogenesis in the KA model. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B, observation of the Nissl-stained sections clearly showed that the neurons in the entorhinal cortex and hippocampus (CA1 and CA3) of each rat in the control group were clustered close together, and the neuronal shape and number were normal. However, compared with the control group, KA treatment disrupted the regular arrangement of neurons and reduced their number in these regions (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversely, compared with KA treatment, NRICM101 or CBZ pretreatment significantly attenuated these alterations in the structures of the entorhinal cortex and hippocampus, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A similar protective effect of NRICM101 was observed through labeling with the neuronal degeneration marker FJB. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D, FJB labeling was never observed in the entorhinal cortex and hippocampus of control rats that did not receive KA. However, a marked increase in the number of FJB-positive neurons in these regions was observed in the KA group compared with that in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On the other hand, rats pretreated with NRICM101 or CBZ did not exhibit any FJB-positive neurons. The number of FJB-positive neurons was significantly less in the NRICM101 or CBZ\u0026thinsp;+\u0026thinsp;KA groups than in the KA group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No significant difference in neuronal damage was observed between the NRICM101 group and the CBZ group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of NIRCM101 on the glutamate, glutamate transporter (GLT-1 and GLAST), and glutamate metabolism-associated enzyme (GS, GDH, and GAD67) levels in the cortex and hippocampus\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExcess glutamate in the brain is known to contribute to the pathogenesis of epilepsy (During and Spencer, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). To investigate whether NRICM101 might modulate the glutamatergic system, contributing to its antiseizure and neuroprotective effects, we evaluated the effect of NRICM101 on glutamate levels in the cortex and hippocampus of rats via HPLC. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the concentration of glutamate was significantly greater in the cortex and hippocampus in the KA group than in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, NRICM101 and CBZ pretreatment of KA-treated rats resulted in a significantly lower glutamate concentration than that in the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and the glutamate concentration was not different from that in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.91). No significant difference was observed in the glutamate concentration between the NRICM101- and CBZ-treated groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Since glutamate elevation in the brain is related to impaired glutamate uptake and metabolism (Eid et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Green et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hotz et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), we analyzed the protein levels of the glutamate transporters GLAST and GLT-1, as well as the glutamate metabolism-associated enzymes glutamine synthetase (GS), glutamate dehydrogenase (GDH), and glutamic acid decarboxylase 67 (GAD67), in the cortex and hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D, the protein expression of GLT-1, GLAST, GS, GDH, and GAD67 in the cortex and hippocampus was significantly lower in the KA group than in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, compared with those in the KA group, the expression of these proteins in the NRICM101 or CBZ groups was significantly increased. No significant differences were observed in the protein levels of GLT-1, GLAST, GS, GDH, or GAD67 of the NRICM101- and CBZ-treated groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects of NRICM101 on astrocyte activation in the cortex and hippocampus\u003c/h2\u003e \u003cp\u003eThe inflammatory response, especially astrogliosis, has been shown to contribute to excess glutamate, and enhanced excitability of the neural network and seizure generation (Devinsky et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bedner et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Astrocyte activation was observed in the entorhinal cortex and hippocampus of KA-treated rats, as assessed using an anti-GFAP antibody. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B show a marked increase in the number of GFAP-labeled cells in the entorhinal cortex and hippocampus (CA1 and CA3) regions in the KA group in contrast to those in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, compared with that in the KA group, the numbers of labeled cells in the NRICM101- and CBZ-treated groups were significantly lower (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, compared with those in the control group, the protein expression of GFAP in the cortex and hippocampus in the KA group was significantly greater (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Alternatively, NRICM101 or CBZ significantly decreased the expression of GFAP in the cortex and hippocampus in comparison with that in the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant difference in GFAP-labeled cells or GFAP expression was observed between the NRICM101 group and the CBZ group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of NRICM101 on the mRNA levels of IL-1β, IL-6, and TNF-α in the cortex and hippocampus\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince elevated levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) have been observed in patients with epilepsy and in several animal models (Vezzani et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Soltani et al., 2022), the expression of IL-1β, IL-6, and TNF-α at the mRNA level was analyzed by qRT-PCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the mRNA levels of IL-1β, IL-6, and TNF-α in the cortex and hippocampus were significantly greater in the KA group than in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In contrast, the NRICM101 and CBZ groups exhibited significantly lower expression of these genes than did the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No statistically significant difference was observed between the NRICM101 group and the CBZ group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of NRICM101 on the protein levels of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 in the cortex and hippocampus\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe neuroimmune inflammatory response mediated by the IL-1β/IL-1R1 and IL-6/p-JAK2/p-STAT3 pathways has been shown to regulate the expression of proinflammatory cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) (Van Vliet et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The protein levels of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 were analyzed via Western blotting. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C, KA treatment substantially increased the protein expression of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 in the cortex and hippocampus in contrast to that in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, NRICM101 or CBZ significantly reduced the protein expression of IL-1β, IL-1R1, IL-6, p-JAK2, and p-STAT3 compared to that in the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). No statistically significant difference was observed between the NRICM101 group and the CBZ group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of NRICM101 on the protein levels of TNF-α, TNFR1, p-IκBα, p65-NFκB, p-RIPK3, and p-MLKL in the cortex and hippocampus\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTNF-α is known to bind to TNFR1 to activate NF-κB, resulting in the upregulation of inflammatory cytokine expression and the phosphorylation of RIPK3 and MLKL, leading to necroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA); these phenomena have been linked to the pathogenesis of epilepsy (Abd EI-Aal et al., 2022; Soltani et al., 2022). NF-κB is localized to the cytoplasm and exists primarily as an inactive p50/p65 heterodimer bound to the IκB inhibitory subunit. IκB phosphorylation causes disassociation from p50/p65 heterodimers, leading to NF-κB activation and subsequent translocation into the nucleus, where it induces immune- and inflammation-related gene expression (Liu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and C, the protein expression levels of TNF-α, TNFR1, cytosolic p-IκBα, p-RIPK3, and p-MLKL in the cortex and hippocampus in the KA group were significantly increased compared to those in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the protein expression levels of TNF-α, TNFR1, p-IκBα, p-RIPK3, and p-MLKL in the cortex and hippocampus were significantly lower in the NRICM101\u0026thinsp;+\u0026thinsp;KA group than in the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the cytosolic p65-NFκB protein level was lower in the cortex and hippocampus of KA-treated rats than in those of control rats, indicating that cytosolic p65-NFκB is translocated to the nucleus. Pretreatment with NRICM101 or CBZ significantly increased the protein expression of cytosolic p65-NFκB compared to that in the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of NRICM on the protein levels of HMGB1 and TLR4 in the cortex and hippocampus\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHMGB1 is a ubiquitous protein that has been reported to be increased in the serum and brain tissue of patients with epilepsy and in the brain tissue in of epilepsy model animal; moreover, HMGB1 promotes the synthesis of inflammatory cytokines by activating TLR4, thereby influencing inflammatory responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) (Kaya et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Yue et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). We observed substantial neuronal damage in the cortex and hippocampus after KA-induced seizures that was concomitant with an increase in the number of activated astrocytes and the expression of inflammatory cytokines. Therefore, we examined whether the neuroprotective effect of NRICM10 is related to HMGB1/TLR4 signaling. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and C, compared with those in the control group, the protein levels of HMGB1 and TLR4 in the cortex and hippocampus were significantly greater in the KA-injected groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, the protein levels of HMGB1 and TLR4 were significantly lower in the NRICM101\u0026thinsp;+\u0026thinsp;KA group than in the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the serum level of HMGB1 was significantly greater in the KA group than in the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, rats in the NRICM101 and KA groups had significantly lower levels of HMGB1 than did those in the KA group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePhytochemical characterization of NRICM101\u003c/h2\u003e \u003cp\u003eRegarding the phytochemical characterization of NRICM101, some standard active compounds were selected on the basis of a review of the literature for identification from the methanol extract. The structures of these compounds were confirmed by 3D fingerprint analysis using the selected standards (approximately 98.99%) (Tsai et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As given, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e shows the chromatogram of NRICM101. The contents of baicalin, oroxylin A-7-O-glucuronide, wogonin-7-O-glucuronide, glycyrrhizic acid, liquiritin, magnolol, and rosmarinic acid in the freeze-dried extract of NRICM101 were 18.34, 1.33, 2.67, 2.42, 1.70, 1.07, and 0.91 mg/g, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of NRICM101 on rat body weight, liver and kidney morphology, and serum ALT and AST levels\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA shows that the body weight of NRICM101 (300 mg/kg, oral gavage)-treated rats was not significantly different from that of control rats (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.84). In addition, compared with those of the control rats, the liver and kidney histopathology of the NRICM101 rats showed no obvious signs of pathological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Additionally, no significant difference in the serum ALT or AST levels was observed between the NRICM101 rats and the control rats (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC). These results imply that NRICM101 administration may be safe.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEpilepsy 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, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eKA 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., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Chang et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Jean et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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\u0026thinsp;+\u0026thinsp;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.\u003c/p\u003e \u003cp\u003eExcess glutamate-mediated neuronal excitation is generally considered a critical factor in the pathological process of epilepsy (During and Spencer, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Soukupova et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). 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, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Rose et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). 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., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Eid et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Green et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hotz et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e. 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., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; van der Hel, 2014; Lin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) 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.\u003c/p\u003e \u003cp\u003eNeuroinflammation has been associated with glutamatergic hyperactivity in epilepsy (Vezzani and Granata, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Vezzani and Viviani, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Vezzani et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). 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., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Devinsky et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). 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., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Tilleux and Hermans, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Perez et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Terrone et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, IL-1β/IL-1R1 pathway activation can enhance glutamatergic NMDA receptor activation, resulting in excitotoxicity and seizures (Viviani et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). 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., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Han et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; 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., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Dhuriya and Sharma, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; 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., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; 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., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eThe HMGB1/TLR4 cascade has been shown to aggravate astrogliosis and augment epileptogenic inflammatory signaling (van Vliet et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). HMGB1 and TLR4 levels are increased in the serum and brain tissue of both epileptic patients and animal models (Luo et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kaya et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yue et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Inhibiting the HMGB1/TLR4 pathway can increase the seizure threshold, alleviate the inflammatory response, and lessen nerve damage after epileptic seizures (Li et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn 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., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Flavonoids have been reported to interact with glutamatergic neurotransmission (Lin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Jean et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pai et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition, baicalin has been shown to produce antiepileptic effects by inhibiting oxidative stress and the inflammatory response (Yang et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAn excessive inflammatory response leads to neuronal damage, resulting in permanent impairment of the structure and function of neural networks, an important underlying cause of the recurrent spontaneous seizures observed in epilepsy. The results obtained from this study revealed that NRICM101 protects model rats against KA-induced seizures and neuronal death and is safe and nontoxic. The suppression of neuroinflammatory trajectories with the consequent maintenance of normal glutamate homeostasis by NRICM101 might explain its antiepileptogenic and neuroprotective effects. Our findings highlight the promising role of NRICM101 in the management of epilepsy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eDesign of the study: Chi-Feng Hung, Su-Jane Wang. Statistical analysis: Chi-Feng Hung, Wei-Che Chiu, Jia-Cih Chen, Wu-Chang Chuang, Su-Jane Wang. Experiment and data collection: Jia-Cih Chen, Wu-Chang Chuang. Writing the manuscript: Su-Jane Wang. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data\u0026nbsp;\u003c/strong\u003eData generated during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was supported by the Taiwan Ministry of Science and Technology (112-2320-B-030-010-MY3) and Cathay General Hospital (110-CGH-FJU-02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe appreciate Sun Ten for providing NRICM101 and their information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003eThe experimental animals were conducted according to the Animal Ethics Committee (IACUC) guidelines issued by the Fu Jen Catholic University (Taipei, Taiwan) (Approval ID: A11206).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003eAll authors have approved the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbd El-Aal SA, El-Abhar HS, Abulfadl YS (2022) Morin offsets PTZ-induced neuronal degeneration and cognitive decrements in rats: The modulation of TNF. Eur J Pharmacol 931:175213-α/TNFR-1/RIPK1,3/MLKL/PGAM5/Drp-1, IL-6/JAK2/STAT3/GFAP and Keap-1/Nrf-2/HO-1 trajectories\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBedner P, Dupper A, H\u0026uuml;ttmann K, M\u0026uuml;ller J, Herde MK, Dublin P, Deshpande T, Schramm J, H\u0026auml;ussler U, Haas CA (2015) Astrocyte uncoupling as a cause of human temporal lobe epilepsy. Brain 138:1208\u0026ndash;1222\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBillakota S, Devinsky O, Kim KW (2020) Why we urgently need improved epilepsy therapies for adult patients. Neuropharmacology 170:107855\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang A, Chang Y, Wang SJ (2022) Rutin prevents seizures in kainic acid-treated rats: evidence of glutamate levels, inflammation and neuronal loss modulation. Food Funct 13:10401\u0026ndash;10414\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Chen X, Liang Y (2023) Meta-analysis of HMGB1 levels in the cerebrospinal fluid and serum of patients with epilepsy. Neurol Sci 44:2329\u0026ndash;2337\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng YD, Lu CC, Hsu YM, Tsai FJ, Bau DT, Tsai SC, Cheng CC, Lin JJ, Huang YY, Juan YN, Chiu YJ, Kuo SC, Yang JS, Wu LT (2011) In Silico and In Vitro studies of taiwan chingguan yihau (NRICM101) on TNF-α/IL-1β-induced human lung cells. Biomedicine 12:56\u0026ndash;71\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDanbolt NC (2001) Glutamate uptake. Prog Neurobiol 65:1\u0026ndash;105\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDevinsky O, Vezzani A, Najjar S, De Lanerolle NC, Rogawski MA (2013) Glia and epilepsy: excitability and inflammation. Trends Neurosci 36:174\u0026ndash;184\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhuriya YK, Sharma D (2018) Necroptosis: a regulated infammatory mode of cell death. J Neuroinfammation 15:199\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuring MJ, Spencer DD (1993) Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain. Lancet 341:1607\u0026ndash;1610\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEid T, Behar K, Bumanglag AV, Lee TS (2012) Role of glutamine synthetase inhibition in epilepsy. Neurochem Res 37:2339\u0026ndash;2350\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedman LK, Pellegrini-Giampietro DE, Sperber EF, Bennett MV, Moshe SL, Zukin RS (1994) Kainate-induced status epilepticus alters glutamate and GABAA receptor gene expression in adult rat hippocampus: an in situ hybridization study. J Neurosci 14:1697\u0026ndash;1707\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen JL, Dos Santos WF, Fontana ACK (2021) Role of glutamate excitotoxicity and glutamate transporter EAAT2 in epilepsy: Opportunities for novel therapeutics development. Biochem Pharmacol 193:114786\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan CL, Ge M, Liu YP, Zhao XM, Wang KL, Chen N, Meng WJ, Hu W, Zhang JG, Li L, Meng FG (2018) LncRNA H19 contributes to hippocampal glial cell activation via JAK/STAT signaling in a rat model of temporal lobe epilepsy. J Neuroinflammation 15:103\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe LY, Hu MB, Li RL, Zhao R, Fan LH, He L, Lu F, Ye X, Huang YL, Wu CJ (2021) Natural medicines for the treatment of epilepsy: Bioactive components, pharmacology and mechanism. Front Pharmacol 12:604\u0026ndash;640\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoda U, Agarwal NB, Vohora D, Parvez S, Raisuddin S (2017) Resveratrol suppressed seizures by attenuating IL-1β, IL1-Ra, IL-6, and TNF-α in the hippocampus and cortex of kindled mice. Nutr Neurosci 20:497\u0026ndash;504\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHotz AL, Jamali A, Rieser NN, Niklaus S, Aydin E, Myren-Svelstad S, Lalla L, Jurisch-Yaksi N, Yaksi E, Neuhauss SCF (2022) Loss of glutamate transporter eaat2a leads to aberrant neuronal excitability, recurrent epileptic seizures, and basal hypoactivity. Glia 70:196\u0026ndash;214\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsieh TY, Chang Y, Wang SJ (2022) Piperine provides neuroprotection against kainic acid-induced neurotoxicity via maintaining NGF signalling pathway. Molecules 27:2638\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eimpact, potential mechanisms, and new innovative treatment options. Pharmacol Rev 72:606\u0026ndash;638\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJean WH, Huang CT, Hsu JH, Chiu KM, Lee MY, Shieh JS, Lin TY, Wang SJ (2022) Anticonvulsive and neuroprotective effects of eupafolin in rats are associated with the inhibition of glutamate overexcitation and upregulation of the Wnt/β-Catenin signaling pathway. ACS Chem Neurosci 13:1594\u0026ndash;1603\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang N, Xu J, Xu Q, Nedergaard M, Kang J (2005) Astrocytic glutamate release-induced transient depolarization and epileptiform discharges in hippocampal CA1 pyramidal neurons. J Neurophysiol 94:4121\u0026ndash;4130\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaya MA, Erin N, Bozkurt O, Erkek N, Duman O, Haspolat S (2021) Changes of HMGB-1 and sTLR4 levels in cerebrospinal fluid of patients with febrile seizures. Epilepsy Res 169:106516\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLason W, Chlebicka M, Rejdak K (2013) Research advances in basic mechanisms of seizures and antiepileptic drug action. Pharmacol Rep 65:787\u0026ndash;801\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026eacute;vesque M, Avoli M (2013) The kainic acid model of temporal lobe epilepsy. Neurosci Biobehav Rev 37:2887\u0026ndash;2899\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi G, Zhang S, Cheng Y, Lu Y, Jia Z, Yang X, Zhang S, Guo W, Pei L (2022) Baicalin suppresses neuron autophagy and apoptosis by regulating astrocyte polarization in pentylenetetrazol-induced epileptic rats and PC12 cells. Brain Res 1774:147723\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Li B, Zhu X, Yin P, Liu J, Huang S, Sun R (2013) Neuroprotective effects of anti-high-mobility group box 1 antibody in juvenile rat hippocampus after kainic acid-induced status epilepticus. NeuroReport 24:785\u0026ndash;790\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin CH, Chen YJ, Lin MW, Chang HJ, Yang XR, Lin CS (2023) ACE2 and a Traditional Chinese Medicine Formula NRICM101 Could Alleviate the Inflammation and Pathogenic Process of Acute Lung Injury. Medicina 59:1554\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin CH, Hsieh CL (2021) Chinese herbal medicine for treating epilepsy. Front Neurosci 15:682821\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin TY, Lu CW, Wang SJ (2016) Luteolin protects the hippocampus against neuron impairments induced by kainic acid in rats. Neurotoxicology 55:48\u0026ndash;57\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin TY, Lu CW, Wang SJ, Huang SK (2015) Protective effect of hispidulin on kainic acid-induced seizures and neurotoxicity in rats. Eur J Pharmacol 755:6\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu T, Zhang L, Joo D, Sun SC (2017) NF-κB signaling in inflammation. Signal Transduct Target Ther 2:17023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD (2021) Analysis of relative gene expression data using real-time quantitative PCR and the 2\u0026thinsp;\u0026ndash; ∆∆CT method. Methods 25:402\u0026ndash;408\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026ouml;scher W, Potschka H, Sisodiya SM, Vezzani A (2020) Drug resistance in epilepsy. Clinical\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu H, Luo M, Chen R, Luo Y, Xi A, Wang K, Xu Z (2023) Efficacy and safety of traditional Chinese medicine for the treatment of epilepsy: A updated meta-analysis of randomized controlled trials. Epilepsy Res 189:107075\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo L, Jin Y, Kim ID, Lee JK (2013) Glycyrrhizin attenuates kainic acid-induced neuronal cell death in the mouse hippocampus. Exp Neurobiol 22:107\u0026ndash;115\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNgugi AK, Kariuki SM, Bottomley C, Kleinschmidt I, Sander JW, Newton CR (2011) Incidence of epilepsy: a systematic review and meta-analysis. Neurology 77:1005\u0026ndash;1012\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePai MS, Wang KC, Yeh KC, Wang SJ (2023) Stabilization of mitochondrial function by chlorogenic acid protects against kainic acid-induced seizures and neuronal cell death in rats. Eur J Pharmacol 961:176197\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaudel YN, Semple BD, Jones NC, Othman I, Shaikh MF (2019) High mobility group box 1 (HMGB1) as a novel frontier in epileptogenesis: from pathogenesis to therapeutic approaches. J Neurochem 151:542\u0026ndash;557\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez EL, Lauritzen F, Wang Y, Lee TS, Kang D, Zaveri HP, Chaudhry FA, Ottersen OP, Bergersen LH, Eid T (2012) Evidence for astrocytes as a potential source of the glutamate excess in temporal lobe epilepsy. Neurobiol Dis 47:331\u0026ndash;337\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerucca P, Gilliam FG (2012) Adverse effects of antiepileptic drugs. Lancet Neurol 11:792\u0026ndash;802\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRacine RJ (1972) Modification of seizure activity by electric stimulation: II. Motor seizure. EEG Clin Neurophysiol 32:281\u0026ndash;294\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRose CR, Felix L, Zeug A, Dietrich D, Reiner A, Henneberger C (2017) Astroglial glutamate signaling and uptake in the hippocampus. Front Mol Neurosci 10:451 \\\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSills GJ, Rogawski MA (2020) Mechanisms of action of currently used antiseizure drugs. Neuropharmacology 168:107966\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh S, Yang YF (2011) Pharmacological Mechanism of NRICM101 for COVID-19 Treatments by Combined Network Pharmacology and Pharmacodynamics. Int J Mol Sci 23:15385\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoltani Khaboushan A, Yazdanpanah N, Rezaei N (2022) Neuroinflammation and proinflammatory cytokines in epileptogenesis. Mol Neurobiol 59:1724\u0026ndash;1743\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoukupova M, Binaschi A, Falcicchia C, Palma E, Roncon P, Zucchini S, Simonato M (2015) Increased extracellular levels of glutamate in the hippocampus of chronically epileptic rats. Neuroscience 301:246\u0026ndash;253\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpigolon G, Veronesi C, Bonny C, Vercelli A (2010) c-Jun n-terminal kinase signaling pathway in excitotoxic cell death following kainic acid-induced status epilepticus. Eur J Neurosci 31:1261\u0026ndash;1272\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSwamy M, Wan Roslina WY, Sirajudeen KNS, Zulkarnain M, Chandran G (2011) Decreased glutamine synthetase, increased citrulline - nitric oxide cycle activities and oxidative stress in different regions of brain in epilepsy rat model. J Physiol Biochem 67:105\u0026ndash;113\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerrone G, Balosso S, Pauletti A, Ravizza T, Vezzani A (2020) Inflammation and reactive oxygen species as disease modifiers in epilepsy. Neuropharmacology 167:107742\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTilleux S, Hermans E (2007) (2007) Neuroinflammation and regulation of glial glutamate uptake in neurological disorders. J Neurosci Res 85: 2059\u0026ndash;2070\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsai KC, Huang YC, Liaw CC, Tsai CI, Chiou CT, Lin CJ, Wei WC, Lin SJ, Tseng YH, Yeh KM, Lin YL, Jan JT, Liang JJ, Liao CC, Chiou WF, Kuo YH, Lee SM, Lee MY, Su YC (2021) A traditional Chinese medicine formula NRICM101 to target COVID-19 through multiple pathways: a bedside-to-bench study. Biomed Pharmacother 133:111037\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Hel WS, Hessel EV, Bos IW, Mulder SD, Verlinde SA, van Eijsden P, Graan PNEl (2014) Persistent reduction of hippocampal glutamine synthetase expression after status epilepticus in immature rats. Eur J Neurosci 40:3711\u0026ndash;3719\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Vliet EA, Aronica E, Vezzani A, Ravizza T (2018) Review: Neuroinflammatory pathways as treatment targets and biomarker candidates in epilepsy: emerging evidence from preclinical and clinical studies. Neuropathol Appl Neurobiol 44:91\u0026ndash;111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVezzani A, Aronica E, Mazarati A, Pittman QJ (2013) Epilepsy and brain inflammation. Exp Neurol 244:11\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVezzani A, Balosso S, Ravizza T (2008) The role of cytokines in the pathophysiology of epilepsy. Brain Behav Immun 22:797\u0026ndash;803\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVezzani A, Balosso S, Ravizza T (2019) Neuroinflammatory pathways as treatment targets and biomarkers in epilepsy. Nat Rev Neurol 15:459\u0026ndash;472\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVezzani A, Granata T (2005) Brain inflammation in epilepsy: experimental and clinical evidence. Epilepsia 46:1724\u0026ndash;1743\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVezzani A, Viviani B (2015) Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability. Neuropharmacology 96:70\u0026ndash;82\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViviani B, Bartesaghi S, Gardoni F, Vezzani A, Behrens MM, Bartfai T, Binaglia M, Corsini E, Di Luca M, Galli CL, Marinovich M (2003) Interleukin-1beta enhances NMDA receptor-mediated intracellular calcium increase through activation of the Src family of kinases. J Neurosci 23:8692\u0026ndash;8700\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei WC, Tsai KC, Liaw CC, Chiou CT, Tseng YH, Liao GY, Lin YC, Chiou WF, Liou KT, Yu IS, Shen YC, Su YC (2023) NRICM101 ameliorates SARS-CoV-2-S1-induced pulmonary injury in K18-hACE2 mice model. Front Pharmacol 14:1125414\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J, Cao M, Peng Y, Dong B, Jiang Y, Hu C, Zhu P, Xing W, Yu L, Xu R, Chen Z (2023) Research progress on the treatment of epilepsy with traditional Chinese medicine. Phytomedicine 120:155022\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Z, Xue T, Zhang Z, Wang X, Xu P, Zhang J, Lei X, Li Y, Xie Y, Wang L, Fang M, Chen Y (2011) Role of signal transducer and activator of transcription-3 in up-regulation of GFAP after epilepsy. Neurochem Res 36:2208\u0026ndash;2215\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Jia Z, Xiao Z, Zhao J, Lu Y, Chu L, Shao H, Pei L, Zhang S, Chen Y (2021) Baicalin Rescues Cognitive Dysfunction, Mitigates Neurodegeneration, and Exerts Anti-Epileptic Effects Through Activating TLR4/MYD88/Caspase-3 Pathway in Rats. Drug Des Devel Ther 15:3163\u0026ndash;3180\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeh KC, Hung CF, Lee HL, Hsieh TY, Wang SJ (2022) Soybean meal extract preserves memory ability by increasing presynaptic function and modulating gut microbiota in rats. Mol Neurobiol 59:1649\u0026ndash;1664\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYue Z, Tang J, Peng S, Cai X, Rong X, Yang L (2023) Serum concentration of high-mobility group box 1, Toll-like receptor 4 as biomarker in epileptic patients. Epilepsy Res 192:107138\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang S, Chen F, Zhai F, Liang S (2022) Role of HMGB1/TLR4 and IL-1β/IL-1R1 signaling pathways in epilepsy. Front Neurol 13:904225\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao J, Wang Y, Xu C, Liu K, Wang Y, Chen L, Wu X, Gao F, Guo Y, Zhu J, Wang S, Nishibori M, Chen Z (2017) Therapeutic potential of an anti-high mobility group box-1 monoclonal antibody in epilepsy. Brain Behav Immun 64:308\u0026ndash;319\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu HL, Wan JB, Wang YT, Li BC, Xiang C, He J, Li P (2014) Medicinal compounds with antiepileptic/anticonvulsant activities. Epilepsia 55:3\u0026ndash;16\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NRICM101, antiseizure, neuroprotection, anti-neuroinflammation, glutamate, kainic acid","lastPublishedDoi":"10.21203/rs.3.rs-3932956/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3932956/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTaiwan Chingguan Yihau (NRICM101) is a Traditional Chinese medicine (TCM) formula used to treat coronavirus disease 2019; however, its impact on epilepsy has not been revealed. Therefore, the present study evaluated the anti-epileptogenic effect of orally administered NRICM101 on kainic acid (KA)-induced seizures in rats and investigated its possible mechanisms of action. Sprague‒Dawley rats were administered NRICM101 (300 mg/kg) by oral gavage for 7 consecutive days before receiving an intraperitoneal injection of KA (15 mg/kg). NRICM101 considerably reduced the seizure behavior and electroencephalographic seizures induced by KA in rats. NRICM101 also significantly decreased the neuronal loss and glutamate increase and increased GLAST, GLT-1, GAD67, GDH and GS levels in the cortex and hippocampus of KA-treated rats. In addition, NRICM101 significantly suppressed astrogliosis (as determined by decreased GFAP expression); neuroinflammatory signaling (as determined by reduced HMGB1, TLR-4, IL-1β, IL-1R, IL-6, p-JAK2, p-STAT3, TNF-α, TNFR1 and p-IκB levels, and increased cytosolic p65-NFκB levels); and necroptosis (as determined by decreased p-RIPK3 and p-MLKL levels) in the cortex and hippocampus of KA-treated rats. The effects of NRICM101 were similar to those of carbamazepine, a well-recognized antiseizure drug. Furthermore, no toxic effects of NRICM101 on the liver and kidney were observed in NRICM101-treated rats. The results indicate that NRICM101 has antiepileptogenic and neuroprotective effects through the suppression of the inflammatory cues (HMGB1/TLR4, Il-1β/IL-1R1, IL-6/p-JAK2/p-STAT3, and TNF-α/TNFR1/NF-κB) and necroptosis signaling pathways (TNF-α/TNFR1/RIP3/MLKL) associated with glutamate level regulation in the brain and is innocuous. Our findings highlight the promising role of NRICM101 in the management of epilepsy.\u003c/p\u003e","manuscriptTitle":"Taiwan Chingguan Yihau (NRICM101) prevents kainic acid-induced seizures in rats by modulating neuroinflammation and the glutamatergic system","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-15 18:38:42","doi":"10.21203/rs.3.rs-3932956/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c327dc7-8baf-4555-b94b-27f21f33082b","owner":[],"postedDate":"February 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-08T19:03:58+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-15 18:38:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3932956","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3932956","identity":"rs-3932956","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}