Naringenin-loaded PCL nanoparticles attenuate glial activation and TNF-α expression in iron-induced post-traumatic epilepsy

Naringenin-loaded PCL nanoparticles attenuate glial activation and TNF-α expression in iron-induced post-traumatic epilepsy | 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 Naringenin-loaded PCL nanoparticles attenuate glial activation and TNF-α expression in iron-induced post-traumatic epilepsy Shyam Sunder Rabidas, Chandra Prakash, Shivani Bhati, Jaydeep Bhattacharya, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7630794/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2026 Read the published version in Neurochemical Research → Version 1 posted 12 You are reading this latest preprint version Abstract Post-traumatic epilepsy (PTE) is the cosequence of traumatic brain damage (TBI), which poses an important health risk for the human population. The underlying mechanism of PTE is complex and appears to be linked to various cellular processes, including oxidative stress and neuroinflammation. Over the years, several studies have reported that most of the available antiseizure medicines are ineffective in preventing PTE. As a result, there is an urgent need to search for alternative treatment options. Naringenin is a flavonoid with multiple pharmacological properties and has shown beneficial effects in several health issues, including neurological disorders. In this study, we investigated the effect of naringenin-loaded PCL nanoparticles (NarNPs) on neuroinflammatory responses in a PTE model. NarNPs were produced using the nanoprecipitation method, and their physicochemical properties were comprehensively examined. To induce epilepsy, FeCl 3 was injected intracortically to rats and naringenin (both free naringenin (NAR) and NarNPs) was administered orally 15 days post-surgery. Epileptic seizures were observed by electroencephalography (EEG) patterns and spectral power analysis of γ-waves. Immunofluorescence analysis was conducted to explore the disease-modifying potential of NarNPs. Our findings demonstrated that NarNPs distinctly reduced epileptiform seizure activity in epileptic rats. The study found that NarNPs lowered the expression of GFAP, IBA1, and TNF-α. The observed ameliorative effects were more pronounced in NarNP-treated rats than in the NAR-treated group. Overall, our data imply that NarNPs have significant antiseizure and disease-modifying potential by attenuating glial activation and TNF-α production in PTE rats. Epilepsy seizures naringenin PCL nanoparticles neuroinflammation astrogliosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Epilepsy is a brain condition with the occurrence of transient, unpredictable and unprovoked seizures resulting from synchronized and excessive firing of neurons. It is a severe neurological disorder that affects roughly 70 million people worldwide, more than 12 million of whom live in India [ 1 ]. Post-traumatic epilepsy (PTE) occurs after traumatic brain injury (TBI) and accounts for approximately 20% of all instances of acquired epilepsy [ 2 ]. The pathophysiology of PTE, leading to the onset and progression of epileptic seizures, is complex and involves multiple pathways such as oxidative stress, neuroinflammation, neurotransmitter imbalance, neurodegeneration, and more. Neuroinflammation is linked to the onset and progression of epilepsy. Evidence from human and animal studies shows that numerous neuroinflammatory factors are activated in various forms of epilepsy [ 3 – 5 ]. Furthermore, recurrent seizures can trigger a neuroinflammatory response, which can lead to increased excitability and neurodegeneration [ 6 , 7 ]. According to some studies, neuroinflammation might lower the seizure threshold, contributing to seizure recurrence [ 6 , 8 ]. The iron-induced epilepsy in rats mimics PTE and has been used for exploring the underlying mechanism and effectiveness of therapeutics. This model can be developed via the intercortical injection of FeCl 3 solution [ 9 ], that develop a chronic epileptogenic focus, spreading throughout the cerebral cortex and subcortical regions [ 10 , 11 ]. Available antiseizure medicines (ASMs) are ineffective in approximately one-third population of total epilepsy cases [ 12 , 13 ]. Additionally, continuous administration of these ASMs can exert side effects including depression, anxiety, memory loss, etc. Hence, the identification of novel therapeutic options is necessary to counter these issues. Flavonoids are polyphenolic bioactive compounds that act as secondary metabolites from plants and have minimal adverse effects [ 14 ]. Preclinical research demonstrated severe pharmacological properties, including anti-inflammatory and neuroprotective effects [ 15 – 17 ]. Naringenin, the primary flavonoid of grapefruit and other citrus fruits, is known to penetrate the blood-brain barrier (BBB) and has numerous pharmacological properties. Regrettably, the bioavailability of naringenin is limited due to its hydrophobic nature, short half-life and rapid conversion to crystalline form, leading to low absorption through the digestive system, which restricting its therapeutic applications [ 18 – 20 ]. Polycaprolactone (PCL) is a semicrystalline aliphatic polyester formed through the ring-opening polymerization of caprolactone monomers. The unique mechanical, chemical, and bioresorbable properties of the polymer have led to its biomedical applications. The PCL has a prolonged circulation in the bloodstream, as it can evade the immune system and avoid rapid clearance [ 21 ]. Moreover, PCL is biocompatible and FDA-approved for various medical applications. Growing research supports the use of polymeric nanoparticles as a drug delivery system to increase the bioavailability. These nanoformulations encapsulated drugs, deliver them in a sustained and/or targeted manner and reduce their toxic effects by protecting nontargeted tissues [ 22 , 23 ]. Therefore, our goal in this study was to synthesize NarNPs and evaluate their therapeutic potential in FeCl 3 -induced experimental PTE in rats. Materials and methods Chemicals Naringenin (#N5893), polycaprolactone (#440752), FeCl 3 (#157740), Pluronic F127 (#P2443), corn oil (#C8267), 4′,6-diamidino-2-phenylindole (DAPI, #9542) and aqueous mounting media (#F4680-25ML) were purchased from Sigma‒Aldrich. Primary antibodies; monoclonal mouse anti-GFAP (#BF 8023, Affinity Biosciences), monoclonal rabbit anti-IBA1 (#17198, Cell Signalling Technology) and polyclonal rabbit anti-TNF-α (#ITT06080, ImmunoTag) were purchased from the mentioned sources. Fluorescence-conjugated secondary antibodies; goat anti-mouse Alexa Fluor 488 (#R37120) and goat anti-rabbit Alexa Fluor 594 (#R37117) were purchased from Invitrogen. All electrodes, connectors, and wires were bought from Plastic One (Roanoke, Virginia, USA). Other chemicals and reagents used were analytical grade and obtained from reputable suppliers in India. Animals and experimentation The Institutional Animal Ethics Committee at Jawaharlal Nehru University (JNU), New Delhi, approved the rat experiment (IAEC code 16/2021). All protocols using rats followed the regulations established by the Committee for the Purpose of Control and Supervision of Animal Experiments. Male Wistar rats aged 2–5 months were obtained from Central Laboratory Animal Resources, JNU, New Delhi. They were housed in polypropylene cages (8 × 12 x 5-inch) with stainless-steel lids. Cages were kept at a temperature of 23 ± 4°C, with a 12/12 h light-dark cycle and free access to water and food. A total of forty rats were divided into five groups, with an equal number of rats in each group. The outline of grouping of animal and experimental design has been depicted in Fig. 1 . SHAM : Rats received intracortical injections of 5 µL of normal saline. EPIL : Rats were subjected to epilepsy induction via the intracortical injection of 5 µL of FeCl 3 solution (100 mM prepared in normal saline). EPIL + NAR : Epileptic rats received free naringenin (20 mg/kg b.wt./day, with corn oil, orally) for 15 days, starting after the 15th day of FeCl 3 injection. EPIL + NarNPs : Epileptic rats received encapsulated naringenin (providing naringenin 20 mg/kg b.wt./day, orally) for 15 days, starting after 15th day of FeCl 3 injection. SHAM + NarNPs : Saline-injected rats received encapsulated naringenin (providing naringenin, 20 mg/kg b.wt./day, orally) for 15 days starting after the 15th day of saline injection. The dose for naringenin administration (20 mg/kg b.wt./day) to experimental rats was as per the previous studies [ 24 , 25 ]. Formulation of NarNPs NarNPs were synthesized via the nanoprecipitation method [ 26 ]. 1 g of PCL polymer was mixed in 50 mL of acetone (organic phase), and then 250 mg of naringenin was added. The aqueous phase was prepared by dissolving 1% Pluronic PF127 in 250 mL of Milli-Q water. The organic phase was added dropwise, with continual stirring at 1000 rpm for 3 h. The acetone was evaporated by stirring the solution at 250 rpm at room temperature overnight. Characterization of NarNPs Size and Zeta potential The particle size (nm) and Zeta potential (mV) of NarNPs were measured using Dynamic Light Scattering (DLS) and a Zeta sizer equipment (Malvern Zetasizer Nano ZS series, UK). The particle size and Zeta potential (mV) were averaged over three observations at room temperature, with mean ± SE. Encapsulation efficiency Aliquots of the synthesized formulation were centrifuged at 15,000x g for 15 min before being washed twice for 5 min each. The resulting pellet was used to measure nanoparticle encapsulation efficiency at 290 nm on a spectrophotometer. The pellet was suspended in dichloromethane and slowly stirred for 10 min before being added to methanol and allowed to evaporate with it. Finally, the resulting naringenin was resuspended in methanol, thoroughly mixed, and centrifuged for 15 min at 15,000 x g. The optical density was measured at 290 nm, and naringenin content was determined using a standard curve. The percentage of encapsulated naringenin was calculated using the formula (Amount of naringenin practically present in the NPs divided by the amount of naringenin theoretically present) x 100. Transmission Electron Microscopy (TEM) The size, shape, and distribution of the NarNPs were confirmed using TEM; pictures were snapped with a field emission electron microscope (JEOL JEM 2100F Tokyo, Japan) at 200 kV and 100x-diluted NarNPs. Atomic Force Microscopy (AFM) NarNPs were also visualized via AFM (Witec GmnH, Germany). The NarNPs were allowed to air dry after being diluted 100X and drop-cast onto a glass slide. Project FOUR software (Witec GmbH, Germany) was used to flatten and analyze each image. Stereotaxic surgery The epilepsy induction and electrode implantation were carried out using 4% isoflurane, as described before [ 27 ]. After placing the rat on the stereotaxic platform, a midline incision was made to expose the cranium. Burr holes (0.5 mm in diameter) were drilled at the brain skull's stereotaxically determined coordinates. A burr hole (1.0 mm anteroposterior to bregma, 1.0 mm lateral from midline, and 2.0 mm ventral) was used to inject 5 µL of FeCl 3 solution (100 mM in normal saline) and sealed with sterile bone wax. In both hemispheres of the cortex, four burr holes were made to implant an epidural cortical electrode (2 mm posterior to the bregma and 2.0 mm lateral to the midline). One screw electrode was also placed in the frontal sinus region, which acts as an animal ground. The burr hole coordinates were determined using the rat brain atlas [ 28 ]. All of the free ends of the electrode wires were fused to a nine-pin connector, and a strong base was constructed using dental acrylic. Electrical activity and spectral power analysis After surgical recovery and completion of the treatment period, EEG signals from rats (n = 5 in each group) were recorded. All rats were habituated to the recording setup, and signals were recorded during the light phase. The extracellular EEG signals were collected as per the previously described protocol [ 29 ]. The electrode signals were filtered for EEG (1 Hz to 100 Hz) and amplified using an amplifier (P511 AC preamplifiers). The signals were then sent to the PolyVIEW 16 Data Acquisition System (Grass Technologies, USA) for visualization and storage on a computer. Synchronous oscillation of EEG signals at different frequencies in the EEG of the neuronal population was calculated via spectral power analysis [ 30 ]. Signals were filtered for gamma bands (30–50 Hz). A Hanning window vector was used to filter EEG recordings to minimize artefact data at each window's boundaries. Fast Fourier Transform analysis was done for 30 s stretches of EEG recordings. The spectral power density for a specific band (unit-Vrms2/Hz) was calculated by dividing the spectral power averaged across each window by a predetermined bandwidth. The relative power of the frequency was calculated by dividing the total power by the absolute power and multiplying by 100. Gamma's relative spectral trend was computed using the GRASS software package's Spectral Trends (Version 2.0). In silico interaction of TNF-α and naringenin To study the interaction between proinflammatory mediator TNF-α and naringenin, we performed molecular docking via CB Dock (PMID: 35609983). The structure of Naringenin was taken from PubChem (CID: 439246), and the structure of TNF-α was obtained from the UniProt online server (ID P16599). The Discovery Studio and chimeraX tools were used for the analysis of the data obtained from CB dock. The protein ligand interactions were examined for hydrogen bonds, hydrophobic interactions and electrostatic interactions. The binding affinity was expressed in terms of binding energy in kcal/mol. Immunofluorescence staining Rats (n = 3 rats each group ) were transcardially perfused with 2% paraformaldehyde (PFA) and normal saline (0.9% NaCl). After overnight post-fixation in 2% PFA, the brain tissues were sunk to 10 and 20% sucrose solutions and preserved in 30% sucrose. A cryostat (Leica CM 1860, Germany) was used for obtaining coronally cut brain sections, which were then collected on gelatin-coated slides and stored at -20°C for later use. Slides were taken out for immunofluorescence analysis and dried at room temperature for 30 min. After three rinses in PBS, tissue sections were immersed in 0.1% Triton X-100 for 10 min and rinsed again. The sections were first treated with 3% normal goat serum (NGS #ab7481, Abcam) for 1 h, then with primary antibodies specific for GFAP, IBA1, and TNF-α at a 1:200 dilution overnight at 4°C. The following day, the sections were rinsed with PBS after being incubated at room temperature for 1 hour. Sections were then rinsed with PBS and incubated with secondary antibodies (1:200 dilution) for 2 h. Finally, the sections were washed with PBS, then incubated with DAPI solution for 10 min to show the nuclei. The sections were covered with Fluoromount™ Aqueous Mounting Media and imaged with a Nikon Eclipse 90i fluorescence microscope (Tokyo, Japan). Statistical analysis All quantitative measures are presented as means ± SE. Statistical significance between groups was determined using one-way analysis of variance (ANOVA), followed by a post-hoc Tukey test for multiple comparisons with GraphPad Prism (8.0.2). Significant differences were defined by p-values of 0.05 or lower. Results Characterization of synthesized NarNPs The particle size of the NarNPs assessed via DLS was approximately 268.16 ± 4.67 nm (Fig. 2 a), indicating that their size (hydrodynamic radii) was within the acceptable range of nanoparticles. The zeta potential of the NarNPs was 21.9 ± 1.02 mV (Fig. 2 b), indicating that the nanoparticle has a negative surface charge due to the terminal carboxylic groups of the PCL polymer. The encapsulation efficiency of narigenin in NarNPs was approximately 45.88 ± 3.79% (Fig. 2 c). In addition, TEM images revealed that the actual particle size of NarNPs was in the range of 64.7–176 nm (Fig. 2 d). The characterization of NarNPs by AFM (Fig. 2 e) revealed a good nanoparticle population within a similar range that was also visualized in the 3D image (Fig. 2 f). NarNPs alleviate seizure activity in epileptic rats Epileptiform seizure activity was examined in the cortex of experimental rats by analyzing the morphology of EEG waves and spectral power analysis of γ-oscillations. As depicted in the representative image of EEG samples of 30 s (Fig. 3 a). We found that EEG paroxysms were composed of single spikes, multiple spikes and spike wave complexes with abnormal patterns of high frequency and amplitude. These EEG paroxysms suggest the abnormal firing of neurons due to their hyperactivity, leading to epileptiform seizure activity. NAR and NarNPs treatment suppressed the epileptiform seizure activity. The EEG paroxysms of epileptic rats administered NAR and NarNPs showed a remarkable decline in epileptiform seizure activity, as evidenced by the short duration of EEG paroxysms. Moreover, the amplitude of these spikes and spike waves was lower than epileptic rats without any treatment. These findings indicate the antiseizure effect of NAR and NarNPs in iron-induced experimental epilepsy. The effect of NarNPs was better compared with NAR-treated rats (Fig. 3 a), suggesting the enhanced antiseizure potential of NarNPs. We did not observe any remarkable change in the EEG samples of NarNPs treated sham rats with sham rats. The EEG recordings of the rats from both groups revealed no epileptiform activity; thus observed no evidence of the occurrence of epileptic seizures. Furthermore, the quantitative extent of epileptiform seizures was examined via spectral power analysis of γ-oscillation (30–50 Hz) of EEG signals (Fig. 3 b). We found a significant increase in the percentage of mean spectral power of epileptic rats (55.69 ± 3.79%) as compared to sham controls (40.78 ± 1.25%, p < 0.01). These changes are in line with the observed epileptiform activity in EEG patterns. The oral administration of NAR (48.75 ± 97%, NS) and NarNPs (46.05 ± 1.43%, p < 0.05) reduced the mean spectral power as compared to epileptic rats. However, the reduction was greater in NarNPs-administered epileptic rats as compared to NAR only administered. Similar to EEG patterns, spectral analysis of γ-oscillation did not reveal any significant change in sham rats administered to rats. NarNPs compared to sham controls. NarNPs attenuate glial activation in epileptic rats The results displayed a lower percentage of GFAP-positive cells, resembling resting astrocytes (Fig. 4 a) in the cortex (8.26 ± 1.67, Fig. 4 b) of sham rats. The epileptic rats showed thickened activated astrocytes (Fig. 4 a) along with a significantly increased percentage (15.49 ± 0.60, Fig. 4 b). The administration of both NAR and NarNPs to epileptic rats significantly restored the percentage of astrocytes. We observed no significant variations in the morphology and percentage of astrocytes between sham rats administered NarNPs and sham controls. Similarly, the appearance of IBA1 positive cells resembled that of resting microglia (ramified shape), and were lower in percentage (7.60 ± 1.51, Fig. 5 b) in sham rats. The percentage of IBA1 positive cells (15.45 ± 2.55, Fig. 5 b) was significantly high and was activated (ameboid shape, Fig. 5 a) in epileptic rats as compared to sham controls, whereas both NAR and NarNPs treatment significantly reduced the percentage of microglia in epileptic rats. No significant change was found in the percentage or morphology of microglia between sham rats administered NarNP and sham controls. Naringenin has a strong binding affinity with TNF-α The molecular docking study was performed to determine the affinity of naringenin with TNF-α. The analysis of the docked complex of TNF-α and naringenin showed the formation of three H-bonds, one electrostatic interaction and three hydrophobic interactions (Fig. 6 ). The binding energy for the interaction between TNF-α and naringenin was − 7 kcal/mol. NarNPs reduce TNF-α expression in epileptic rats The immunostaining results of TNF-α expression has been presented in the Fig. 7 . Epileptic rats showed higher TNF-α expression (3.87 ± 0.81 times, p value < 0.05, Fig. 7 b) than sham controls. These findings indicate that activation of astrocytes and microglia may trigger the production of TNF-α. Oral administration of NAR and NarNPs to epileptic rats restored TNF-α expression in the cortex. TNF-α expression was lowered to 1.21 ± 0.26 (p < 0.05) and 1.21 ± .56 (p < 0.05), respectively. We observed no significant difference between NarNPs administered to sham rats and sham controls. Discussion Biodegradable polymer-based nanoformulations are popular in neuroscience research due to their ability to cross the BBB, lower drug requirements and slow and sustained release [ 31 , 32 ]. In the current study, NarNPs were synthesized via the nanoprecipitation method and their physicochemical characteristics were examined by DLS, TEM and AFM. The nanoformulation enabled the rapid and dependable creation of a scaffold with a homogenous and spherical shape that was evident from the AFM images. Several previous investigations have also produced such PCL-based nanoformulation by this approach [ 33 – 36 ]. We promise that oral administration of NarNPs can enable delivery of naringenin to the brain and protection from epilepsy-associated damage. Hence, we evaluated the effect of NarNPs on EEG patterns and the spectral power analysis of γ-oscillations in epileptic rats. EEG signals were analyzed to ascertain the presence of epileptic seizures and identify the brain regions where seizures originate [ 37 ]. The γ-oscillations (35–45 Hz) of EEG signals are associated with informational processing and cognitive functions [ 38 ]. In humans, γ-wave frequency is majorly associated with the somatosensory cortical region [ 39 ]. In our study, EEG signals from epileptic rats consisted of sharp waves and spike wave complexes of high frequency and amplitude. These unusual trends are consistent with prior studies[ 40 , 41 ] which seem to imply the occurrence of epileptiform seizures in iron-injected rats, which signifing the development of electric field potential in cortical neurons. The results also showed a considerable increase in the percentage spectral power of γ-waves. A prior study found that kainic acid boosts β and γ-oscillations in the neocortex during spike-wave discharges [ 42 ]. A recent study[ 43 ] found that amplitude envelope correlations from interictal EEG signals show frequency-dependent increase γ frequency bands (40-200Hz) in epileptogenic cortex. The epileptic rats treated with NAR and NarNPs exhibited fewer single spikes, polyspikes, and spike waves, along with a reduced spectral power of γ-oscillations. These effects were more evident in the NarNP group compared to NAR group, indicating an improved antiepileptic potential of naringenin in nanoformulation. Earlier studies have shown that modification of naringenin 7-O-methyl ether improved its therapeutic efficacy in PTZ-induced seizures [ 44 ]. Similarly, synthesised naringenin-conjugated graphene oxide nanoparticles also suppressed convulsant behavior in adult zebrafish [ 45 ]. Astrocytes and microglia are crucial and contribute to cellular homeostasis, immune response and maintenance of the BBB. Astrocytes are a common type of glial cell vital for maintaining brain homeostasis [ 46 ]. Reactive astrocytes increase the expression of complement cascade genes and produce proinflammatory mediators such as IL-1β, TNF-α, and NO [ 47 ]. Microglia are ubiquitously distributed in the brain and are implicated in the modulation of neuroinflammatory response. Stimuli such as brain injury, infection, and oxidative stress reactivate microglia, which in turn produce proinflammatory cytokines and chemokines. Both clinical and experimental research showed that these brain cells play a key role in the pathophysiology of neurological disorders, including epilepsy [ 48 – 51 ]. Astrocytes can precipitate seizures by releasing TNF-α themselves or in combination with glutamate [ 52 ]. As, they are known to inhibit the release of the GABA precursor (glutamine) or promote the loss of glutamine synthetase, which in turn increases synaptic glutamate levels while decreasing GABA levels [ 53 , 54 ]. The data revealed a higher number of astrocytes in the cortex of epileptic rats, which were structurally similar to reactive astrocytes, implying that they are activated in PTE. Similar to astrocytes, the number of microglia was also amplified, along with their morphological similarity to reactive microglia (ameboid shape). Previously, several animal studies have examined the expression patterns and examined the morphological features of astrocytes and microglia in different epilepsy models[ 55 , 56 ] and sequential activation of glial cells is implicated in epileptogenesis in various experimental models of epilepsy, including status epilepticus [ 57 ]. During KA-induced status epilepticus, microglia rapidly become reactive and release TNF-α, which affects astrocytes, disrupts gap junctions, and contributes to temporal lobe epilepsy without influencing the progression of hippocampal sclerosis [ 55 ]. Prior evidence demonstrated that TNF-α contributes to epilepsy progression through astrocyte-linked mechanisms, including apoptosis, impaired synaptic function, and increased glutamate production [ 52 ]. Our study presented that epileptic rats treated with NAR and NarNPs had fewer astrocytes and microglia, indicating inhibition of glial activation. These findings are consistent with earlier studies indicating that naringenin suppresses neuroprotein and activates glial cells [ 58 ]. A recent study found that lower microglial activation coincided with a shift in phenotype from proinflammatory M1 to anti-inflammatory [ 59 ]. These data suggest that attenuation of glial cells by naringenin may be responsible for reducing seizures in PTE rats. The release of cytokines like TNF-α leads to neuronal hyperexcitability by inhibiting the reuptake of glutamate by astrocytes, thus increasing glutamatergic neurotransmission, reducing GABA-mediated inhibition and decreasing chloride currents and expression of GABA-A receptors [ 60 ]. Molecular docking revealed naringenin's high affinity for TNF-α known as key regulator of the pro-inflammatory cytokines cascade. Earlier reports also showed modulation of neuroinflammatory response by naringenin and suggested that naringenin can promote lysosome-dependent cytokine degradation [ 61 ]. Our study found increased TNF-α levels in the brains of epileptic rats, indicating that activation of astrocytes and microglia may have resulted in TNF-α release. The oral administration of NAR and NarNPs restored the expression of TNF-α, where NarNPs treatment had a greater effect compared to NAR treatment. These results indicate enhanced anti-inflammatory effect in NarNPs. Previously, a study formulated gelatin-coated PCL nanoparticles and reported their anti-inflammatory effect on oxygen-glucose-deprived human mesenchymal stem cells [ 62 ]. Based on our findings, we hypothesize (Fig. 8 ) that alleviation of seizure activity in epileptic rats by NarNPs may be due to reduced glial activation and TNF-α production in the cortex of PTE rats. Conclusion Overall, the study concluded that encapsulating naringenin in PCL nanoparticles significantly increased its efficacy. The formulated NarNPs showed a significant inhibitory effect on seizure activity compared to free naringenin. The antiseizure activity of NarNPs was mediated via alleviating glial activation and lowering the release of TNF-α, a proinflammatory mediator. Declarations Conflicts of interest The authors declare that there is no competing interest. Ethics statement All procedures involving animals were duly approved by the Institutional Animal Ethics Committee of JNU, New Delhi (IAEC code: 16/2021) and performed as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India Funding The financial support was provided by the Indian Council of Medical Research (5/5–5/4GIA/Trauma/2020-NCD-I) to DS and SS. Senior Research Fellowship was awarded by the Department of Biotechnology and Council of Scientific and Industrial Research to SSR (DBT/JRF/BET-18/1/2018/AL/181) and SB (09/0263(11758)/2021-EMR-I), respectively. DHR-Young Scientist grant was sanctioned to CP (R.12018/04/2024-HR) by the Department of Health Research. Author Contribution DS, JB and SSR conceptualized the idea. SSR synthesized nanoparticles, performed electrophysiology, in silco and immunofluorescence studies. SSR and SB characterized the nanoparticles. SSR synthesized the initial draft. SSR and CP performed the animal experiments and drafted the manuscript. JB, SS and DS critically reviewed and edited the final manuscript. All authors have approved the final version of the manuscript. Acknowledgement The authors acknowledge the Central Instrument Facility, School of Life Sciences, and Central Laboratory Animal Resources, JNU, for facilitating the experimentation. The authors also acknowledge the Advanced Instrumentation Research Facility, JNU, for providing instrument facilities. Data Availability The data will be available upon a reasonable request. References Amudhan S, Gururaj G, Satishchandra P (2015) Epilepsy in India I: Epidemiology and public health. Ann Indian Acad Neurol 18:263. https://doi.org/10.4103/0972-2327.160093 Ali I, Silva JC, Liu S, et al (2019) Targeting neurodegeneration to prevent post-traumatic epilepsy. 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08:17:28","extension":"png","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":819123,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/4449ba1b1c9e425cdcc65525.png"},{"id":92573832,"identity":"0c223fb9-d5d3-4cc0-ae48-94a2ae941e96","added_by":"auto","created_at":"2025-10-01 08:09:28","extension":"png","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":465440,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/8da4abe45261da0225e111f9.png"},{"id":92575022,"identity":"5991232b-c501-4411-9fd6-9dafc6327edb","added_by":"auto","created_at":"2025-10-01 08:17:27","extension":"xml","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133188,"visible":true,"origin":"","legend":"","description":"","filename":"add71afc1390440caa4730d95a9d4ea61structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/6981546ed7b8636052f9f5e4.xml"},{"id":92573829,"identity":"bdbc2d42-f837-41bb-a972-ccd2e0d0f030","added_by":"auto","created_at":"2025-10-01 08:09:27","extension":"html","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144707,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/de3f125ef0464b384edea82f.html"},{"id":92575013,"identity":"82572da3-0034-4532-9f5e-6e4e9a2ca237","added_by":"auto","created_at":"2025-10-01 08:17:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1509218,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of animal experimental design to evaluate the effect of naringenin in iron induced model of epilepsy \u003cstrong\u003e(a)\u003c/strong\u003e Five groups of experimental rats with treatment paradigm \u003cstrong\u003e(b)\u003c/strong\u003e time line for the animal experimental studies. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/b1f077e53daf7bfc2ff22b31.jpg"},{"id":92573795,"identity":"5c1c4321-dad9-4f42-a075-6c9929125f65","added_by":"auto","created_at":"2025-10-01 08:09:27","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3266317,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of naringenin-loaded PCL nanoparticles. \u003cstrong\u003e(a)\u003c/strong\u003e DLS size \u003cstrong\u003e(b)\u003c/strong\u003e Zeta potential \u003cstrong\u003e(c) \u003c/strong\u003eEncapsulation efficiency \u003cstrong\u003e(d),\u003c/strong\u003e TEM image (Scale bars: 100 nm) \u003cstrong\u003e(e)\u003c/strong\u003e AFM image and its \u003cstrong\u003e(f)\u003c/strong\u003e 3D visualization (Scale bars: 0.6 µm). Data is presented as mean ± SE.\u003c/p\u003e","description":"","filename":"Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/88b53b85b419608bd1b630af.jpg"},{"id":92576145,"identity":"5b5104a3-99fb-4d32-b2ce-544f9487eeba","added_by":"auto","created_at":"2025-10-01 08:25:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3432298,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NarNPs on the epileptiform activity of iron-injected rats \u003cstrong\u003e(a) \u003c/strong\u003eRepresentative EEG samples of 30 s duration from the cortex of experimental rats on the 30\u003csup\u003eth\u003c/sup\u003e day post-stereotaxic surgery. \u003cstrong\u003e(b)\u003c/strong\u003e The graph shows the relative mean spectral power of the y-oscillation (30-50Hz). Data represent the mean ± SE (n = 5). **p\u0026lt;0.01 significantly different from sham and \u003csup\u003e\u003cstrong\u003e##\u003c/strong\u003e\u003c/sup\u003ep\u0026lt;0.01, significantly different from epileptic rats.\u003c/p\u003e","description":"","filename":"Fig3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/e41e89052f9af67cc45f42ce.jpg"},{"id":92573827,"identity":"57072475-c614-4200-8f52-68d608e2e10a","added_by":"auto","created_at":"2025-10-01 08:09:27","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4497491,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NarNPs on the astroglial activation in the cortex. \u003cstrong\u003e(a) \u003c/strong\u003ePhotomicrographs show GFAP-positive cells (green), DAPI-stained nuclei (blue) and their overlay from the cortical region of experimental rats. \u003cstrong\u003e(b)\u003c/strong\u003e The bar graph shows the percentage of GFAP-positive cells. Data represent the mean ± SE (n = 3). **p\u0026lt;0.01, significantly different from sham control rats.\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003ep\u0026lt;0.05,\u003cstrong\u003e \u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e##\u003c/strong\u003e\u003c/sup\u003ep\u0026lt;0.01, significantly different from epileptic rats.\u003c/p\u003e","description":"","filename":"Fig4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/0b8935e333f641ef1e2c05ab.jpg"},{"id":92573800,"identity":"0e781a49-22cf-4109-a899-70f7a1c3d9d9","added_by":"auto","created_at":"2025-10-01 08:09:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3737627,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NarNPs on the microglial activation in the cortex. \u003cstrong\u003e(a)\u003c/strong\u003e Photomicrographs show IBA1-positive cells (red), DAPI-stained nuclei (blue) and their overlay from the cortical region of experimental rats. \u003cstrong\u003e(b) \u003c/strong\u003eThe bar graph shows the percentage of IBA1-positive cells. Data represent the mean ± SE (n = 3). **p\u0026lt;0.01, significantly different from sham control rats. ##p\u0026lt;0.01, significantly different from epileptic rats.\u003c/p\u003e","description":"","filename":"Fig5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/f20e7a59e58723ac06f7bce3.jpg"},{"id":92573812,"identity":"9be69ba9-b111-42f9-aeca-11c655c6e001","added_by":"auto","created_at":"2025-10-01 08:09:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3120723,"visible":true,"origin":"","legend":"\u003cp\u003eDocking study of interaction between TNF-α and naringenin: \u003cstrong\u003e(a)\u003c/strong\u003e structure of TNF-α obtained from UniProt (ID: P16599). \u003cstrong\u003e(b)\u003c/strong\u003eMolecular docking between TNF-α and Naringenin (CID: 439246). \u003cstrong\u003e(c)\u003c/strong\u003e 2D interaction diagram showing the interacting residues of the protein with the ligand. \u003cstrong\u003e(d)\u003c/strong\u003e Showing the detailed list of protein-ligand interactions and the bond type.\u003c/p\u003e","description":"","filename":"Fig6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/92e1afcf85afb06fc3da2c6b.jpg"},{"id":92575015,"identity":"1bde6c39-e50d-4e56-b307-7bd5078b4cd5","added_by":"auto","created_at":"2025-10-01 08:17:27","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3786282,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of NarNPs on the TNF-α release in the cortex. \u003cstrong\u003e(a)\u003c/strong\u003e Photomicrographs show TNF-α (Red), DAPI-stained nuclei and their overlay from the cortical region of experimental rats. \u003cstrong\u003e(b)\u003c/strong\u003e The bar graph shows relative TNF-α expression. Data represent the mean ± SE (n = 3). *p\u0026lt;0.05, significantly different from sham control rats.\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003ep\u0026lt;0.05, significantly different from epileptic rats.\u003c/p\u003e","description":"","filename":"Fig7.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/b635970079ec9a5041ec9041.jpg"},{"id":92576149,"identity":"7829123e-5ff2-4894-a02d-348dd0ac25f0","added_by":"auto","created_at":"2025-10-01 08:25:28","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2277414,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of a hypothetical mechanism of antiepileptic and anti-inflammatory effects of NarNPs on iron-induced post-traumatic epilepsy in rats. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"Fig8.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/df5f3ee830f67ee787539848.jpg"},{"id":103765659,"identity":"3ba55e85-ae47-42a7-8fde-0176ffe06913","added_by":"auto","created_at":"2026-03-02 16:06:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26439242,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7630794/v1/b716c945-b7a4-4bda-9aaf-6bc578ee9297.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Naringenin-loaded PCL nanoparticles attenuate glial activation and TNF-α expression in iron-induced post-traumatic epilepsy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy is a brain condition with the occurrence of transient, unpredictable and unprovoked seizures resulting from synchronized and excessive firing of neurons. It is a severe neurological disorder that affects roughly 70\u0026nbsp;million people worldwide, more than 12\u0026nbsp;million of whom live in India [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Post-traumatic epilepsy (PTE) occurs after traumatic brain injury (TBI) and accounts for approximately 20% of all instances of acquired epilepsy [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The pathophysiology of PTE, leading to the onset and progression of epileptic seizures, is complex and involves multiple pathways such as oxidative stress, neuroinflammation, neurotransmitter imbalance, neurodegeneration, and more. Neuroinflammation is linked to the onset and progression of epilepsy. Evidence from human and animal studies shows that numerous neuroinflammatory factors are activated in various forms of epilepsy [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, recurrent seizures can trigger a neuroinflammatory response, which can lead to increased excitability and neurodegeneration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. According to some studies, neuroinflammation might lower the seizure threshold, contributing to seizure recurrence [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe iron-induced epilepsy in rats mimics PTE and has been used for exploring the underlying mechanism and effectiveness of therapeutics. This model can be developed via the intercortical injection of FeCl\u003csub\u003e3\u003c/sub\u003e solution [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], that develop a chronic epileptogenic focus, spreading throughout the cerebral cortex and subcortical regions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Available antiseizure medicines (ASMs) are ineffective in approximately one-third population of total epilepsy cases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, continuous administration of these ASMs can exert side effects including depression, anxiety, memory loss, etc. Hence, the identification of novel therapeutic options is necessary to counter these issues.\u003c/p\u003e\u003cp\u003eFlavonoids are polyphenolic bioactive compounds that act as secondary metabolites from plants and have minimal adverse effects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Preclinical research demonstrated severe pharmacological properties, including anti-inflammatory and neuroprotective effects [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Naringenin, the primary flavonoid of grapefruit and other citrus fruits, is known to penetrate the blood-brain barrier (BBB) and has numerous pharmacological properties. Regrettably, the bioavailability of naringenin is limited due to its hydrophobic nature, short half-life and rapid conversion to crystalline form, leading to low absorption through the digestive system, which restricting its therapeutic applications [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePolycaprolactone (PCL) is a semicrystalline aliphatic polyester formed through the ring-opening polymerization of caprolactone monomers. The unique mechanical, chemical, and bioresorbable properties of the polymer have led to its biomedical applications. The PCL has a prolonged circulation in the bloodstream, as it can evade the immune system and avoid rapid clearance [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, PCL is biocompatible and FDA-approved for various medical applications. Growing research supports the use of polymeric nanoparticles as a drug delivery system to increase the bioavailability. These nanoformulations encapsulated drugs, deliver them in a sustained and/or targeted manner and reduce their toxic effects by protecting nontargeted tissues [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, our goal in this study was to synthesize NarNPs and evaluate their therapeutic potential in FeCl\u003csub\u003e3\u003c/sub\u003e-induced experimental PTE in rats.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemicals\u003c/h2\u003e\u003cp\u003eNaringenin (#N5893), polycaprolactone (#440752), FeCl\u003csub\u003e3\u003c/sub\u003e (#157740), Pluronic F127 (#P2443), corn oil (#C8267), 4\u0026prime;,6-diamidino-2-phenylindole (DAPI, #9542) and aqueous mounting media (#F4680-25ML) were purchased from Sigma‒Aldrich. Primary antibodies; monoclonal mouse anti-GFAP (#BF 8023, Affinity Biosciences), monoclonal rabbit anti-IBA1 (#17198, Cell Signalling Technology) and polyclonal rabbit anti-TNF-α (#ITT06080, ImmunoTag) were purchased from the mentioned sources. Fluorescence-conjugated secondary antibodies; goat anti-mouse Alexa Fluor 488 (#R37120) and goat anti-rabbit Alexa Fluor 594 (#R37117) were purchased from Invitrogen. All electrodes, connectors, and wires were bought from Plastic One (Roanoke, Virginia, USA). Other chemicals and reagents used were analytical grade and obtained from reputable suppliers in India.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimals and experimentation\u003c/h3\u003e\n\u003cp\u003e The Institutional Animal Ethics Committee at Jawaharlal Nehru University (JNU), New Delhi, approved the rat experiment (IAEC code 16/2021). All protocols using rats followed the regulations established by the Committee for the Purpose of Control and Supervision of Animal Experiments. Male Wistar rats aged 2\u0026ndash;5 months were obtained from Central Laboratory Animal Resources, JNU, New Delhi. They were housed in polypropylene cages (8 \u0026times; 12 x 5-inch) with stainless-steel lids. Cages were kept at a temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u0026deg;C, with a 12/12 h light-dark cycle and free access to water and food.\u003c/p\u003e\u003cp\u003eA total of forty rats were divided into five groups, with an equal number of rats in each group. The outline of grouping of animal and experimental design has been depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSHAM\u003c/b\u003e: Rats received intracortical injections of 5 \u0026micro;L of normal saline.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eEPIL\u003c/b\u003e: Rats were subjected to epilepsy induction via the intracortical injection of 5 \u0026micro;L of FeCl\u003csub\u003e3\u003c/sub\u003e solution (100 mM prepared in normal saline).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eEPIL\u0026thinsp;+\u0026thinsp;NAR\u003c/b\u003e: Epileptic rats received free naringenin (20 mg/kg b.wt./day, with corn oil, orally) for 15 days, starting after the 15th day of FeCl\u003csub\u003e3\u003c/sub\u003e injection.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eEPIL\u0026thinsp;+\u0026thinsp;NarNPs\u003c/b\u003e: Epileptic rats received encapsulated naringenin (providing naringenin 20 mg/kg b.wt./day, orally) for 15 days, starting after 15th day of FeCl\u003csub\u003e3\u003c/sub\u003e injection.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSHAM\u0026thinsp;+\u0026thinsp;NarNPs\u003c/b\u003e: Saline-injected rats received encapsulated naringenin (providing naringenin, 20 mg/kg b.wt./day, orally) for 15 days starting after the 15th day of saline injection.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eThe dose for naringenin administration (20 mg/kg b.wt./day) to experimental rats was as per the previous studies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eFormulation of NarNPs\u003c/h3\u003e\n\u003cp\u003eNarNPs were synthesized via the nanoprecipitation method [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. 1 g of PCL polymer was mixed in 50 mL of acetone (organic phase), and then 250 mg of naringenin was added. The aqueous phase was prepared by dissolving 1% Pluronic PF127 in 250 mL of Milli-Q water. The organic phase was added dropwise, with continual stirring at 1000 rpm for 3 h. The acetone was evaporated by stirring the solution at 250 rpm at room temperature overnight.\u003c/p\u003e\n\u003ch3\u003eCharacterization of NarNPs\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eSize and Zeta potential\u003c/h2\u003e\u003cp\u003eThe particle size (nm) and Zeta potential (mV) of NarNPs were measured using Dynamic Light Scattering (DLS) and a Zeta sizer equipment (Malvern Zetasizer Nano ZS series, UK). The particle size and Zeta potential (mV) were averaged over three observations at room temperature, with mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEncapsulation efficiency\u003c/h2\u003e\u003cp\u003eAliquots of the synthesized formulation were centrifuged at 15,000x g for 15 min before being washed twice for 5 min each. The resulting pellet was used to measure nanoparticle encapsulation efficiency at 290 nm on a spectrophotometer. The pellet was suspended in dichloromethane and slowly stirred for 10 min before being added to methanol and allowed to evaporate with it. Finally, the resulting naringenin was resuspended in methanol, thoroughly mixed, and centrifuged for 15 min at 15,000 x g. The optical density was measured at 290 nm, and naringenin content was determined using a standard curve. The percentage of encapsulated naringenin was calculated using the formula (Amount of naringenin practically present in the NPs divided by the amount of naringenin theoretically present) x 100.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTransmission Electron Microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eThe size, shape, and distribution of the NarNPs were confirmed using TEM; pictures were snapped with a field emission electron microscope (JEOL JEM 2100F Tokyo, Japan) at 200 kV and 100x-diluted NarNPs.\u003c/p\u003e\n\u003ch3\u003eAtomic Force Microscopy (AFM)\u003c/h3\u003e\n\u003cp\u003eNarNPs were also visualized via AFM (Witec GmnH, Germany). The NarNPs were allowed to air dry after being diluted 100X and drop-cast onto a glass slide. Project FOUR software (Witec GmbH, Germany) was used to flatten and analyze each image.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStereotaxic surgery\u003c/h2\u003e\u003cp\u003eThe epilepsy induction and electrode implantation were carried out using 4% isoflurane, as described before [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. After placing the rat on the stereotaxic platform, a midline incision was made to expose the cranium. Burr holes (0.5 mm in diameter) were drilled at the brain skull's stereotaxically determined coordinates. A burr hole (1.0 mm anteroposterior to bregma, 1.0 mm lateral from midline, and 2.0 mm ventral) was used to inject 5 \u0026micro;L of FeCl\u003csub\u003e3\u003c/sub\u003e solution (100 mM in normal saline) and sealed with sterile bone wax. In both hemispheres of the cortex, four burr holes were made to implant an epidural cortical electrode (2 mm posterior to the bregma and 2.0 mm lateral to the midline). One screw electrode was also placed in the frontal sinus region, which acts as an animal ground. The burr hole coordinates were determined using the rat brain atlas [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. All of the free ends of the electrode wires were fused to a nine-pin connector, and a strong base was constructed using dental acrylic.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eElectrical activity and spectral power analysis\u003c/h2\u003e\u003cp\u003eAfter surgical recovery and completion of the treatment period, EEG signals from rats (n\u0026thinsp;=\u0026thinsp;5 in each group) were recorded. All rats were habituated to the recording setup, and signals were recorded during the light phase. The extracellular EEG signals were collected as per the previously described protocol [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The electrode signals were filtered for EEG (1 Hz to 100 Hz) and amplified using an amplifier (P511 AC preamplifiers). The signals were then sent to the PolyVIEW 16 Data Acquisition System (Grass Technologies, USA) for visualization and storage on a computer.\u003c/p\u003e\u003cp\u003eSynchronous oscillation of EEG signals at different frequencies in the EEG of the neuronal population was calculated via spectral power analysis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Signals were filtered for gamma bands (30\u0026ndash;50 Hz). A Hanning window vector was used to filter EEG recordings to minimize artefact data at each window's boundaries. Fast Fourier Transform analysis was done for 30 s stretches of EEG recordings. The spectral power density for a specific band (unit-Vrms2/Hz) was calculated by dividing the spectral power averaged across each window by a predetermined bandwidth. The relative power of the frequency was calculated by dividing the total power by the absolute power and multiplying by 100. Gamma's relative spectral trend was computed using the GRASS software package's Spectral Trends (Version 2.0).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eIn silico interaction of TNF-α and naringenin\u003c/h2\u003e\u003cp\u003eTo study the interaction between proinflammatory mediator TNF-α and naringenin, we performed molecular docking via CB Dock (PMID: 35609983). The structure of Naringenin was taken from PubChem (CID: 439246), and the structure of TNF-α was obtained from the UniProt online server (ID P16599). The Discovery Studio and chimeraX tools were used for the analysis of the data obtained from CB dock. The protein ligand interactions were examined for hydrogen bonds, hydrophobic interactions and electrostatic interactions. The binding affinity was expressed in terms of binding energy in kcal/mol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003eRats (n\u0026thinsp;=\u0026thinsp;3 rats each group ) were transcardially perfused with 2% paraformaldehyde (PFA) and normal saline (0.9% NaCl). After overnight post-fixation in 2% PFA, the brain tissues were sunk to 10 and 20% sucrose solutions and preserved in 30% sucrose. A cryostat (Leica CM 1860, Germany) was used for obtaining coronally cut brain sections, which were then collected on gelatin-coated slides and stored at -20\u0026deg;C for later use. Slides were taken out for immunofluorescence analysis and dried at room temperature for 30 min. After three rinses in PBS, tissue sections were immersed in 0.1% Triton X-100 for 10 min and rinsed again. The sections were first treated with 3% normal goat serum (NGS #ab7481, Abcam) for 1 h, then with primary antibodies specific for GFAP, IBA1, and TNF-α at a 1:200 dilution overnight at 4\u0026deg;C. The following day, the sections were rinsed with PBS after being incubated at room temperature for 1 hour. Sections were then rinsed with PBS and incubated with secondary antibodies (1:200 dilution) for 2 h. Finally, the sections were washed with PBS, then incubated with DAPI solution for 10 min to show the nuclei. The sections were covered with Fluoromount\u0026trade; Aqueous Mounting Media and imaged with a Nikon Eclipse 90i fluorescence microscope (Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll quantitative measures are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SE. Statistical significance between groups was determined using one-way analysis of variance (ANOVA), followed by a post-hoc Tukey test for multiple comparisons with GraphPad Prism (8.0.2). Significant differences were defined by p-values of 0.05 or lower.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCharacterization of synthesized NarNPs\u003c/h2\u003e\u003cp\u003eThe particle size of the NarNPs assessed via DLS was approximately 268.16\u0026thinsp;\u0026plusmn;\u0026thinsp;4.67 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), indicating that their size (hydrodynamic radii) was within the acceptable range of nanoparticles. The zeta potential of the NarNPs was 21.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), indicating that the nanoparticle has a negative surface charge due to the terminal carboxylic groups of the PCL polymer. The encapsulation efficiency of narigenin in NarNPs was approximately 45.88\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). In addition, TEM images revealed that the actual particle size of NarNPs was in the range of 64.7\u0026ndash;176 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The characterization of NarNPs by AFM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) revealed a good nanoparticle population within a similar range that was also visualized in the 3D image (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eNarNPs alleviate seizure activity in epileptic rats\u003c/h2\u003e\u003cp\u003eEpileptiform seizure activity was examined in the cortex of experimental rats by analyzing the morphology of EEG waves and spectral power analysis of γ-oscillations. As depicted in the representative image of EEG samples of 30 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). We found that EEG paroxysms were composed of single spikes, multiple spikes and spike wave complexes with abnormal patterns of high frequency and amplitude. These EEG paroxysms suggest the abnormal firing of neurons due to their hyperactivity, leading to epileptiform seizure activity. NAR and NarNPs treatment suppressed the epileptiform seizure activity. The EEG paroxysms of epileptic rats administered NAR and NarNPs showed a remarkable decline in epileptiform seizure activity, as evidenced by the short duration of EEG paroxysms. Moreover, the amplitude of these spikes and spike waves was lower than epileptic rats without any treatment. These findings indicate the antiseizure effect of NAR and NarNPs in iron-induced experimental epilepsy. The effect of NarNPs was better compared with NAR-treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), suggesting the enhanced antiseizure potential of NarNPs. We did not observe any remarkable change in the EEG samples of NarNPs treated sham rats with sham rats. The EEG recordings of the rats from both groups revealed no epileptiform activity; thus observed no evidence of the occurrence of epileptic seizures.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, the quantitative extent of epileptiform seizures was examined via spectral power analysis of γ-oscillation (30\u0026ndash;50 Hz) of EEG signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We found a significant increase in the percentage of mean spectral power of epileptic rats (55.69\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79%) as compared to sham controls (40.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These changes are in line with the observed epileptiform activity in EEG patterns. The oral administration of NAR (48.75\u0026thinsp;\u0026plusmn;\u0026thinsp;97%, NS) and NarNPs (46.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reduced the mean spectral power as compared to epileptic rats. However, the reduction was greater in NarNPs-administered epileptic rats as compared to NAR only administered. Similar to EEG patterns, spectral analysis of γ-oscillation did not reveal any significant change in sham rats administered to rats. NarNPs compared to sham controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eNarNPs attenuate glial activation in epileptic rats\u003c/h2\u003e\u003cp\u003eThe results displayed a lower percentage of GFAP-positive cells, resembling resting astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) in the cortex (8.26\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) of sham rats. The epileptic rats showed thickened activated astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) along with a significantly increased percentage (15.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The administration of both NAR and NarNPs to epileptic rats significantly restored the percentage of astrocytes. We observed no significant variations in the morphology and percentage of astrocytes between sham rats administered NarNPs and sham controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, the appearance of IBA1 positive cells resembled that of resting microglia (ramified shape), and were lower in percentage (7.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) in sham rats. The percentage of IBA1 positive cells (15.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) was significantly high and was activated (ameboid shape, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) in epileptic rats as compared to sham controls, whereas both NAR and NarNPs treatment significantly reduced the percentage of microglia in epileptic rats. No significant change was found in the percentage or morphology of microglia between sham rats administered NarNP and sham controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eNaringenin has a strong binding affinity with TNF-α\u003c/h2\u003e\u003cp\u003eThe molecular docking study was performed to determine the affinity of naringenin with TNF-α. The analysis of the docked complex of TNF-α and naringenin showed the formation of three H-bonds, one electrostatic interaction and three hydrophobic interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The binding energy for the interaction between TNF-α and naringenin was \u0026minus;\u0026thinsp;7 kcal/mol.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eNarNPs reduce TNF-α expression in epileptic rats\u003c/h2\u003e\u003cp\u003eThe immunostaining results of TNF-α expression has been presented in the Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. Epileptic rats showed higher TNF-α expression (3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 times, p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb) than sham controls. These findings indicate that activation of astrocytes and microglia may trigger the production of TNF-α. Oral administration of NAR and NarNPs to epileptic rats restored TNF-α expression in the cortex. TNF-α expression was lowered to 1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 1.21\u0026thinsp;\u0026plusmn;\u0026thinsp;.56 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), respectively. We observed no significant difference between NarNPs administered to sham rats and sham controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBiodegradable polymer-based nanoformulations are popular in neuroscience research due to their ability to cross the BBB, lower drug requirements and slow and sustained release [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the current study, NarNPs were synthesized via the nanoprecipitation method and their physicochemical characteristics were examined by DLS, TEM and AFM. The nanoformulation enabled the rapid and dependable creation of a scaffold with a homogenous and spherical shape that was evident from the AFM images. Several previous investigations have also produced such PCL-based nanoformulation by this approach [\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We promise that oral administration of NarNPs can enable delivery of naringenin to the brain and protection from epilepsy-associated damage.\u003c/p\u003e\u003cp\u003eHence, we evaluated the effect of NarNPs on EEG patterns and the spectral power analysis of γ-oscillations in epileptic rats. EEG signals were analyzed to ascertain the presence of epileptic seizures and identify the brain regions where seizures originate [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The γ-oscillations (35\u0026ndash;45 Hz) of EEG signals are associated with informational processing and cognitive functions [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In humans, γ-wave frequency is majorly associated with the somatosensory cortical region [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In our study, EEG signals from epileptic rats consisted of sharp waves and spike wave complexes of high frequency and amplitude. These unusual trends are consistent with prior studies[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] which seem to imply the occurrence of epileptiform seizures in iron-injected rats, which signifing the development of electric field potential in cortical neurons. The results also showed a considerable increase in the percentage spectral power of γ-waves. A prior study found that kainic acid boosts β and γ-oscillations in the neocortex during spike-wave discharges [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. A recent study[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] found that amplitude envelope correlations from interictal EEG signals show frequency-dependent increase γ frequency bands (40-200Hz) in epileptogenic cortex.\u003c/p\u003e\u003cp\u003eThe epileptic rats treated with NAR and NarNPs exhibited fewer single spikes, polyspikes, and spike waves, along with a reduced spectral power of γ-oscillations. These effects were more evident in the NarNP group compared to NAR group, indicating an improved antiepileptic potential of naringenin in nanoformulation. Earlier studies have shown that modification of naringenin 7-O-methyl ether improved its therapeutic efficacy in PTZ-induced seizures [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Similarly, synthesised naringenin-conjugated graphene oxide nanoparticles also suppressed convulsant behavior in adult zebrafish [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAstrocytes and microglia are crucial and contribute to cellular homeostasis, immune response and maintenance of the BBB. Astrocytes are a common type of glial cell vital for maintaining brain homeostasis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Reactive astrocytes increase the expression of complement cascade genes and produce proinflammatory mediators such as IL-1β, TNF-α, and NO [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Microglia are ubiquitously distributed in the brain and are implicated in the modulation of neuroinflammatory response. Stimuli such as brain injury, infection, and oxidative stress reactivate microglia, which in turn produce proinflammatory cytokines and chemokines. Both clinical and experimental research showed that these brain cells play a key role in the pathophysiology of neurological disorders, including epilepsy [\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Astrocytes can precipitate seizures by releasing TNF-α themselves or in combination with glutamate [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. As, they are known to inhibit the release of the GABA precursor (glutamine) or promote the loss of glutamine synthetase, which in turn increases synaptic glutamate levels while decreasing GABA levels [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe data revealed a higher number of astrocytes in the cortex of epileptic rats, which were structurally similar to reactive astrocytes, implying that they are activated in PTE. Similar to astrocytes, the number of microglia was also amplified, along with their morphological similarity to reactive microglia (ameboid shape). Previously, several animal studies have examined the expression patterns and examined the morphological features of astrocytes and microglia in different epilepsy models[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and sequential activation of glial cells is implicated in epileptogenesis in various experimental models of epilepsy, including status epilepticus [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. During KA-induced status epilepticus, microglia rapidly become reactive and release TNF-α, which affects astrocytes, disrupts gap junctions, and contributes to temporal lobe epilepsy without influencing the progression of hippocampal sclerosis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Prior evidence demonstrated that TNF-α contributes to epilepsy progression through astrocyte-linked mechanisms, including apoptosis, impaired synaptic function, and increased glutamate production [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Our study presented that epileptic rats treated with NAR and NarNPs had fewer astrocytes and microglia, indicating inhibition of glial activation. These findings are consistent with earlier studies indicating that naringenin suppresses neuroprotein and activates glial cells [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. A recent study found that lower microglial activation coincided with a shift in phenotype from proinflammatory M1 to anti-inflammatory [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. These data suggest that attenuation of glial cells by naringenin may be responsible for reducing seizures in PTE rats.\u003c/p\u003e\u003cp\u003eThe release of cytokines like TNF-α leads to neuronal hyperexcitability by inhibiting the reuptake of glutamate by astrocytes, thus increasing glutamatergic neurotransmission, reducing GABA-mediated inhibition and decreasing chloride currents and expression of GABA-A receptors [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Molecular docking revealed naringenin's high affinity for TNF-α known as key regulator of the pro-inflammatory cytokines cascade. Earlier reports also showed modulation of neuroinflammatory response by naringenin and suggested that naringenin can promote lysosome-dependent cytokine degradation [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Our study found increased TNF-α levels in the brains of epileptic rats, indicating that activation of astrocytes and microglia may have resulted in TNF-α release. The oral administration of NAR and NarNPs restored the expression of TNF-α, where NarNPs treatment had a greater effect compared to NAR treatment. These results indicate enhanced anti-inflammatory effect in NarNPs. Previously, a study formulated gelatin-coated PCL nanoparticles and reported their anti-inflammatory effect on oxygen-glucose-deprived human mesenchymal stem cells [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Based on our findings, we hypothesize (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) that alleviation of seizure activity in epileptic rats by NarNPs may be due to reduced glial activation and TNF-α production in the cortex of PTE rats.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, the study concluded that encapsulating naringenin in PCL nanoparticles significantly increased its efficacy. The formulated NarNPs showed a significant inhibitory effect on seizure activity compared to free naringenin. The antiseizure activity of NarNPs was mediated via alleviating glial activation and lowering the release of TNF-α, a proinflammatory mediator.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of interest\u003c/h2\u003e\u003cp\u003eThe authors declare that there is no competing interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics statement\u003c/h2\u003e\u003cp\u003e All procedures involving animals were duly approved by the Institutional Animal Ethics Committee of JNU, New Delhi (IAEC code: 16/2021) and performed as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe financial support was provided by the Indian Council of Medical Research (5/5\u0026ndash;5/4GIA/Trauma/2020-NCD-I) to DS and SS. Senior Research Fellowship was awarded by the Department of Biotechnology and Council of Scientific and Industrial Research to SSR (DBT/JRF/BET-18/1/2018/AL/181) and SB (09/0263(11758)/2021-EMR-I), respectively. DHR-Young Scientist grant was sanctioned to CP (R.12018/04/2024-HR) by the Department of Health Research.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDS, JB and SSR conceptualized the idea. SSR synthesized nanoparticles, performed electrophysiology, in silco and immunofluorescence studies. SSR and SB characterized the nanoparticles. SSR synthesized the initial draft. SSR and CP performed the animal experiments and drafted the manuscript. JB, SS and DS critically reviewed and edited the final manuscript. All authors have approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge the Central Instrument Facility, School of Life Sciences, and Central Laboratory Animal Resources, JNU, for facilitating the experimentation. The authors also acknowledge the Advanced Instrumentation Research Facility, JNU, for providing instrument facilities.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data will be available upon a reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmudhan S, Gururaj G, Satishchandra P (2015) Epilepsy in India I: Epidemiology and public health. Ann Indian Acad Neurol 18:263. https://doi.org/10.4103/0972-2327.160093\u003c/li\u003e\n\u003cli\u003eAli I, Silva JC, Liu S, et al (2019) Targeting neurodegeneration to prevent post-traumatic epilepsy. 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Pharmacol Res 135:122\u0026ndash;126. https://doi.org/10.1016/j.phrs.2018.08.002\u003c/li\u003e\n\u003cli\u003eAhmad A, Fauzia E, Kumar M, et al (2019) Gelatin-Coated Polycaprolactone Nanoparticle-Mediated Naringenin Delivery Rescue Human Mesenchymal Stem Cells from Oxygen Glucose Deprivation-Induced Inflammatory Stress. ACS Biomater Sci Eng 5:683\u0026ndash;695. https://doi.org/10.1021/acsbiomaterials.8b01081\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Epilepsy, seizures, naringenin, PCL nanoparticles, neuroinflammation, astrogliosis","lastPublishedDoi":"10.21203/rs.3.rs-7630794/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7630794/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePost-traumatic epilepsy (PTE) is the cosequence of traumatic brain damage (TBI), which poses an important health risk for the human population. The underlying mechanism of PTE is complex and appears to be linked to various cellular processes, including oxidative stress and neuroinflammation. Over the years, several studies have reported that most of the available antiseizure medicines are ineffective in preventing PTE. As a result, there is an urgent need to search for alternative treatment options. Naringenin is a flavonoid with multiple pharmacological properties and has shown beneficial effects in several health issues, including neurological disorders. In this study, we investigated the effect of naringenin-loaded PCL nanoparticles (NarNPs) on neuroinflammatory responses in a PTE model. NarNPs were produced using the nanoprecipitation method, and their physicochemical properties were comprehensively examined. To induce epilepsy, FeCl\u003csub\u003e3\u003c/sub\u003e was injected intracortically to rats and naringenin (both free naringenin (NAR) and NarNPs) was administered orally 15 days post-surgery. Epileptic seizures were observed by electroencephalography (EEG) patterns and spectral power analysis of γ-waves. Immunofluorescence analysis was conducted to explore the disease-modifying potential of NarNPs. Our findings demonstrated that NarNPs distinctly reduced epileptiform seizure activity in epileptic rats. The study found that NarNPs lowered the expression of GFAP, IBA1, and TNF-α. The observed ameliorative effects were more pronounced in NarNP-treated rats than in the NAR-treated group. Overall, our data imply that NarNPs have significant antiseizure and disease-modifying potential by attenuating glial activation and TNF-α production in PTE rats.\u003c/p\u003e","manuscriptTitle":"Naringenin-loaded PCL nanoparticles attenuate glial activation and TNF-α expression in iron-induced post-traumatic epilepsy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 08:09:21","doi":"10.21203/rs.3.rs-7630794/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-13T16:06:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-13T15:24:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-12T15:47:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T09:51:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"76791348049783443846658847686416997027","date":"2025-09-22T13:47:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159305053793589267392802147780797025082","date":"2025-09-22T06:05:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148158166230177944887504248922825588917","date":"2025-09-22T04:45:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72282612809411116122270363285640979917","date":"2025-09-19T21:13:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-19T13:56:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-18T19:55:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-17T03:34:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2025-09-16T13:02:59+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"447d86bb-6978-4506-af8b-a4e9643b445f","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:03:42+00:00","versionOfRecord":{"articleIdentity":"rs-7630794","link":"https://doi.org/10.1007/s11064-026-04707-9","journal":{"identity":"neurochemical-research","isVorOnly":false,"title":"Neurochemical Research"},"publishedOn":"2026-02-23 15:58:40","publishedOnDateReadable":"February 23rd, 2026"},"versionCreatedAt":"2025-10-01 08:09:21","video":"","vorDoi":"10.1007/s11064-026-04707-9","vorDoiUrl":"https://doi.org/10.1007/s11064-026-04707-9","workflowStages":[]},"version":"v1","identity":"rs-7630794","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7630794","identity":"rs-7630794","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}