The neuroprotective effect of carbon dots from Crinis Carbonisatus (carbonized human hair) against epilepsy

The neuroprotective effect of carbon dots from Crinis Carbonisatus (carbonized human hair) against 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 The neuroprotective effect of carbon dots from Crinis Carbonisatus (carbonized human hair) against epilepsy 杰 胡, 凯 程, 小科 王, 易凡 张, 新荣 田, 燕 黄, 晨心 他, 西文 张, 彭 锹, 金宇 马, 小汉 库, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6202061/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 Background Epilepsy is a brain neurological disease with a high incidence and recurrent attacks. Currently, there is still a lack of simple, long-term prevention and control measures. Crinis Carbonisatus (named “Xue-yu-tan” in Chinese) is forged from healthy human hair and is widely used in traditional Chinese medicine to treat epilepsy, hemostasis, stroke and other diseases. Previous studies have successfully isolated and characterized carbon dots derived from Crinis Carbonisatus (CrCi-CDs), confirming their pharmacological activity in treating ischemic stroke and demonstrating neuroprotective effects against neural injury. Building on these findings, this study aims to explore the potential therapeutic effects of CrCi-CDs on acute epilepsy. Methods Clean, healthy human hair was calcined in a muffle furnace at 350°C for 1 hour and then decocted in deionized water and filtered to obtain a solution of CrCi-CDs. We used Pentylenetetrazole (PTZ), Pilocarpine (PILO) and Penicillin (PNC) to simulate clinical epileptogenic factors to establish three acute epilepsy models in mice and investigate the anti-epileptic effect of CrCi-CDs. We explored whether CrCi-CDs can reduce nerve excitability, improve nerve tissue inflammation, and oxidative stress levels, thereby reducing nervous system damage and improving epileptic symptoms. Based on the classic neuronal apoptosis pathway, we preliminarily explored the anti-epileptic mechanism of CrCi-CDs. Results In this study, we successfully isolated CrCi-CDs by referring to the previous method. CrCi-CDs is spherical in shape, well dispersed in aqueous solution, with uniform and consistent particle size distribution, and contains a large number of hydroxyl, amino and carbonyl/carboxy groups on the surface. The antiepileptic effects of CrCi-CDs were evaluated using Pentylenetetrazole (PTZ), Pilocarpine (PILO) and Penicillin (PNC)-induced epileptic mouse models. After CrCi-CDs intervention, the latency period of epileptic mice in each group was prolonged, and their spatial learning and memory abilities were improved. In addition, nerve damage in the hippocampus of epileptic mice was reduced by the CrCi-CDs intervention, the imbalance of neurotransmitters such as Glutamic acid (GLU) and Gamma-Aminobutyric acid (GABA) was regulated, the levels of inflammatory factors such as Interleukin-1β(IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-α(TNF-α) and Interleukin-18 (IL-18), and oxidative stress such as malondialdehyde (MDA) and superoxide dismutase (SOD) was improved. The above results showed that the improvement effect of high-dose CrCi-CDs was the most significant. Initial mechanistic investigations suggest that CrCi-CDs may ameliorate epileptic damage by suppressing neuronal apoptosis in brain tissue through modulation of the Bax/Bcl-2/Caspase-3 signaling pathway. Conclusions CrCi-CDs show significant anti-epileptic potential, which may be achieved through multiple pathways including regulating neurotransmitter balance, inhibiting neuroinflammation and oxidative stress. This study lays the foundation for the clinical application of CrCi-CDs and further drug development. Epilepsy Carbon Dots Apoptosis Neuroprotection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Introduction Epilepsy is a recurrent neurological disorder characterized by recurrent, transient, and stereotyped episodes of central nervous system dysfunction [ 1 ] . This condition not only induces neurobiological alterations but also exerts effects on cognitive, psychological, and social dimensions of health. The World Health Organization (WHO) has identified epilepsy as a priority neurological and psychiatric disorder within global prevention and treatment initiatives [ 2 ] . Currently, epilepsy affects estimated 50 million individuals worldwide, with an overall lifetime prevalence of 7.6% and a prevalence of active epilepsy at 6.38%. Despite its widespread impact, nearly 80% of individuals with epilepsy globally lack access to antiepileptic medication, with the majority of these cases concentrated in developing nations [ 3 ] . The prevailing consensus regarding the specific pathogenesis of epilepsy centers on the disruption of the equilibrium between excitation and inhibition within the central nervous system (CNS), which is closely associated with aberrant neurotransmitter signaling and neuroinflammatory processes [ 4 – 6 ] . Among neurotransmitters, amino acid neurotransmitters, particularly glutamic acid (Glu, an excitatory neurotransmitter) and gamma-aminobutyric acid (GABA, an inhibitory neurotransmitter), are most prominently implicated in epileptic seizures [ 7 ] . An imbalance in Glu/GABA levels—characterized by an elevation in Glu and a reduction in GABA—can precipitate neuronal hyperexcitation, induce excitatory neurotoxicity, result in neuronal damage, and ultimately provoke acute epileptic episodes [ 8 – 9 ] . Additionally, neuroinflammation during epileptic seizures represents a critical pathological factor. Seizures have been shown to elevate the levels of pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and transforming growth factor-β1 (TGF-β1). The resulting inflammatory milieu exacerbates CNS injury, thereby contributing to the initiation and progression of refractory epilepsy [ 10 – 12 ] . In the management of epilepsy, although a variety of therapeutic modalities—including pharmacological interventions, surgical procedures, lifestyle adjustments, and gene-based therapies—are available, monotherapy with antiepileptic drugs (AEDs) remains the cornerstone of treatment [ 13 – 14 ] . Different classes of AEDs exert their antiepileptic effects by targeting diverse pathogenic mechanisms underlying epilepsy, such as ion channel modulation, synaptic transmission regulation, and inflammatory factor inhibition [ 15 – 16 ] . However, prolonged administration of AEDs is associated with a spectrum of adverse effects, encompassing neurological disturbances, visual and auditory deficits, and hepatorenal toxicity. Consequently, there is an imperative need to develop a novel, sustainable, and minimally invasive anti-epileptic therapeutic strategy [ 17 ] . In recent years, carbon dots (CDs)—nanostructures characterized by a carbon-based core, sub-10 nm dimensions, and surface passivation groups—have attracted considerable interest within the field of nanomaterials [ 18 – 19 ] . These CDs have been extensively explored for diverse applications spanning bioimaging, sensor development, catalytic processes, energy conversion/storage systems, and biomedical technologies [ 20 – 21 ] . Our investigation has revealed the presence of a distinct category of CDs in “Tan Yao”, a traditional Chinese medicinal preparation, wherein natural herbal constituents facilitate the in situ synthesis of CDs during the preparation process [ 22 ] . Following this discovery, multiple distinct classes of CDs were successfully extracted and isolated from varied herbal precursors. These CDs exhibit notable pharmacological activities, including immunomodulatory functions, anti-inflammatory efficacy, and hemostatic properties [ 23 – 30 ] . Crinis Carbonisatus (CrCi), referred to as “Xue-yu-tan” in traditional Chinese medicine (TCM), is a highly significant therapeutic substance within the TCM pharmacopeia. It is synthesized through a process involving cleaning, drying, and carbonization of healthy human hair. As shown in Fig. 1 A, the silk manuscript excavated from a Han Dynasty tomb demonstrates the use of calcined hair (prototype of “Xue-yu-tan”) in emergency hemostasis. Figure 1 B reveals that as early as the Eastern Han Dynasty, Shennong Bencaojing (Divine Farmer's Materia Medica), a pharmacological compendium, documented the application of “Xue-yu-tan” in treating various ailments including hemorrhage, stroke, and epilepsy. The medicinal application of “Xue-yu-tan” has been extensively documented in classical TCM literature spanning over two millennia, with references to its therapeutic efficacy appearing in medical texts from various Chinese dynasties. In prior research, our team successfully isolated and characterized carbon dots (CDs) derived from “Xue-yu-tan” (CrCi-CDs), thereby elucidating the therapeutic potential of Blood Residue Charcoal in the context of ischemic stroke [ 33 ] . Preliminary experimental findings indicate that CrCi-CDs modulate cerebral neurotransmitter levels and exhibit a spectrum of pharmacological activities, including sedative, analgesic, and anxiolytic effects. Furthermore, CrCi-CDs demonstrate pronounced neuroprotective properties, attenuating inflammatory responses, excitotoxic damage, and neuronal apoptosis subsequent to cerebral ischemia. These empirical observations substantiate the hypothesis that CrCi-CDs may possess antiepileptic efficacy, potentially mediated through mechanisms analogous to those implicated in stroke therapy. In this study, we used three distinct acute epilepsy models to investigate the potential of CrCi-CDs in mitigating neuroinflammation and oxidative stress induced by epileptic seizures. Furthermore, we examined whether CrCi-CDs exhibit antiepileptic properties by attenuating neuronal damage and cell loss. The equilibrium between neural excitation and inhibition was assessed through the quantification of seizure duration and the measurement of amino acid neurotransmitter levels. Spatial learning and memory deficits were evaluated using the Morris water maze behavioral paradigm. Neuroinflammatory damage was analyzed by measuring the concentrations of inflammatory mediators and conducting histopathological examinations of brain tissue sections. These comprehensive assessments were conducted to elucidate the antiepileptic efficacy of CrCi-CDs and to explore the underlying mechanisms contributing to their potential therapeutic effects. Materials and methods Chemicals Dialysis membranes with a molecular weight of 1000 Da were purchased from Beijing Ruida Henghui Technology Development Co., Ltd. (Beijing, China). Pentylenetetrazol (PTZ) and Sodium Valproate (VPA) was purchased from Sigma-Aldrich Co., Ltd. (USA), Pilocarpine (PILO) was purchased from APExBIO Technology Co., Ltd. (USA), Penicillin sodium for injection (PNC) was purchased from North China Pharmaceutical Co., Ltd.(Shijiazhuang, China), and Other analytical reagents were purchased from Wuhan Saiwei'er Biological Technology Co., Ltd. (Wuhan, China). All the experiments were performed using deionized water (DW). Animals Animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals that was approved by the Committee of Ethics of Animal Experimentation of the Beijing University of Chinese Medicine. Male C57BL/6 rats (weighing 20.0 ± 2.0 g) were purchased from Beijing Sibeifu Biotechnology Co., Ltd. (Beijing, China). These animals were housed under the following conditions: temperature, (24.0 ± 1.0) °C; relative humidity, 55–65%, and a 12-h light/dark cycle, with ad libitum access to food and water. Synthesis of CrCi-CDs The method for the separation and extraction of CrCi-CDs is coincident to our previous work [ 31 ] . In brief, after cleaning the collected healthy human hair to remove grease, it is dried in an oven at 60°C for 24 hours. After drying, the hair is calcined by a muffle furnace. The calcination conditions are as follows: a 5-minute ramp to 70°C, held for 20 minutes, then a 25-minute ramp to 350°C, and held for 1 hour. Once the hair has cooled to room temperature, it is pulverized using a small high-speed pulverizer. Subsequently, DW is added and the mixture is heated in a water bath at 100°C for 1 hour. The resulting solution is first filtered through a 0.22 µm organic membrane filter, and then dialyzed using a 1000-Da dialysis membrane for 72 hours to obtain the CrCi-CDs solution. Sample characterization Photoluminescence experiments were conducted with a Shimadzu RF5-5301 PC spectrofluorimeter (Shimadzu, Japan). UV-vis absorption spectra were obtained using a TU-1991 UV-vis spectrophotometer. Fourier transform infrared spectroscopy (FTIR) was measured in the range of 500–4000 cm − 1 using a Nicolet 6700 FTIR spectrophotometer. Transmission electron microscopy analyses to study morphology and mean diameter of the resultant samples were carried out on a JEM-2100F (FEI, USA), operating at an accelerating voltage of 200kV. Experimental protocol Experimental protocol Because this experiment adopted three acute epilepsy mouse models, three batches of 144 C57BL/6 male mice were used. After weighing 48 mice in each batch, they were randomly divided into 6 groups (8 mice in each group), namely, blank group (Control), model group (Model), VPA group (VPA), CrCi-CDs high (High), medium (Medium) and low (Low) dose groups. The blank group and model group were given DW by gavage, the positive drug group was given VPA by gavage (200 mg/kg), and the CrCi-CDs high, medium and low dose groups were given CrCi-CDs solution by gavage (Dosing concentrations were 6 mg/kg, 3 mg/kg, and 1.5 mg/kg, respectively), and the drugs were given continuously for 7 days. Animals modeling On the 7th day of the experiment, 1 hour after the administration of each group, the first batch of mice except the blank group were intraperitoneally injected with PTZ (65 mg/kg), and the blank group was intraperitoneally injected with an equal amount of saline. Similarly, the second batch of mice and the third batch were intraperitoneally injected with PILO 280 mg/kg (i.e., 7 million units/kg) and PNC 4.2 g/kg respectively. Behavioral observation After the injection, we placed the mice in a transparent box for observation for 30 min. The epileptic seizure grade of the mice was evaluated according to the Racine scale [ 34 ] , and the latency to clonic convulsions (equivalent to Racine scores of 1–3) and tonic-clonic seizures (equivalent to Racine scores of 4–5) were recorded for each mouse. Morris water maze test PTZ-induced epileptic mice from each group were selected for the Morris water maze test. The water maze apparatus comprised a circular pool measuring 90 cm in diameter and 50 cm in height, along with a platform measuring 9 cm in diameter and 30 cm in height. The safety platform was positioned at the midpoint of the outer third of a designated quadrant within the water maze. Four plastic cards of distinct shapes and colors were affixed to the walls of the four quadrants to serve as spatial reference cues. The place navigation test was conducted daily at 9:00 AM over four consecutive days. Each mouse underwent four training sessions per day. During each session, a quadrant was randomly chosen, and the mouse was introduced into the pool facing the wall. The latency for the mouse to locate and ascend the submerged safety platform was recorded within a 90-second time frame. On the fifth day, the spatial exploration test was performed. The submerged safety platform was removed, and the quadrant opposite to its original location was designated as the entry point for the mice. The number of crossings through the area previously occupied by the safety platform was recorded over a 60-second period. HE stain ༆ Nissl stain The fixed brain tissue was dehydrated, transparent, waxed, and embedded, and then coronal sections were made with a thickness of 3 µm. Subsequently, xylene and ethanol were used for dewaxing, and HE staining and Nissl staining were performed to observe the pathological conditions of the neuronal cells in the hippocampus of the mouse brain tissue under an optical microscope. Enzyme linked immunosorbent assay The frozen brain tissue was removed and ground at low temperature, then cooled and centrifuged to obtain the brain tissue homogenate supernatant; the corresponding ELISA kits were used to detect the levels of amino acid neurotransmitters (GABA, Glu), inflammatory factors (TNF-α, IL-1β, IL-6, IL-18), and oxidative stress indicators (SOD, MDA) in the brain tissue. Western blot analysis Total protein from mouse brain tissue was extracted with cold RIPA buffer containing 1% protease inhibitors, and its concentration was quantified using a bi-creatine (BCA) kit. Equal amounts of protein from brain tissue were then separated by SDS-PAGE electrophoresis, and then transferred to NC membranes after blocking with 5% skim milk at room temperature for 1.5 h. Proteins were then detected overnight at 4°C using the corresponding primary antibodies (GAPDH, Bax, Bcl-2, and Cleaved-caspase-3), followed by incubation with secondary antibodies for 1 h at room temperature. After washing with TBST, the target proteins in the NC membrane were observed using an automated chemiluminescence image analysis system using enhanced chemiluminescence. Results Characterization of CrCi-CDs As shown in Fig. 2A, TEM observation shows that CrCi-CDs are spherical, well dispersed in aqueous solution, and have uniform particle size distribution, ranging from 2–3 nm, with an average particle size of less than 10 nm (Fig. 2B). The Tyndall effect of its aqueous solution indicates that the CrCi-CDs aqueous solution is a colloidal solution, with relatively uniform CrCi-CDs particle sizes and a certain degree of metastability. Figure 2B shows that there is an obvious lattice in a single spherical nanoparticle, and the lattice spacing of CrCi-CDs is 0.228 nm, which is consistent with the crystal plane of graphene C. Figure 2C shows that the XRD spectrum of CrCi-CDs had distinct diffraction peaks (2θ = 29.399°), indicating that CrCi-CDs were attributed to amorphous carbons arranged in a considerably random fashion.The UV-Vis spectrum of CrCi-CDs shows a smooth curve with a broad and weak absorption region around 320 nm (Fig. 1D), which may be related to the n-π* transition of the C = O bond in CrCi-CDs or the π-π* transition of the C = C bond. Figure 2E is the fluorescence excitation emission spectrum of the CrCi-CDs aqueous solution. At an excitation wavelength of 334 nm, the emission peak of CrCi-CDs is located at around 402 nm. Under irradiation with a 365 nm ultraviolet lamp, the CrCi-CDs solution emits bright blue fluorescence. The FTIR absorption spectrum of CrCi-CDs (Fig. 2F) shows that the absorption peaks of different functional groups appear at 3440 cm − 1 , 2924 cm − 1 , 1630 cm − 1 , 1401 cm − 1 , 1262 cm − 1 , 1101 cm − 1 , and 863 cm − 1 , respectively. The strong absorption peak at 3440 cm − 1 may be related to the stretching vibration of O-H bond and N-H bond, the absorption peak at 2924 cm − 1 may come from the stretching vibration of -CH bond in -CH3 and -CH2 groups, the absorption peak at 1630 cm − 1 indicates the presence of C = O bond in the surface group of CrCi-CDs, the absorption peak at 1401 cm − 1 may be caused by the stretching vibration of C–N bond, the bending vibration of N–H bond and C–H bond, the absorption peak at 1262 cm − 1 indicates the presence of C-OH bond, the absorption peak at 1101 cm − 1 is related to the vibration of C-O-C bond, and the absorption peak at 863 cm − 1 may come from the bending vibration of C-H bond on benzene ring. Therefore, we believe that the surface of CrCi-CDs has abundant hydroxyl, amino and carbonyl/carboxylate groups. XPS spectra can be used to analyze the elemental composition and coordination information of CrCi-CDs. As shown in Fig. 3A, the full scan XPS spectrum reveals that the nano-component mainly contains C (285.08 eV), N (400.08 eV), and O (532.08 eV) elements, accounting for 70.7%, 6.13%, and 23.17% respectively. The detailed coordination characteristics of each element are analyzed in Fig. 3B, 3C, and 3D.In the high-resolution XPS spectrum of C (Fig. 3B), four coordination modes of C are observed: C-C (284.8 eV), C-N (285.3 eV), C-O (286 eV), and C = O (287.7 eV). In the high-resolution XPS spectrum of O (Fig. 3C), the peaks at 531.0 eV and 532 eV correspond to C-O and C = O respectively. Similarly, in the high-resolution XPS spectrum of N (Fig. 3D), the peaks at 399.5 eV and 400.5 eV indicate the presence of C-N and C = N bonds in CrCi-CDs. The characterization data of CrCi-CDs obtained in the above experiments are consistent with the results of our previous work, which proves that we have obtained a stable and uniform CrCi-CDs extraction and separation method. CrCi-CDs attenuate seizure severity in epileptic mice As shown in Fig. 4, compared with the model group, VPA and high, medium, and low doses of CrCi-CDs increased seizure latency in the three epilepsy mouse models to varying degrees, thereby mitigating seizure severity. Taking the PILO-induced epilepsy mouse model (Fig. 4C and D) as an example, the latency of clonic convulsions in the VPA group (944.30 ± 138.90 s), CrCi-CDs high-dose group (817.10 ± 221.00 s), and CrCi-CDs medium-dose group (636.40 ± 156.90 s) was significantly prolonged (p < 0.01). Figure 4D shows that, compared with the model group (1942.00 ± 245.50 s), VPA (3380.00 ± 367.70 s) and CrCi-CDs high-dose (3126.00 ± 464.80 s) intervention in the acute epilepsy mouse model significantly prolonged the latency of tonic convulsions in mice (p < 0.01). We also observed similar trends in other epilepsy models, indicating that different doses of CrCi-CDs ameliorate epilepsy, with high doses of CrCi-CDs demonstrating the most significant therapeutic efficacy. CrCi-CDs improve spatial learning and memory in epileptic mice The Morris water maze is a classic experiment used to evaluate the spatial learning and memory abilities of experimental animals and is widely used in numerous research fields related to memory. Our study used the PTZ-induced epilepsy mouse model to evaluate the effects of CrCi-CDs on spatial learning and memory in acute epileptic mice. As shown in Fig. 5A, the swimming trajectory diagram revealed that the blank group mice exhibited the best spatial learning ability, while the movement trajectory of the model group mice indicated severe impairment in their spatial memory ability. VPA and varying doses of CrCi-CDs intervention alleviated the impairment of spatial learning ability in epileptic mice to some extent, with VPA and high doses of CrCi-CDs demonstrating the most significant improvement. Figure 5B and 4C show that the escape latency and swimming distance of mice in the model group were significantly increased compared to those in the blank group ( p < 0.01), indicating that the spatial learning ability of mice in the model group was severely impaired. VPA and CrCi-CDs reduced the escape latency and swimming distance to varying degrees. With the intervention of VPA and high-dose CrCi-CDs, the escape latency of mice was significantly reduced from day 1 to day 4 compared to the model group ( p < 0.01). With the intervention of high-dose CrCi-CDs, the swimming distance of mice on days 3 and 4 was significantly reduced compared to the model group ( p < 0.01). These findings demonstrate that interventions with the VPA group and varying doses of CrCi-CDs alleviated the impairment of spatial learning ability in epileptic mice. The spatial exploration experiment conducted on the fifth day was used to evaluate the spatial memory ability of experimental animals. Figure 6A shows that the model group mice exhibited the poorest memory of the location of the underwater safety platform, and the mice predominantly searched in the quadrant opposite the platform. The blank group mice frequently crossed the location and quadrant containing the safety platform, indicating that their spatial memory was not impaired. Following intervention with VPA and varying doses of CrCi-CDs, the impairment of spatial memory in epileptic mice was alleviated to varying degrees. Figure 6B and 5C show that compared to the blank group (11.62 ± 3.39 s; 4.00 ± 0.89), the escape latency of the model group mice (36.75 ± 4.90 s) in the spatial exploration experiment was significantly increased ( p < 0.01), and the number of crossings in the platform area (1.50 ± 0.55) was significantly reduced ( p < 0.01), indicating that the epilepsy model mice had severe memory impairment. Following drug intervention, VPA significantly reduced the escape latency (20.86 ± 3.55 s) and increased the number of crossings in the platform area (3.67 ± 0.52, p < 0.01), and high-dose CrCi-CDs also significantly reduced the escape latency (22.63 ± 3.68 s, p < 0.01) and increased the number of crossings in the platform area (3.50 ± 1.05, p < 0.05). Effects of CrCi-CDs on brain tissue morphology in epileptic mice Figure 7A and 7B show that in the PTZ-induced epilepsy mouse model, the hippocampal tissue cells in the model group exhibited a more chaotic arrangement compared to those in the control group, including gaps, cell swelling, uneven cytoplasmic staining, vacuoles, and even cell rupture and loss. Following administration of VPA and varying doses of CrCi-CDs, the extent of tissue damage was alleviated to varying degrees. Figure 7C and 7D show that PTZ induced a significant reduction in Nissl bodies within the cytoplasm of hippocampal neurons in mice, and VPA and CrCi-CDs ameliorated this phenomenon. HE and Nissl staining of brain tissue sections from PILO- and PNC-induced epileptic mice (Figs. 8 and 9) also yielded similar results, indicating that CrCi-CDs can mitigate neural tissue damage in epileptic mice induced by PTZ, PILO, and PNC. CrCi-CDs ameliorate the level imbalance of Glu/GABA in brain tissue of epileptic mice The imbalance in GABA and Glu expression, which disrupts the brain's excitatory/inhibitory balance, is considered a central mechanism in epilepsy. Our experiments confirmed this phenomenon in the PTZ-, PILO-, and PNC-induced epilepsy models, as shown in Fig. 9. The levels of the excitatory neurotransmitter Glu (PTZ: 0.56 ± 0.05 µmol/g; PILO: 0.56 ± 0.08 µmol/g; PNC: 0.59 ± 0.03 µmol/g) in the brain tissue of the model group mice were significantly upregulated ( p < 0.01) compared to the control group, and the levels of the inhibitory neurotransmitter GABA (PTZ: 1.13 ± 0.28 µmol/L; PILO: 1.07 ± 0.23 µmol/L; PNC: 1.09 ± 0.20 µmol/L) were significantly decreased ( p < 0.01). Following intervention with varying doses of CrCi-CDs, we observed that the dysregulated Glu and GABA levels were normalized. Using the PTZ-induced epilepsy mouse model (Fig. 10A and 10B) as an example, high-dose CrCi-CDs reduced Glu levels (0.42 ± 0.09 µmol/g, p < 0.05) and significantly increased GABA levels (1.67 ± 0.19 µmol/L, p < 0.01). In the PILO- and PNC-induced models, we also observed a similar trend (Fig. 10C and 10D, 10E and 10F), which aligned with the results of previous behavioral experiments. This demonstrates that high-dose CrCi-CDs are most effective in increasing GABA levels and reducing Glu levels in brain tissue, effectively ameliorating the Glu/GABA imbalance in epilepsy models induced by PTZ, PILO, and PNC. CrCi-CDs attenuate brain tissue inflammation in epileptic mice Key cytokines produced by glial cells and neurons activate multiple inflammatory pathways, resulting in detrimental synaptic alterations and neuronal overexcitation, which contribute to neurotoxicity and exacerbate epileptic symptoms [35] . As shown in Fig. 11, the levels of inflammatory factors in the brain tissue of the model group were significantly elevated compared to those in the control group, irrespective of whether the epilepsy model was induced by PTZ, PILO, or PNC. Using the PTZ-induced epilepsy model as an example (Fig. 11A), the levels of inflammatory factors in the model group (TNF-α: 124.96 ± 11.14 pg/mL; IL-1β: 20.56 ± 2.45 pg/mL; IL-6: 25.09 ± 2.18 pg/mL; IL-18: 29.14 ± 3.78 pg/mL) were significantly elevated compared to those in the control group (TNF-α: 69.78 ± 12.42 pg/mL; IL-1β: 12.00 ± 3.27 pg/mL; IL-6: 17.32 ± 2.87 pg/mL; IL-18: 16.08 ± 2.36 pg/mL; p < 0.01). After intervention with different doses of CrCi-CDs, the levels of each factor decreased to varying degrees, among which the high dose of CrCi-CDs was the most significant [TNF-α: 85.64 ± 17.46 pg/mL, IL-6: 19.64 ± 2.89 pg/mL, IL-18: 18.72 ± 4.09 pg/mL( p < 0.01), IL-1β: 14.80 ± 2.47 pg/mL( p < 0.05)] in the PTZ model.The high-dose CrCi-CDs in the PILO and PNC model also had similar effects. In the PILO model, like [IL-1β: 13.21 ± 3.22 pg/mL, IL-6: 19.69 ± 2.33 pg/mL( p < 0.01), TNF-α: 95.28 ± 13.18 pg/mL, IL-18: 19.08 ± 3.03 pg/mL( p < 0.05)], and in the PNC model, like [TNF-α: 86.18 ± 14.01 pg/mL, IL-6: 17.13 ± 3.67 pg/mL, IL-18: 18.49 ± 3.62 pg/mL( p < 0.01), IL-1β: 14.80 ± 2.47 pg/mL( p < 0.05)]. Therefore, we believe that CrCi-CDs can reduce the level of neuroinflammation, thereby improving the damage of neural tissue, and the high-dose group has the best effect among all dose groups. CrCi-CDs improve oxidative stress levels in brain tissue of epileptic mice During an epileptic seizure, excessive reactive oxygen species are produced. Excessive reactive oxygen species can damage cell membranes, leading to increased membrane permeability and inducing the production of harmful intermediates such as malondialdehyde (MDA). MDA can interact with DNA and proteins, inducing mutagenesis and reducing cell survival, thereby exacerbating cellular damage. Therefore, it is considered a reliable indicator of oxidative damage in epilepsy [36] . In addition, the reduced activity of antioxidant systems, such as superoxide dismutase (SOD), exacerbates the damage caused by reactive oxygen species to the brain [37] . As shown in Fig. 12, in the PTZ-, PILO-, and PNC-induced epilepsy mouse models, we observed that the levels of SOD in the brain tissue of the model group mice (PTZ model: 10.87 ± 2.61 ng/mL; PILO model: 10.69 ± 1.95 ng/mL; PNC model: 12.73 ± 2.97 ng/mL; p < 0.01) were significantly reduced, while the levels of MDA were significantly elevated (PTZ model: 4.42 ± 0.41 nmol/mL; PILO model: 4.00 ± 0.46 nmol/mL; PNC model: 4.02 ± 0.51 nmol/mL; p < 0.01) compared to the control group. Following intervention with VPA and varying doses of CrCi-CDs, the levels of SOD and MDA were normalized. In the PTZ model, compared to the model group, the MDA levels in the brain tissue of the VPA group and the high-, medium-, and low-dose CrCi-CDs groups (VPA: 2.51 ± 0.42 nmol/mL; high-dose group: 3.07 ± 0.52 nmol/mL; medium-dose group: 3.32 ± 0.48 nmol/mL; low-dose group: 3.21 ± 0.49 nmol/mL; p < 0.01) were significantly reduced. Compared to the model group, VPA and high-dose CrCi-CDs (VPA: 17.09 ± 3.41 ng/mL; high-dose group: 18.41 ± 3.02 ng/mL; p < 0.01) significantly increased the activity of SOD in the brain tissue of mice following intervention. The PILO and PNC models exhibited similar trends, demonstrating that varying doses of CrCi-CDs exert differing degrees of inhibitory effects on oxidative stress levels in the brain tissue of epileptic mice, with the high dose being the most effective. CrCi-CDs inhibited the expression of proteins in the neuronal apoptosis pathway During epileptic seizures, excessive neuronal excitation, coupled with concurrent neural tissue inflammation and dysregulated oxidative stress, can induce neuronal damage, ultimately leading to the apoptosis of nerve cells, as evidenced by the pathological sections presented in Figs. 7, 8, and 9. Within the Bcl-2 family, Bcl-2 and Bax serve as pivotal regulators in the process of cellular apoptosis. Additionally, the Caspase protein family plays a critical role in apoptosis, functioning as an essential component within the apoptotic cascade. Consequently, this study investigates the anti-apoptotic effects of CrCi-CDs on nerve cells in epilepsy models induced by PTZ, PILO, and PNC, with a specific focus on the Bax/Bcl-2/Caspase-3 signaling pathway. In the PTZ-induced epilepsy model, as shown in Fig. 13A, compared with the blank group, the levels of Bax (0.84 ± 0.04), the ratio of Bax/Bcl-2 (8.76 ± 0.85) and Cleaved-caspase-3 (0.63 ± 0.05) in the model group mice were significantly increased ( p < 0.01), and the expression of Bcl-2 (0.10 ± 0.01) was significantly decreased ( p < 0.01), indicating that a large number of neurons apoptosis occurred after PTZ-induced epileptic seizures in mice. Compared with the model group, high-dose CrCi-CDs significantly decreased the expression of Bax (0.32 ± 0.10), the ratio of Bax/Bcl-2 (0.66 ± 0.17), and the expression of Cleaved-caspase-3 (0.21 ± 0.02) in the mouse brain tissue ( p < 0.01), and upregulated the expression of Bcl-2 (0.49 ± 0.05) ( p < 0.01). As shown in Fig. 13B, in the PILO-induced model, high-dose CrCi-CDs influenced the expression of proteins (Bax: 0.35 ± 0.07, Bcl-2: 0.57 ± 0.04, Caspase-3: 0.22 ± 0.04, Bax/Bcl-2: 0.63 ± 0.17) significantly( p < 0.01). In the PNC-induced model, high-dose CrCi-CDs also showed similar effects (Bax: 0.31 ± 0.07, Bcl-2: 0.60 ± 0.06, Caspase-3: 0.26 ± 0.02, Bax/Bcl-2: 0.52 ± 0.12)(Fig. 13C) in the expression of proteins( p < 0.01). Therefore, after acute epileptic seizures induced by PTZ, PILO, or PNC in mice, the expression of pro-apoptotic proteins Bax and Cleaved-caspase-3 in brain tissue increased significantly, while the expression of anti-apoptotic proteins Bcl-2 was significantly downregulated. This indicates that neurons in the brain tissue were in a state of massive apoptosis. Following CrCi-CDs administration, the expression of pro-apoptotic proteins was significantly downregulated, while the expression of anti-apoptotic proteins was upregulated. These findings suggest that CrCi-CDs effectively inhibit neuronal apoptosis in the brain tissue of epileptic mice. Discussion In traditional Chinese medicine theory, “Xue-yu-tan” is a medicine with unique properties. The precursor of "Xue-yu-tan" is healthy human hair, which initially lacked medicinal properties but acquires therapeutic effects after processing. The anti-epileptic effect of "Xue-yu-tan" was documented as early as 2,000 years ago in traditional Chinese medicine texts. Nevertheless, the investigation into its active compounds and underlying mechanisms has remained ongoing, with no definitive conclusions reached to date. Advancements in nanotechnology have facilitated the isolation and extraction of a uniquely structured carbon dot, designated as CrCi-CDs, from "Xue-yu-tan." Subsequent analysis has revealed its distinctive characteristics as a nanomaterial and its potential therapeutic applications. Previous research has primarily established the therapeutic efficacy of CrCi-CDs in the context of ischemic brain injury. The findings revealed that CrCi-CDs effectively suppress the activity of excitatory neurotransmitters following cerebral ischemia and mitigate the extensive neural tissue inflammation induced by cerebral ischemia-reperfusion. These observations suggest that CrCi-CDs facilitate the release of inhibitory neurotransmitters while promoting the depletion of excitatory neurotransmitters in neurological disorders. Consequently, this leads to adaptive physiological changes, including reduced neural excitability and diminished neurotoxicity, thereby conferring a neuroprotective effect. Epilepsy, a neurological disorder characterized by a multifactorial pathogenesis, is fundamentally associated with the overexcitation of the central nervous system. This overexcitation, a hallmark of epileptic seizures, is predominantly attributed to an imbalance between excitatory and inhibitory neurotransmitters, coupled with neural tissue inflammation. Given these mechanisms, it is plausible that CrCi-CDs may also exert a protective influence against epileptic neural injury, analogous to their demonstrated efficacy in ischemic brain injury. In this study, three distinct epilepsy models were utilized to investigate the anti-epileptic properties of CrCi-CDs. Following a comprehensive evaluation of various epilepsy modeling methodologies, cost-effectiveness, and alignment with clinical practices, the acute epilepsy model was identified as an efficient, cost-effective, and clinically relevant approach for assessing the anti-epileptic efficacy of CrCi-CDs and conducting preliminary mechanistic investigations [ 38 ] . The mechanisms through which the three drugs induce epilepsy are depicted in Fig. 14 . PTZ, a GABA receptor antagonist, competes with GABA for binding to postsynaptic GABA receptors, thereby inhibiting neuronal chloride channel function, reducing the neuronal excitability threshold, and precipitating epileptic seizures. The PTZ-induced acute epilepsy model is highly valuable for the initial screening of anti-epileptic compounds and mechanistic research [ 39 ] . PILO exerts a pronounced excitatory effect on the central nervous system. Upon the induction of epileptic seizures, it elevates Glu levels in brain tissue, and the resultant epilepsy displays distinct temporal characteristics that closely mirror the progression of human temporal lobe epilepsy [ 40 – 41 ] . PNC, another widely utilized convulsant, is employed to induce acute epilepsy. It inhibits the postsynaptic inhibitory effects of GABA while promoting the release of presynaptic excitatory neurotransmitters, thereby facilitating epileptic activity. The PNC-induced epilepsy model is widely recognized as a robust representation of human absence epilepsy and is extensively employed in related research [ 42 – 43 ] . Consequently, for this experimental study, we selected the acute epilepsy models induced by PTZ, PILO, and PNC to evaluate the anti-epileptic effects of CrCi-CDs. Our observations demonstrate that CrCi-CDs significantly enhance the behavioral outcomes of epileptic mouse models, particularly by extending the latency of paroxysmal and tonic convulsions. These findings underscore the sedative properties of CrCi-CDs, consistent with prior research. Further validation was achieved through the analysis of brain tissue sections and neurotransmitter levels, which substantiated the results of behavioral experiments. In the Morris water maze experiment, PTZ-induced epileptic mice displayed a marked reduction in spatial learning and memory capabilities, indicative of damage to specific brain regions. CrCi-CDs ameliorate these cognitive deficits in a dose-dependent manner, with the most substantial improvements observed at higher doses. Additionally, CrCi-CDs effectively rectify the Glu/GABA imbalance in epileptic mice induced by PTZ, PILO, and PNC, demonstrating dose-dependent efficacy. Glu and GABA, essential amino acid neurotransmitters, play pivotal roles in neurotransmission. Elevated Glu levels coupled with diminished GABA concentrations induce excitotoxicity, leading to neuronal damage, cell loss, and the initiation of neuroinflammation and oxidative stress imbalance. CrCi-CDs attenuate epileptic seizures by mitigating neurotoxicity associated with neuronal overactivation. Furthermore, in epileptic seizures induced by PTZ, PILO, and PNC, neuroexcitotoxicity, resulting from neurotransmitter imbalance, not only precipitates neuronal destruction and loss but also initiates neuroinflammation and oxidative stress imbalance, thereby exacerbating neural tissue damage. Our observations indicate that CrCi-CDs effectively reduce the levels of inflammatory factors (TNF-α, IL-1β, IL-6, IL-18) in epileptic neural tissues, ameliorate oxidative stress by enhancing SOD activity and reducing MDA levels, and restore the balance between excitatory and inhibitory neurotransmitters. These findings align with prior evidence demonstrating the free radical scavenging capacity carbon dots derived from natural sources [ 44 – 46 ] and the neuroprotective efficacy of CrCi-CDs through the mitigation of neuroexcitotoxicity and inflammation [ 33 ] . In the context of neuronal destruction and apoptosis induced by neuroexcitotoxicity, the Bcl-2 family of proteins plays a pivotal role in the regulation of cellular apoptosis. The dysregulation of Bcl-2 family proteins is widely regarded as a primary mechanism underlying endogenous apoptosis [ 47 – 48 ] . The Bcl-2 family can be categorized into two distinct groups: anti-apoptotic members (e.g., Bcl-2, Bcl-XL, Bcl-W) and pro-apoptotic members (e.g., Bax, Bcl-Xs). Upon neuronal detection of external environmental stimuli, Bax becomes activated, resulting in alterations to the permeability of the mitochondrial outer membrane. This process facilitates the release of cytochrome C through the pores of the mitochondrial lipid membrane, thereby initiating downstream cascade reactions [ 49 ] . The Caspase family is intricately associated with cellular apoptosis, with Caspase-3 serving as the principal executor within this family. Caspase-3 not only represents the convergence point of multiple apoptotic pathways but also constitutes the terminal pathway for the execution of apoptosis, underscoring its critical role in the apoptotic mechanism. The activation of Caspase-3 signifies the irreversible progression of cellular apoptosis [ 50 ] . In this study, whether PTZ or PNC competitively binds to GABA receptors, or PILO binds to M1 muscarinic receptors, our findings demonstrate that all three epilepsy models ultimately result in heightened neuronal excitability, thereby activating the Bax/Bcl-2/Caspase-3 pathway and precipitating neuronal apoptosis. The intervention of CrCi-CDs has been shown to suppress the expression of pro-apoptotic proteins Bax and Caspase-3, while upregulating the expression of the anti-apoptotic protein Bcl-2, thereby mitigating neuronal apoptosis induced by epileptic seizures. This observation further underscores the broad neuroprotective efficacy of CrCi-CDs. The findings of this study indicate that CrCi-CDs are capable of modulating the imbalance between excitatory and inhibitory neurotransmitters, suppressing the activation of neuroinflammatory pathways, and conferring a neuroprotective effect in murine models of epilepsy. Future research endeavors will focus on elucidating the upstream molecular mechanisms underlying the neuroprotective properties of CrCi-CDs, optimizing their in vivo biodistribution, and investigating their therapeutic potential alongside other pharmacological activities. Presently, AEDs (antiepileptic drugs) represent the cornerstone of epilepsy management, primarily functioning through the reduction of neuronal hyperexcitability and the inhibition of aberrant electrical discharges. Nevertheless, the associated adverse effects, including neurological disturbances and hepatorenal toxicity, remain significant concerns. CrCi-CDs, which are derived from natural sources, demonstrate a favorable safety profile. Both prior research and the current study have established that CrCi-CDs exhibit a markedly enhanced neuroprotective efficacy, thereby suggesting their potential applicability as an adjunctive therapeutic agent in the clinical management of epilepsy. Conclusion In conclusion, leveraging insights from previous research, this study achieved the successful synthesis of CrCi-CDs utilizing human hair as a precursor material. The anti-epileptic efficacy of CrCi-CDs was systematically validated through the employment of three distinct murine epilepsy models, each induced by differing etiological factors. The therapeutic mechanisms underlying the anti-epileptic effects of CrCi-CDs appear to be attributable to the modulation of neurotransmitter homeostasis, the attenuation of neuroinflammatory responses and oxidative stress, and the suppression of apoptotic pathways within neural cells. Furthermore, this study substantiates the broad-spectrum neuroprotective properties of CrCi-CDs, thereby establishing a foundational basis for their prospective integration into clinical therapeutic strategies. Declarations Ethics approval and consent to participate The animal protocols were approved by the Committee of Ethics of Animal Experimentation of the Beijing University of Chinese Medicine. Consent for publication All the co-authors were aware of this submission and approve for publication. Competing interests The authors have no other relevant afliations or fnancial involvement with any organization or entity with a fnancial interest in or fnancial confict with the subject matter or materials discussed in the manuscript apart from those disclosed. Funding This work was supported by Beijing Municipal Natural Science Foundation - Haidian Original Innovation Joint Fund Sponsored Projects (L222132), National Natural Science Foundation of China (82204914) and Fundamental Research Funds for the Central Universities (China) (2024-JYB-JBZD-0400, 2024-JYB-JBZD-023, 2024-JYB-JBZD-045). Author Contribution Experiments were designed by YZ, HK and YZ, and conducted by JH, XKW, KC, YFZ and XRT. 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Asadi M, Taghizadeh S, Kaviani E, et al. Caspase-3: Structure, function, and biotechnological aspects. Biotechnol Appl Biochem. 2022;69(4):1633–45. Additional Declarations No competing interests reported. Supplementary Files GA.png 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-6202061","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":427764788,"identity":"b24960dd-7c1b-4021-87e1-a5bc0f7d3d98","order_by":0,"name":"杰 胡","email":"","orcid":"","institution":"Beijing University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"杰","middleName":"","lastName":"胡","suffix":""},{"id":427764792,"identity":"4a9112c8-166a-4e27-938f-ca4b18f2bf52","order_by":1,"name":"凯 程","email":"","orcid":"","institution":"Beijing University of Chinese 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10:12:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1133057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The silk manuscript inscribed with the \u003cem\u003eWushi'er Bingfang\u003c/em\u003e (Prescriptions for Fifty-Two Ailments) unearthed from the Mawangdui Han Tombs\u003csup\u003e[31]\u003c/sup\u003e. (\u003cstrong\u003eB) \u003c/strong\u003eThe facsimile reproduction of the Qing Dynasty compiled edition of the \u003cem\u003eShennong Bencaojing\u003c/em\u003e (Divine Farmer's Materia Medica), a pharmacological compendium from the Eastern Han period\u003csup\u003e[32]\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/c3102f8b57ac21aabb0354ab.png"},{"id":78512502,"identity":"6bfcc063-e36b-4338-9b03-c426ec1ef0de","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":693031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eTransmission electron microscopy (TEM) image of CrCi-CDs and tyndall effect of CrCi-CDs aqueous solution. (\u003cstrong\u003eB) \u003c/strong\u003eHigh resolution transmission electron microscopy (HRTEM) image and measurement of lattice spacing of CrCi-CDs. (\u003cstrong\u003eC) \u003c/strong\u003eXRD pattern of CrCi-CDs. (\u003cstrong\u003eD)\u003c/strong\u003e Ultraviolet-visible absorption spectra (UV-Vis) of CrCi-CDs. (\u003cstrong\u003eE)\u003c/strong\u003e Fluorescence spectrum (FL) of CrCi-CDs. \u003cstrong\u003eF\u003c/strong\u003eFourier transform infrared spectroscopy (FTIR) of CrCi-CDs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/3ab7f7cf25b9fdefc1e08950.png"},{"id":78512503,"identity":"dee342bc-b5f1-4de6-842a-c13324116c1f","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":202481,"visible":true,"origin":"","legend":"\u003cp\u003eXPS characterization of CrCi-CDs. \u003cstrong\u003e(A)\u003c/strong\u003eFull-scan XPS spectrum of CrCi-CDs. The high resolution XPS spectra of \u003cstrong\u003e(B)\u003c/strong\u003eC 1s, \u003cstrong\u003e(C)\u003c/strong\u003e O 1s, and\u003cstrong\u003e(D)\u003c/strong\u003eN 1s.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/5d4e7eac7902401c6bdf6b86.png"},{"id":78512507,"identity":"98552469-2db0-4a54-bb3f-df1e476f17ee","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":121644,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of CrCi-CDs on PTZ, PILO and PNC-induced acute seizure models in mice. (\u003cstrong\u003eA＆B)\u003c/strong\u003eEffect of CrCi-CDs on latency to clonic convulsion (\u003cstrong\u003eA\u003c/strong\u003e) and latency to tonic-clonic seizures (\u003cstrong\u003eB\u003c/strong\u003e) in mice with PTZ-induced epilepsy. (\u003cstrong\u003eC＆D)\u003c/strong\u003eEffect of CrCi-CDs on latency to clonic convulsion (\u003cstrong\u003eC\u003c/strong\u003e) and latency to tonic-clonic seizures (\u003cstrong\u003eD\u003c/strong\u003e) in mice with PNC-induced epilepsy. \u003cstrong\u003eE＆F \u003c/strong\u003eEffect of CrCi-CDs on latency to clonic convulsion (\u003cstrong\u003eE\u003c/strong\u003e) and latency to tonic-clonic seizures (\u003cstrong\u003eF\u003c/strong\u003e) in mice with PNC-induced epilepsy.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/5efb7f486114efb4e5e91673.png"},{"id":78512504,"identity":"fd161520-1754-433d-a33a-9e0a697a29b4","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":301371,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CrCi-CDs on spatial learning ability of PTZ-induced epileptic mice. (\u003cstrong\u003eA) \u003c/strong\u003eThe trajectory diagrams of place navigation test. (\u003cstrong\u003eB)\u003c/strong\u003e The effects of CrCi-CDs on escape latency of place navigation test. (\u003cstrong\u003eC)\u003c/strong\u003e The effects of CrCi-CDs on swimming distance of place navigation test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/a501e33fdac909d48566950e.png"},{"id":78513033,"identity":"364b38a0-f1e6-40a0-8adc-b0d2e313c17b","added_by":"auto","created_at":"2025-03-14 10:12:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":274295,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CrCi-CDs on spatial memory in PTZ-induced epileptic mice. (\u003cstrong\u003eA) \u003c/strong\u003eThe trajectory diagrams of spatial exploration test. (\u003cstrong\u003eB)\u003c/strong\u003e The effects of CrCi-CDs on escape latency of spatial exploration test. (\u003cstrong\u003eC)\u003c/strong\u003e The effects of CrCi-CDs on times of crossing platform area of spatial exploration test. ##\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs control group. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs model group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/e003a88eb010ae0318fb6ee2.png"},{"id":78513225,"identity":"18e0981d-f3ff-4d5a-8335-132dd62676da","added_by":"auto","created_at":"2025-03-14 10:20:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1078656,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CrCi-CDs on the pathological morphology of the dentate gyrus and CA1 region of the hippocampus in the brain tissue of epileptic mice induced by PTZ. (\u003cstrong\u003eA)\u003c/strong\u003eHE staining of the dentate gyrus. (\u003cstrong\u003eB)\u003c/strong\u003e HE staining of the CA1 region. (\u003cstrong\u003eC)\u003c/strong\u003eNissl staining of the dentate gyrus. (\u003cstrong\u003eD)\u003c/strong\u003e Nissl staining of the CA1 region.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/282d3cc31ae979260212da30.png"},{"id":78512519,"identity":"dcf929f8-58c3-4ec7-b0aa-ec9b20a4aa90","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1060298,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CrCi-CDs on the pathological morphology of the dentate gyrus and CA1 region of the hippocampus in the brain tissue of epileptic mice induced by PILO. (\u003cstrong\u003eA)\u003c/strong\u003eHE staining of the dentate gyrus. (\u003cstrong\u003eB)\u003c/strong\u003e HE staining of the CA1 region. (\u003cstrong\u003eC)\u003c/strong\u003eNissl staining of the dentate gyrus. (\u003cstrong\u003eD)\u003c/strong\u003e Nissl staining of the CA1 region.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/b87f7af5ca8e3bde7179424b.png"},{"id":78512516,"identity":"33acb96e-96d3-4932-ad1d-ef1cb34412dd","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1031126,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CrCi-CDs on the pathological morphology of the dentate gyrus and CA1 region of the hippocampus in the brain tissue of epileptic mice induced by PNC. (\u003cstrong\u003eA)\u003c/strong\u003e HE staining of the dentate gyrus. (\u003cstrong\u003eB)\u003c/strong\u003e HE staining of the CA1 region. (\u003cstrong\u003eC)\u003c/strong\u003eNissl staining of the dentate gyrus. (\u003cstrong\u003eD)\u003c/strong\u003e Nissl staining of the CA1 region.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/0af030549907d9c83599e562.png"},{"id":78513224,"identity":"b3e413cc-2148-4505-a9e3-bb8fbca45196","added_by":"auto","created_at":"2025-03-14 10:20:00","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":106266,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CrCi-CDs on Glu and GABA levels in the brain tissue of mice with epilepsy induced by PTZ, PILO and PNC. (\u003cstrong\u003eA＆B)\u003c/strong\u003e Effects of CrCi-CDs on Glu (\u003cstrong\u003eA\u003c/strong\u003e) and GABA (\u003cstrong\u003eB\u003c/strong\u003e) levels in the brain tissue of mice with epilepsy induced by PTZ. (\u003cstrong\u003eC＆D)\u003c/strong\u003e Effects of CrCi-CDs on Glu (\u003cstrong\u003eC\u003c/strong\u003e) and GABA (\u003cstrong\u003eD\u003c/strong\u003e) levels in the brain tissue of mice with epilepsy induced by PILO. (\u003cstrong\u003eE＆F)\u003c/strong\u003e Effects of CrCi-CDs on Glu (\u003cstrong\u003eE\u003c/strong\u003e) and GABA (\u003cstrong\u003eF\u003c/strong\u003e) levels in the brain tissue of mice with epilepsy induced by PNC. \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs control group. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs model group.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/9223596d3cb0f78a0005bd16.png"},{"id":78512509,"identity":"3ebace6e-3db8-49f7-9668-d68c91006095","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":225666,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of CrCi-CDs on the levels of inflammatory factors in the brain tissue of mice with epilepsy. caused by PTZ, PILO and PNC. (\u003cstrong\u003eA) \u003c/strong\u003eEffects of CrCi-CDs on TNF-α (\u003cstrong\u003ea\u003c/strong\u003e), IL-1β (\u003cstrong\u003eb\u003c/strong\u003e), IL-6(\u003cstrong\u003ec\u003c/strong\u003e) and IL-18(\u003cstrong\u003ed\u003c/strong\u003e) levels in the brain tissue of mice with epilepsy induced by PTZ. (\u003cstrong\u003eB) \u003c/strong\u003eEffects of CrCi-CDs on TNF-α (\u003cstrong\u003ea\u003c/strong\u003e), IL-1β (\u003cstrong\u003eb\u003c/strong\u003e), IL-6(\u003cstrong\u003ec\u003c/strong\u003e) and IL-18(\u003cstrong\u003ed\u003c/strong\u003e) levels in the brain tissue of mice with epilepsy induced by PILO. (\u003cstrong\u003eC) \u003c/strong\u003eEffects of CrCi-CDs on TNF-α (\u003cstrong\u003ea\u003c/strong\u003e), IL-1β (\u003cstrong\u003eb\u003c/strong\u003e), IL-6(\u003cstrong\u003ec\u003c/strong\u003e) and IL-18(\u003cstrong\u003ed\u003c/strong\u003e) levels in the brain tissue of mice with epilepsy induced by PNC. \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs control group. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs model group.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/eeced50a1b0343417c121112.png"},{"id":78513039,"identity":"b29d5857-4198-4034-838c-6f5594d5b553","added_by":"auto","created_at":"2025-03-14 10:12:00","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":92780,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CrCi-CDs on oxidative stress (MDA, SOD) levels in brain tissue of mice with epilepsy induced by PTZ and PILO. (\u003cstrong\u003eA＆B)\u003c/strong\u003eEffects of CrCi-CDs on MDA (\u003cstrong\u003eA\u003c/strong\u003e) and SOD (\u003cstrong\u003eB\u003c/strong\u003e) levels in brain tissue of mice with epilepsy induced by PTZ. (\u003cstrong\u003eC＆D)\u003c/strong\u003eEffects of CrCi-CDs on MDA (\u003cstrong\u003eC\u003c/strong\u003e) and SOD (\u003cstrong\u003eD\u003c/strong\u003e) levels in brain tissue of mice with epilepsy induced by PILO. (\u003cstrong\u003eE＆F)\u003c/strong\u003eEffects of CrCi-CDs on MDA (\u003cstrong\u003eE\u003c/strong\u003e) and SOD (\u003cstrong\u003eF\u003c/strong\u003e) levels in brain tissue of mice with epilepsy induced by PNC. ##\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 vs control group. **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01 and *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs model group.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/366b0d6b2eb4aa227a3d6537.png"},{"id":78512520,"identity":"6aefc54c-a176-4cb2-aca6-914eae38fa46","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":195428,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of CrCi-CDs on the expression of proteins related to Bax/Bcl-2/Caspase-3 signaling pathway in brain tissue of epileptic mice induced by PTZ, PILO and PNC. \u003cstrong\u003eA\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e), \u003cstrong\u003eB \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) and \u003cstrong\u003eC \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Representative immunoblot analysis and densitometry. \u003cstrong\u003eA \u003c/strong\u003e(\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e), \u003cstrong\u003eB\u003c/strong\u003e (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003e d\u003c/strong\u003e), \u003cstrong\u003eC\u003c/strong\u003e (\u003cstrong\u003eb\u003c/strong\u003e,\u003cstrong\u003e c\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e) Quantification of Bax, Bcl-2 and Cleaved-caspase-3 proteins. \u003cstrong\u003eA\u003c/strong\u003e (\u003cstrong\u003ee\u003c/strong\u003e), \u003cstrong\u003eB\u003c/strong\u003e (\u003cstrong\u003ee\u003c/strong\u003e), \u003cstrong\u003eC\u003c/strong\u003e (\u003cstrong\u003ee\u003c/strong\u003e) The ratios of Bax and Bcl-2 levels. ##\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 vs control group. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs model group.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/80b1bee33e552ea03e30f10e.png"},{"id":78514043,"identity":"666a7868-a806-4a0a-97be-9de5cddf04bc","added_by":"auto","created_at":"2025-03-14 10:28:00","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":1014058,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of the pathogenesis of epilepsy caused by PTZ, PILO, and PNC, respectively. (\u003cstrong\u003eA)\u003c/strong\u003ePostsynaptic inhibition in the normal brain. (\u003cstrong\u003eB＆C＆D) \u003c/strong\u003eMechanism of epilepsy caused by post-neural excitotoxicity induced by PTZ(\u003cstrong\u003eB\u003c/strong\u003e), PILO(\u003cstrong\u003eC\u003c/strong\u003e), and PNC(\u003cstrong\u003eD\u003c/strong\u003e). Drawn By Figdraw.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/d93425f95d91761f5245a1b3.png"},{"id":79066817,"identity":"d24d5048-ccba-4d75-a9ab-9382d5013503","added_by":"auto","created_at":"2025-03-24 04:32:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8378863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/78653eb0-179d-4237-8f8c-33ef5772339d.pdf"},{"id":78512501,"identity":"12f7e3c8-f81d-4930-90f9-700b20fdd573","added_by":"auto","created_at":"2025-03-14 10:04:00","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":326068,"visible":true,"origin":"","legend":"","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-6202061/v1/fc03b41f05664de524706102.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"The neuroprotective effect of carbon dots from Crinis Carbonisatus (carbonized human hair) against epilepsy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy is a recurrent neurological disorder characterized by recurrent, transient, and stereotyped episodes of central nervous system dysfunction\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. This condition not only induces neurobiological alterations but also exerts effects on cognitive, psychological, and social dimensions of health. The World Health Organization (WHO) has identified epilepsy as a priority neurological and psychiatric disorder within global prevention and treatment initiatives\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Currently, epilepsy affects estimated 50\u0026nbsp;million individuals worldwide, with an overall lifetime prevalence of 7.6% and a prevalence of active epilepsy at 6.38%. Despite its widespread impact, nearly 80% of individuals with epilepsy globally lack access to antiepileptic medication, with the majority of these cases concentrated in developing nations\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe prevailing consensus regarding the specific pathogenesis of epilepsy centers on the disruption of the equilibrium between excitation and inhibition within the central nervous system (CNS), which is closely associated with aberrant neurotransmitter signaling and neuroinflammatory processes\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Among neurotransmitters, amino acid neurotransmitters, particularly glutamic acid (Glu, an excitatory neurotransmitter) and gamma-aminobutyric acid (GABA, an inhibitory neurotransmitter), are most prominently implicated in epileptic seizures\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. An imbalance in Glu/GABA levels\u0026mdash;characterized by an elevation in Glu and a reduction in GABA\u0026mdash;can precipitate neuronal hyperexcitation, induce excitatory neurotoxicity, result in neuronal damage, and ultimately provoke acute epileptic episodes\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Additionally, neuroinflammation during epileptic seizures represents a critical pathological factor. Seizures have been shown to elevate the levels of pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and transforming growth factor-β1 (TGF-β1). The resulting inflammatory milieu exacerbates CNS injury, thereby contributing to the initiation and progression of refractory epilepsy\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the management of epilepsy, although a variety of therapeutic modalities\u0026mdash;including pharmacological interventions, surgical procedures, lifestyle adjustments, and gene-based therapies\u0026mdash;are available, monotherapy with antiepileptic drugs (AEDs) remains the cornerstone of treatment\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Different classes of AEDs exert their antiepileptic effects by targeting diverse pathogenic mechanisms underlying epilepsy, such as ion channel modulation, synaptic transmission regulation, and inflammatory factor inhibition\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, prolonged administration of AEDs is associated with a spectrum of adverse effects, encompassing neurological disturbances, visual and auditory deficits, and hepatorenal toxicity. Consequently, there is an imperative need to develop a novel, sustainable, and minimally invasive anti-epileptic therapeutic strategy\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, carbon dots (CDs)\u0026mdash;nanostructures characterized by a carbon-based core, sub-10 nm dimensions, and surface passivation groups\u0026mdash;have attracted considerable interest within the field of nanomaterials\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. These CDs have been extensively explored for diverse applications spanning bioimaging, sensor development, catalytic processes, energy conversion/storage systems, and biomedical technologies\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Our investigation has revealed the presence of a distinct category of CDs in \u0026ldquo;Tan Yao\u0026rdquo;, a traditional Chinese medicinal preparation, wherein natural herbal constituents facilitate the in situ synthesis of CDs during the preparation process\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Following this discovery, multiple distinct classes of CDs were successfully extracted and isolated from varied herbal precursors. These CDs exhibit notable pharmacological activities, including immunomodulatory functions, anti-inflammatory efficacy, and hemostatic properties\u003csup\u003e[\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28 CR29\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCrinis Carbonisatus (CrCi), referred to as \u0026ldquo;Xue-yu-tan\u0026rdquo; in traditional Chinese medicine (TCM), is a highly significant therapeutic substance within the TCM pharmacopeia. It is synthesized through a process involving cleaning, drying, and carbonization of healthy human hair. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, the silk manuscript excavated from a Han Dynasty tomb demonstrates the use of calcined hair (prototype of \u0026ldquo;Xue-yu-tan\u0026rdquo;) in emergency hemostasis. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB reveals that as early as the Eastern Han Dynasty, \u003cem\u003eShennong Bencaojing\u003c/em\u003e (Divine Farmer's Materia Medica), a pharmacological compendium, documented the application of \u0026ldquo;Xue-yu-tan\u0026rdquo; in treating various ailments including hemorrhage, stroke, and epilepsy. The medicinal application of \u0026ldquo;Xue-yu-tan\u0026rdquo; has been extensively documented in classical TCM literature spanning over two millennia, with references to its therapeutic efficacy appearing in medical texts from various Chinese dynasties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn prior research, our team successfully isolated and characterized carbon dots (CDs) derived from \u0026ldquo;Xue-yu-tan\u0026rdquo; (CrCi-CDs), thereby elucidating the therapeutic potential of Blood Residue Charcoal in the context of ischemic stroke\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Preliminary experimental findings indicate that CrCi-CDs modulate cerebral neurotransmitter levels and exhibit a spectrum of pharmacological activities, including sedative, analgesic, and anxiolytic effects. Furthermore, CrCi-CDs demonstrate pronounced neuroprotective properties, attenuating inflammatory responses, excitotoxic damage, and neuronal apoptosis subsequent to cerebral ischemia. These empirical observations substantiate the hypothesis that CrCi-CDs may possess antiepileptic efficacy, potentially mediated through mechanisms analogous to those implicated in stroke therapy.\u003c/p\u003e \u003cp\u003eIn this study, we used three distinct acute epilepsy models to investigate the potential of CrCi-CDs in mitigating neuroinflammation and oxidative stress induced by epileptic seizures. Furthermore, we examined whether CrCi-CDs exhibit antiepileptic properties by attenuating neuronal damage and cell loss. The equilibrium between neural excitation and inhibition was assessed through the quantification of seizure duration and the measurement of amino acid neurotransmitter levels. Spatial learning and memory deficits were evaluated using the Morris water maze behavioral paradigm. Neuroinflammatory damage was analyzed by measuring the concentrations of inflammatory mediators and conducting histopathological examinations of brain tissue sections. These comprehensive assessments were conducted to elucidate the antiepileptic efficacy of CrCi-CDs and to explore the underlying mechanisms contributing to their potential therapeutic effects.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eDialysis membranes with a molecular weight of 1000 Da were purchased from Beijing Ruida Henghui Technology Development Co., Ltd. (Beijing, China). Pentylenetetrazol (PTZ) and Sodium Valproate (VPA) was purchased from Sigma-Aldrich Co., Ltd. (USA), Pilocarpine (PILO) was purchased from APExBIO Technology Co., Ltd. (USA), Penicillin sodium for injection (PNC) was purchased from North China Pharmaceutical Co., Ltd.(Shijiazhuang, China), and Other analytical reagents were purchased from Wuhan Saiwei'er Biological Technology Co., Ltd. (Wuhan, China). All the experiments were performed using deionized water (DW).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003e Animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals that was approved by the Committee of Ethics of Animal Experimentation of the Beijing University of Chinese Medicine. Male C57BL/6 rats (weighing 20.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 g) were purchased from Beijing Sibeifu Biotechnology Co., Ltd. (Beijing, China). These animals were housed under the following conditions: temperature, (24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0) \u0026deg;C; relative humidity, 55\u0026ndash;65%, and a 12-h light/dark cycle, with ad libitum access to food and water.\u003c/p\u003e\n\u003ch3\u003eSynthesis of CrCi-CDs\u003c/h3\u003e\n\u003cp\u003eThe method for the separation and extraction of CrCi-CDs is coincident to our previous work\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In brief, after cleaning the collected healthy human hair to remove grease, it is dried in an oven at 60\u0026deg;C for 24 hours. After drying, the hair is calcined by a muffle furnace. The calcination conditions are as follows: a 5-minute ramp to 70\u0026deg;C, held for 20 minutes, then a 25-minute ramp to 350\u0026deg;C, and held for 1 hour. Once the hair has cooled to room temperature, it is pulverized using a small high-speed pulverizer. Subsequently, DW is added and the mixture is heated in a water bath at 100\u0026deg;C for 1 hour. The resulting solution is first filtered through a 0.22 \u0026micro;m organic membrane filter, and then dialyzed using a 1000-Da dialysis membrane for 72 hours to obtain the CrCi-CDs solution.\u003c/p\u003e\n\u003ch3\u003eSample characterization\u003c/h3\u003e\n\u003cp\u003ePhotoluminescence experiments were conducted with a Shimadzu RF5-5301 PC spectrofluorimeter (Shimadzu, Japan). UV-vis absorption spectra were obtained using a TU-1991 UV-vis spectrophotometer. Fourier transform infrared spectroscopy (FTIR) was measured in the range of 500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using a Nicolet 6700 FTIR spectrophotometer. Transmission electron microscopy analyses to study morphology and mean diameter of the resultant samples were carried out on a JEM-2100F (FEI, USA), operating at an accelerating voltage of 200kV.\u003c/p\u003e\n\u003ch3\u003eExperimental protocol\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eExperimental protocol\u003c/div\u003e \u003cp\u003eBecause this experiment adopted three acute epilepsy mouse models, three batches of 144 C57BL/6 male mice were used. After weighing 48 mice in each batch, they were randomly divided into 6 groups (8 mice in each group), namely, blank group (Control), model group (Model), VPA group (VPA), CrCi-CDs high (High), medium (Medium) and low (Low) dose groups. The blank group and model group were given DW by gavage, the positive drug group was given VPA by gavage (200 mg/kg), and the CrCi-CDs high, medium and low dose groups were given CrCi-CDs solution by gavage (Dosing concentrations were 6 mg/kg, 3 mg/kg, and 1.5 mg/kg, respectively), and the drugs were given continuously for 7 days.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimals modeling\u003c/h2\u003e \u003cp\u003eOn the 7th day of the experiment, 1 hour after the administration of each group, the first batch of mice except the blank group were intraperitoneally injected with PTZ (65 mg/kg), and the blank group was intraperitoneally injected with an equal amount of saline. Similarly, the second batch of mice and the third batch were intraperitoneally injected with PILO 280 mg/kg (i.e., 7\u0026nbsp;million units/kg) and PNC 4.2 g/kg respectively.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBehavioral observation\u003c/h3\u003e\n\u003cp\u003eAfter the injection, we placed the mice in a transparent box for observation for 30 min. The epileptic seizure grade of the mice was evaluated according to the Racine scale\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e, and the latency to clonic convulsions (equivalent to Racine scores of 1\u0026ndash;3) and tonic-clonic seizures (equivalent to Racine scores of 4\u0026ndash;5) were recorded for each mouse.\u003c/p\u003e\n\u003ch3\u003eMorris water maze test\u003c/h3\u003e\n\u003cp\u003ePTZ-induced epileptic mice from each group were selected for the Morris water maze test. The water maze apparatus comprised a circular pool measuring 90 cm in diameter and 50 cm in height, along with a platform measuring 9 cm in diameter and 30 cm in height. The safety platform was positioned at the midpoint of the outer third of a designated quadrant within the water maze. Four plastic cards of distinct shapes and colors were affixed to the walls of the four quadrants to serve as spatial reference cues.\u003c/p\u003e \u003cp\u003eThe place navigation test was conducted daily at 9:00 AM over four consecutive days. Each mouse underwent four training sessions per day. During each session, a quadrant was randomly chosen, and the mouse was introduced into the pool facing the wall. The latency for the mouse to locate and ascend the submerged safety platform was recorded within a 90-second time frame. On the fifth day, the spatial exploration test was performed. The submerged safety platform was removed, and the quadrant opposite to its original location was designated as the entry point for the mice. The number of crossings through the area previously occupied by the safety platform was recorded over a 60-second period.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHE stain ༆ Nissl stain\u003c/h2\u003e \u003cp\u003eThe fixed brain tissue was dehydrated, transparent, waxed, and embedded, and then coronal sections were made with a thickness of 3 \u0026micro;m. Subsequently, xylene and ethanol were used for dewaxing, and HE staining and Nissl staining were performed to observe the pathological conditions of the neuronal cells in the hippocampus of the mouse brain tissue under an optical microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme linked immunosorbent assay\u003c/h2\u003e \u003cp\u003eThe frozen brain tissue was removed and ground at low temperature, then cooled and centrifuged to obtain the brain tissue homogenate supernatant; the corresponding ELISA kits were used to detect the levels of amino acid neurotransmitters (GABA, Glu), inflammatory factors (TNF-α, IL-1β, IL-6, IL-18), and oxidative stress indicators (SOD, MDA) in the brain tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eTotal protein from mouse brain tissue was extracted with cold RIPA buffer containing 1% protease inhibitors, and its concentration was quantified using a bi-creatine (BCA) kit. Equal amounts of protein from brain tissue were then separated by SDS-PAGE electrophoresis, and then transferred to NC membranes after blocking with 5% skim milk at room temperature for 1.5 h. Proteins were then detected overnight at 4\u0026deg;C using the corresponding primary antibodies (GAPDH, Bax, Bcl-2, and Cleaved-caspase-3), followed by incubation with secondary antibodies for 1 h at room temperature. After washing with TBST, the target proteins in the NC membrane were observed using an automated chemiluminescence image analysis system using enhanced chemiluminescence.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003eCharacterization of CrCi-CDs\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;2A, TEM observation shows that CrCi-CDs are spherical, well dispersed in aqueous solution, and have uniform particle size distribution, ranging from 2–3 nm, with an average particle size of less than 10 nm (Fig.\u0026nbsp;2B). The Tyndall effect of its aqueous solution indicates that the CrCi-CDs aqueous solution is a colloidal solution, with relatively uniform CrCi-CDs particle sizes and a certain degree of metastability. Figure\u0026nbsp;2B shows that there is an obvious lattice in a single spherical nanoparticle, and the lattice spacing of CrCi-CDs is 0.228 nm, which is consistent with the crystal plane of graphene C.\u003c/p\u003e\n \u003cp\u003eFigure\u0026nbsp;2C shows that the XRD spectrum of CrCi-CDs had distinct diffraction peaks (2θ = 29.399°), indicating that CrCi-CDs were attributed to amorphous carbons arranged in a considerably random fashion.The UV-Vis spectrum of CrCi-CDs shows a smooth curve with a broad and weak absorption region around 320 nm (Fig.\u0026nbsp;1D), which may be related to the n-π* transition of the C = O bond in CrCi-CDs or the π-π* transition of the C = C bond.\u003c/p\u003e\n \u003cp\u003eFigure\u0026nbsp;2E is the fluorescence excitation emission spectrum of the CrCi-CDs aqueous solution. At an excitation wavelength of 334 nm, the emission peak of CrCi-CDs is located at around 402 nm. Under irradiation with a 365 nm ultraviolet lamp, the CrCi-CDs solution emits bright blue fluorescence.\u003c/p\u003e\n \u003cp\u003eThe FTIR absorption spectrum of CrCi-CDs (Fig.\u0026nbsp;2F) shows that the absorption peaks of different functional groups appear at 3440 cm\u003csup\u003e− 1\u003c/sup\u003e, 2924 cm\u003csup\u003e− 1\u003c/sup\u003e, 1630 cm\u003csup\u003e− 1\u003c/sup\u003e, 1401 cm\u003csup\u003e− 1\u003c/sup\u003e, 1262 cm\u003csup\u003e− 1\u003c/sup\u003e, 1101 cm\u003csup\u003e− 1\u003c/sup\u003e, and 863 cm\u003csup\u003e− 1\u003c/sup\u003e, respectively. The strong absorption peak at 3440 cm\u003csup\u003e− 1\u003c/sup\u003e may be related to the stretching vibration of O-H bond and N-H bond, the absorption peak at 2924 cm\u003csup\u003e− 1\u003c/sup\u003e may come from the stretching vibration of -CH bond in -CH3 and -CH2 groups, the absorption peak at 1630 cm\u003csup\u003e− 1\u003c/sup\u003e indicates the presence of C = O bond in the surface group of CrCi-CDs, the absorption peak at 1401 cm\u003csup\u003e− 1\u003c/sup\u003e may be caused by the stretching vibration of C–N bond, the bending vibration of N–H bond and C–H bond, the absorption peak at 1262 cm\u003csup\u003e− 1\u003c/sup\u003e indicates the presence of C-OH bond, the absorption peak at 1101 cm\u003csup\u003e− 1\u003c/sup\u003e is related to the vibration of C-O-C bond, and the absorption peak at 863 cm\u003csup\u003e− 1\u003c/sup\u003e may come from the bending vibration of C-H bond on benzene ring. Therefore, we believe that the surface of CrCi-CDs has abundant hydroxyl, amino and carbonyl/carboxylate groups.\u003c/p\u003e\n \u003cp\u003eXPS spectra can be used to analyze the elemental composition and coordination information of CrCi-CDs. As shown in Fig.\u0026nbsp;3A, the full scan XPS spectrum reveals that the nano-component mainly contains C (285.08 eV), N (400.08 eV), and O (532.08 eV) elements, accounting for 70.7%, 6.13%, and 23.17% respectively. The detailed coordination characteristics of each element are analyzed in Fig.\u0026nbsp;3B, 3C, and 3D.In the high-resolution XPS spectrum of C (Fig.\u0026nbsp;3B), four coordination modes of C are observed: C-C (284.8 eV), C-N (285.3 eV), C-O (286 eV), and C = O (287.7 eV). In the high-resolution XPS spectrum of O (Fig.\u0026nbsp;3C), the peaks at 531.0 eV and 532 eV correspond to C-O and C = O respectively. Similarly, in the high-resolution XPS spectrum of N (Fig.\u0026nbsp;3D), the peaks at 399.5 eV and 400.5 eV indicate the presence of C-N and C = N bonds in CrCi-CDs.\u003c/p\u003e\n \u003cp\u003eThe characterization data of CrCi-CDs obtained in the above experiments are consistent with the results of our previous work, which proves that we have obtained a stable and uniform CrCi-CDs extraction and separation method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003eCrCi-CDs attenuate seizure severity in epileptic mice\u003c/h2\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;4, compared with the model group, VPA and high, medium, and low doses of CrCi-CDs increased seizure latency in the three epilepsy mouse models to varying degrees, thereby mitigating seizure severity. Taking the PILO-induced epilepsy mouse model (Fig.\u0026nbsp;4C and D) as an example, the latency of clonic convulsions in the VPA group (944.30 ± 138.90 s), CrCi-CDs high-dose group (817.10 ± 221.00 s), and CrCi-CDs medium-dose group (636.40 ± 156.90 s) was significantly prolonged (p \u0026lt; 0.01). Figure\u0026nbsp;4D shows that, compared with the model group (1942.00 ± 245.50 s), VPA (3380.00 ± 367.70 s) and CrCi-CDs high-dose (3126.00 ± 464.80 s) intervention in the acute epilepsy mouse model significantly prolonged the latency of tonic convulsions in mice (p \u0026lt; 0.01). We also observed similar trends in other epilepsy models, indicating that different doses of CrCi-CDs ameliorate epilepsy, with high doses of CrCi-CDs demonstrating the most significant therapeutic efficacy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eCrCi-CDs improve spatial learning and memory in epileptic mice\u003c/h2\u003e\n \u003cp\u003eThe Morris water maze is a classic experiment used to evaluate the spatial learning and memory abilities of experimental animals and is widely used in numerous research fields related to memory. Our study used the PTZ-induced epilepsy mouse model to evaluate the effects of CrCi-CDs on spatial learning and memory in acute epileptic mice. As shown in Fig.\u0026nbsp;5A, the swimming trajectory diagram revealed that the blank group mice exhibited the best spatial learning ability, while the movement trajectory of the model group mice indicated severe impairment in their spatial memory ability. VPA and varying doses of CrCi-CDs intervention alleviated the impairment of spatial learning ability in epileptic mice to some extent, with VPA and high doses of CrCi-CDs demonstrating the most significant improvement.\u003c/p\u003e\n \u003cp\u003eFigure\u0026nbsp;5B and 4C show that the escape latency and swimming distance of mice in the model group were significantly increased compared to those in the blank group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), indicating that the spatial learning ability of mice in the model group was severely impaired. VPA and CrCi-CDs reduced the escape latency and swimming distance to varying degrees. With the intervention of VPA and high-dose CrCi-CDs, the escape latency of mice was significantly reduced from day 1 to day 4 compared to the model group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). With the intervention of high-dose CrCi-CDs, the swimming distance of mice on days 3 and 4 was significantly reduced compared to the model group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). These findings demonstrate that interventions with the VPA group and varying doses of CrCi-CDs alleviated the impairment of spatial learning ability in epileptic mice.\u003c/p\u003e\n \u003cp\u003eThe spatial exploration experiment conducted on the fifth day was used to evaluate the spatial memory ability of experimental animals. Figure\u0026nbsp;6A shows that the model group mice exhibited the poorest memory of the location of the underwater safety platform, and the mice predominantly searched in the quadrant opposite the platform. The blank group mice frequently crossed the location and quadrant containing the safety platform, indicating that their spatial memory was not impaired. Following intervention with VPA and varying doses of CrCi-CDs, the impairment of spatial memory in epileptic mice was alleviated to varying degrees.\u003c/p\u003e\n \u003cp\u003eFigure\u0026nbsp;6B and 5C show that compared to the blank group (11.62 ± 3.39 s; 4.00 ± 0.89), the escape latency of the model group mice (36.75 ± 4.90 s) in the spatial exploration experiment was significantly increased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), and the number of crossings in the platform area (1.50 ± 0.55) was significantly reduced (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), indicating that the epilepsy model mice had severe memory impairment. Following drug intervention, VPA significantly reduced the escape latency (20.86 ± 3.55 s) and increased the number of crossings in the platform area (3.67 ± 0.52, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), and high-dose CrCi-CDs also significantly reduced the escape latency (22.63 ± 3.68 s, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) and increased the number of crossings in the platform area (3.50 ± 1.05, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eEffects of CrCi-CDs on brain tissue morphology in epileptic mice\u003c/h2\u003e\n \u003cp\u003eFigure\u0026nbsp;7A and 7B show that in the PTZ-induced epilepsy mouse model, the hippocampal tissue cells in the model group exhibited a more chaotic arrangement compared to those in the control group, including gaps, cell swelling, uneven cytoplasmic staining, vacuoles, and even cell rupture and loss. Following administration of VPA and varying doses of CrCi-CDs, the extent of tissue damage was alleviated to varying degrees. Figure\u0026nbsp;7C and 7D show that PTZ induced a significant reduction in Nissl bodies within the cytoplasm of hippocampal neurons in mice, and VPA and CrCi-CDs ameliorated this phenomenon. HE and Nissl staining of brain tissue sections from PILO- and PNC-induced epileptic mice (Figs.\u0026nbsp;8 and 9) also yielded similar results, indicating that CrCi-CDs can mitigate neural tissue damage in epileptic mice induced by PTZ, PILO, and PNC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eCrCi-CDs ameliorate the level imbalance of Glu/GABA in brain tissue of epileptic mice\u003c/h2\u003e\n \u003cp\u003eThe imbalance in GABA and Glu expression, which disrupts the brain's excitatory/inhibitory balance, is considered a central mechanism in epilepsy. Our experiments confirmed this phenomenon in the PTZ-, PILO-, and PNC-induced epilepsy models, as shown in Fig.\u0026nbsp;9. The levels of the excitatory neurotransmitter Glu (PTZ: 0.56 ± 0.05 µmol/g; PILO: 0.56 ± 0.08 µmol/g; PNC: 0.59 ± 0.03 µmol/g) in the brain tissue of the model group mice were significantly upregulated (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) compared to the control group, and the levels of the inhibitory neurotransmitter GABA (PTZ: 1.13 ± 0.28 µmol/L; PILO: 1.07 ± 0.23 µmol/L; PNC: 1.09 ± 0.20 µmol/L) were significantly decreased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n \u003cp\u003eFollowing intervention with varying doses of CrCi-CDs, we observed that the dysregulated Glu and GABA levels were normalized. Using the PTZ-induced epilepsy mouse model (Fig.\u0026nbsp;10A and 10B) as an example, high-dose CrCi-CDs reduced Glu levels (0.42 ± 0.09 µmol/g, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and significantly increased GABA levels (1.67 ± 0.19 µmol/L, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). In the PILO- and PNC-induced models, we also observed a similar trend (Fig.\u0026nbsp;10C and 10D, 10E and 10F), which aligned with the results of previous behavioral experiments. This demonstrates that high-dose CrCi-CDs are most effective in increasing GABA levels and reducing Glu levels in brain tissue, effectively ameliorating the Glu/GABA imbalance in epilepsy models induced by PTZ, PILO, and PNC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eCrCi-CDs attenuate brain tissue inflammation in epileptic mice\u003c/h2\u003e\n \u003cp\u003e\u0026nbsp;Key cytokines produced by glial cells and neurons activate multiple inflammatory pathways, resulting in detrimental synaptic alterations and neuronal overexcitation, which contribute to neurotoxicity and exacerbate epileptic symptoms\u003csup\u003e[35]\u003c/sup\u003e. As shown in Fig.\u0026nbsp;11, the levels of inflammatory factors in the brain tissue of the model group were significantly elevated compared to those in the control group, irrespective of whether the epilepsy model was induced by PTZ, PILO, or PNC. Using the PTZ-induced epilepsy model as an example (Fig.\u0026nbsp;11A), the levels of inflammatory factors in the model group (TNF-α: 124.96 ± 11.14 pg/mL; IL-1β: 20.56 ± 2.45 pg/mL; IL-6: 25.09 ± 2.18 pg/mL; IL-18: 29.14 ± 3.78 pg/mL) were significantly elevated compared to those in the control group (TNF-α: 69.78 ± 12.42 pg/mL; IL-1β: 12.00 ± 3.27 pg/mL; IL-6: 17.32 ± 2.87 pg/mL; IL-18: 16.08 ± 2.36 pg/mL; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n \u003cp\u003eAfter intervention with different doses of CrCi-CDs, the levels of each factor decreased to varying degrees, among which the high dose of CrCi-CDs was the most significant [TNF-α: 85.64 ± 17.46 pg/mL, IL-6: 19.64 ± 2.89 pg/mL, IL-18: 18.72 ± 4.09 pg/mL(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), IL-1β: 14.80 ± 2.47 pg/mL(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)] in the PTZ model.The high-dose CrCi-CDs in the PILO and PNC model also had similar effects. In the PILO model, like [IL-1β: 13.21 ± 3.22 pg/mL, IL-6: 19.69 ± 2.33 pg/mL(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), TNF-α: 95.28 ± 13.18 pg/mL, IL-18: 19.08 ± 3.03 pg/mL(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)], and in the PNC model, like [TNF-α: 86.18 ± 14.01 pg/mL, IL-6: 17.13 ± 3.67 pg/mL, IL-18: 18.49 ± 3.62 pg/mL(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), IL-1β: 14.80 ± 2.47 pg/mL(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05)]. Therefore, we believe that CrCi-CDs can reduce the level of neuroinflammation, thereby improving the damage of neural tissue, and the high-dose group has the best effect among all dose groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003eCrCi-CDs improve oxidative stress levels in brain tissue of epileptic mice\u003c/h2\u003e\n \u003cp\u003e\u0026nbsp;During an epileptic seizure, excessive reactive oxygen species are produced. Excessive reactive oxygen species can damage cell membranes, leading to increased membrane permeability and inducing the production of harmful intermediates such as malondialdehyde (MDA). MDA can interact with DNA and proteins, inducing mutagenesis and reducing cell survival, thereby exacerbating cellular damage. Therefore, it is considered a reliable indicator of oxidative damage in epilepsy\u003csup\u003e[36]\u003c/sup\u003e. In addition, the reduced activity of antioxidant systems, such as superoxide dismutase (SOD), exacerbates the damage caused by reactive oxygen species to the brain\u003csup\u003e[37]\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;12, in the PTZ-, PILO-, and PNC-induced epilepsy mouse models, we observed that the levels of SOD in the brain tissue of the model group mice (PTZ model: 10.87 ± 2.61 ng/mL; PILO model: 10.69 ± 1.95 ng/mL; PNC model: 12.73 ± 2.97 ng/mL; p \u0026lt; 0.01) were significantly reduced, while the levels of MDA were significantly elevated (PTZ model: 4.42 ± 0.41 nmol/mL; PILO model: 4.00 ± 0.46 nmol/mL; PNC model: 4.02 ± 0.51 nmol/mL; p \u0026lt; 0.01) compared to the control group.\u003c/p\u003e\n \u003cp\u003eFollowing intervention with VPA and varying doses of CrCi-CDs, the levels of SOD and MDA were normalized. In the PTZ model, compared to the model group, the MDA levels in the brain tissue of the VPA group and the high-, medium-, and low-dose CrCi-CDs groups (VPA: 2.51 ± 0.42 nmol/mL; high-dose group: 3.07 ± 0.52 nmol/mL; medium-dose group: 3.32 ± 0.48 nmol/mL; low-dose group: 3.21 ± 0.49 nmol/mL; p \u0026lt; 0.01) were significantly reduced. Compared to the model group, VPA and high-dose CrCi-CDs (VPA: 17.09 ± 3.41 ng/mL; high-dose group: 18.41 ± 3.02 ng/mL; p \u0026lt; 0.01) significantly increased the activity of SOD in the brain tissue of mice following intervention. The PILO and PNC models exhibited similar trends, demonstrating that varying doses of CrCi-CDs exert differing degrees of inhibitory effects on oxidative stress levels in the brain tissue of epileptic mice, with the high dose being the most effective.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003eCrCi-CDs inhibited the expression of proteins in the neuronal apoptosis pathway\u003c/h2\u003e\n \u003cp\u003e\u0026nbsp;During epileptic seizures, excessive neuronal excitation, coupled with concurrent neural tissue inflammation and dysregulated oxidative stress, can induce neuronal damage, ultimately leading to the apoptosis of nerve cells, as evidenced by the pathological sections presented in Figs.\u0026nbsp;7, 8, and 9. Within the Bcl-2 family, Bcl-2 and Bax serve as pivotal regulators in the process of cellular apoptosis. Additionally, the Caspase protein family plays a critical role in apoptosis, functioning as an essential component within the apoptotic cascade. Consequently, this study investigates the anti-apoptotic effects of CrCi-CDs on nerve cells in epilepsy models induced by PTZ, PILO, and PNC, with a specific focus on the Bax/Bcl-2/Caspase-3 signaling pathway.\u003c/p\u003e\n \u003cp\u003eIn the PTZ-induced epilepsy model, as shown in Fig.\u0026nbsp;13A, compared with the blank group, the levels of Bax (0.84 ± 0.04), the ratio of Bax/Bcl-2 (8.76 ± 0.85) and Cleaved-caspase-3 (0.63 ± 0.05) in the model group mice were significantly increased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), and the expression of Bcl-2 (0.10 ± 0.01) was significantly decreased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), indicating that a large number of neurons apoptosis occurred after PTZ-induced epileptic seizures in mice. Compared with the model group, high-dose CrCi-CDs significantly decreased the expression of Bax (0.32 ± 0.10), the ratio of Bax/Bcl-2 (0.66 ± 0.17), and the expression of Cleaved-caspase-3 (0.21 ± 0.02) in the mouse brain tissue (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), and upregulated the expression of Bcl-2 (0.49 ± 0.05) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;13B, in the PILO-induced model, high-dose CrCi-CDs influenced the expression of proteins (Bax: 0.35 ± 0.07, Bcl-2: 0.57 ± 0.04, Caspase-3: 0.22 ± 0.04, Bax/Bcl-2: 0.63 ± 0.17) significantly(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). In the PNC-induced model, high-dose CrCi-CDs also showed similar effects (Bax: 0.31 ± 0.07, Bcl-2: 0.60 ± 0.06, Caspase-3: 0.26 ± 0.02, Bax/Bcl-2: 0.52 ± 0.12)(Fig.\u0026nbsp;13C) in the expression of proteins(\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n \u003cp\u003eTherefore, after acute epileptic seizures induced by PTZ, PILO, or PNC in mice, the expression of pro-apoptotic proteins Bax and Cleaved-caspase-3 in brain tissue increased significantly, while the expression of anti-apoptotic proteins Bcl-2 was significantly downregulated. This indicates that neurons in the brain tissue were in a state of massive apoptosis. Following CrCi-CDs administration, the expression of pro-apoptotic proteins was significantly downregulated, while the expression of anti-apoptotic proteins was upregulated. These findings suggest that CrCi-CDs effectively inhibit neuronal apoptosis in the brain tissue of epileptic mice.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn traditional Chinese medicine theory, \u0026ldquo;Xue-yu-tan\u0026rdquo; is a medicine with unique properties. The precursor of \"Xue-yu-tan\" is healthy human hair, which initially lacked medicinal properties but acquires therapeutic effects after processing. The anti-epileptic effect of \"Xue-yu-tan\" was documented as early as 2,000 years ago in traditional Chinese medicine texts. Nevertheless, the investigation into its active compounds and underlying mechanisms has remained ongoing, with no definitive conclusions reached to date. Advancements in nanotechnology have facilitated the isolation and extraction of a uniquely structured carbon dot, designated as CrCi-CDs, from \"Xue-yu-tan.\" Subsequent analysis has revealed its distinctive characteristics as a nanomaterial and its potential therapeutic applications.\u003c/p\u003e \u003cp\u003ePrevious research has primarily established the therapeutic efficacy of CrCi-CDs in the context of ischemic brain injury. The findings revealed that CrCi-CDs effectively suppress the activity of excitatory neurotransmitters following cerebral ischemia and mitigate the extensive neural tissue inflammation induced by cerebral ischemia-reperfusion. These observations suggest that CrCi-CDs facilitate the release of inhibitory neurotransmitters while promoting the depletion of excitatory neurotransmitters in neurological disorders. Consequently, this leads to adaptive physiological changes, including reduced neural excitability and diminished neurotoxicity, thereby conferring a neuroprotective effect. Epilepsy, a neurological disorder characterized by a multifactorial pathogenesis, is fundamentally associated with the overexcitation of the central nervous system. This overexcitation, a hallmark of epileptic seizures, is predominantly attributed to an imbalance between excitatory and inhibitory neurotransmitters, coupled with neural tissue inflammation. Given these mechanisms, it is plausible that CrCi-CDs may also exert a protective influence against epileptic neural injury, analogous to their demonstrated efficacy in ischemic brain injury.\u003c/p\u003e \u003cp\u003eIn this study, three distinct epilepsy models were utilized to investigate the anti-epileptic properties of CrCi-CDs. Following a comprehensive evaluation of various epilepsy modeling methodologies, cost-effectiveness, and alignment with clinical practices, the acute epilepsy model was identified as an efficient, cost-effective, and clinically relevant approach for assessing the anti-epileptic efficacy of CrCi-CDs and conducting preliminary mechanistic investigations\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. The mechanisms through which the three drugs induce epilepsy are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e14\u003c/span\u003e.\u003c/p\u003e \u003cp\u003ePTZ, a GABA receptor antagonist, competes with GABA for binding to postsynaptic GABA receptors, thereby inhibiting neuronal chloride channel function, reducing the neuronal excitability threshold, and precipitating epileptic seizures. The PTZ-induced acute epilepsy model is highly valuable for the initial screening of anti-epileptic compounds and mechanistic research\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. PILO exerts a pronounced excitatory effect on the central nervous system. Upon the induction of epileptic seizures, it elevates Glu levels in brain tissue, and the resultant epilepsy displays distinct temporal characteristics that closely mirror the progression of human temporal lobe epilepsy\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. PNC, another widely utilized convulsant, is employed to induce acute epilepsy. It inhibits the postsynaptic inhibitory effects of GABA while promoting the release of presynaptic excitatory neurotransmitters, thereby facilitating epileptic activity. The PNC-induced epilepsy model is widely recognized as a robust representation of human absence epilepsy and is extensively employed in related research\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. Consequently, for this experimental study, we selected the acute epilepsy models induced by PTZ, PILO, and PNC to evaluate the anti-epileptic effects of CrCi-CDs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur observations demonstrate that CrCi-CDs significantly enhance the behavioral outcomes of epileptic mouse models, particularly by extending the latency of paroxysmal and tonic convulsions. These findings underscore the sedative properties of CrCi-CDs, consistent with prior research. Further validation was achieved through the analysis of brain tissue sections and neurotransmitter levels, which substantiated the results of behavioral experiments. In the Morris water maze experiment, PTZ-induced epileptic mice displayed a marked reduction in spatial learning and memory capabilities, indicative of damage to specific brain regions. CrCi-CDs ameliorate these cognitive deficits in a dose-dependent manner, with the most substantial improvements observed at higher doses.\u003c/p\u003e \u003cp\u003eAdditionally, CrCi-CDs effectively rectify the Glu/GABA imbalance in epileptic mice induced by PTZ, PILO, and PNC, demonstrating dose-dependent efficacy. Glu and GABA, essential amino acid neurotransmitters, play pivotal roles in neurotransmission. Elevated Glu levels coupled with diminished GABA concentrations induce excitotoxicity, leading to neuronal damage, cell loss, and the initiation of neuroinflammation and oxidative stress imbalance. CrCi-CDs attenuate epileptic seizures by mitigating neurotoxicity associated with neuronal overactivation.\u003c/p\u003e \u003cp\u003eFurthermore, in epileptic seizures induced by PTZ, PILO, and PNC, neuroexcitotoxicity, resulting from neurotransmitter imbalance, not only precipitates neuronal destruction and loss but also initiates neuroinflammation and oxidative stress imbalance, thereby exacerbating neural tissue damage. Our observations indicate that CrCi-CDs effectively reduce the levels of inflammatory factors (TNF-α, IL-1β, IL-6, IL-18) in epileptic neural tissues, ameliorate oxidative stress by enhancing SOD activity and reducing MDA levels, and restore the balance between excitatory and inhibitory neurotransmitters. These findings align with prior evidence demonstrating the free radical scavenging capacity carbon dots derived from natural sources\u003csup\u003e[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e and the neuroprotective efficacy of CrCi-CDs through the mitigation of neuroexcitotoxicity and inflammation\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the context of neuronal destruction and apoptosis induced by neuroexcitotoxicity, the Bcl-2 family of proteins plays a pivotal role in the regulation of cellular apoptosis. The dysregulation of Bcl-2 family proteins is widely regarded as a primary mechanism underlying endogenous apoptosis\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. The Bcl-2 family can be categorized into two distinct groups: anti-apoptotic members (e.g., Bcl-2, Bcl-XL, Bcl-W) and pro-apoptotic members (e.g., Bax, Bcl-Xs). Upon neuronal detection of external environmental stimuli, Bax becomes activated, resulting in alterations to the permeability of the mitochondrial outer membrane. This process facilitates the release of cytochrome C through the pores of the mitochondrial lipid membrane, thereby initiating downstream cascade reactions\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. The Caspase family is intricately associated with cellular apoptosis, with Caspase-3 serving as the principal executor within this family. Caspase-3 not only represents the convergence point of multiple apoptotic pathways but also constitutes the terminal pathway for the execution of apoptosis, underscoring its critical role in the apoptotic mechanism. The activation of Caspase-3 signifies the irreversible progression of cellular apoptosis\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, whether PTZ or PNC competitively binds to GABA receptors, or PILO binds to M1 muscarinic receptors, our findings demonstrate that all three epilepsy models ultimately result in heightened neuronal excitability, thereby activating the Bax/Bcl-2/Caspase-3 pathway and precipitating neuronal apoptosis. The intervention of CrCi-CDs has been shown to suppress the expression of pro-apoptotic proteins Bax and Caspase-3, while upregulating the expression of the anti-apoptotic protein Bcl-2, thereby mitigating neuronal apoptosis induced by epileptic seizures. This observation further underscores the broad neuroprotective efficacy of CrCi-CDs.\u003c/p\u003e \u003cp\u003eThe findings of this study indicate that CrCi-CDs are capable of modulating the imbalance between excitatory and inhibitory neurotransmitters, suppressing the activation of neuroinflammatory pathways, and conferring a neuroprotective effect in murine models of epilepsy. Future research endeavors will focus on elucidating the upstream molecular mechanisms underlying the neuroprotective properties of CrCi-CDs, optimizing their in vivo biodistribution, and investigating their therapeutic potential alongside other pharmacological activities. Presently, AEDs (antiepileptic drugs) represent the cornerstone of epilepsy management, primarily functioning through the reduction of neuronal hyperexcitability and the inhibition of aberrant electrical discharges. Nevertheless, the associated adverse effects, including neurological disturbances and hepatorenal toxicity, remain significant concerns. CrCi-CDs, which are derived from natural sources, demonstrate a favorable safety profile. Both prior research and the current study have established that CrCi-CDs exhibit a markedly enhanced neuroprotective efficacy, thereby suggesting their potential applicability as an adjunctive therapeutic agent in the clinical management of epilepsy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, leveraging insights from previous research, this study achieved the successful synthesis of CrCi-CDs utilizing human hair as a precursor material. The anti-epileptic efficacy of CrCi-CDs was systematically validated through the employment of three distinct murine epilepsy models, each induced by differing etiological factors. The therapeutic mechanisms underlying the anti-epileptic effects of CrCi-CDs appear to be attributable to the modulation of neurotransmitter homeostasis, the attenuation of neuroinflammatory responses and oxidative stress, and the suppression of apoptotic pathways within neural cells. Furthermore, this study substantiates the broad-spectrum neuroprotective properties of CrCi-CDs, thereby establishing a foundational basis for their prospective integration into clinical therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e The animal protocols were approved by the Committee of Ethics of Animal Experimentation of the Beijing University of Chinese Medicine.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003e All the co-authors were aware of this submission and approve for publication.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors have no other relevant afliations or fnancial involvement with any organization or entity with a fnancial interest in or fnancial confict with the subject matter or materials discussed in the manuscript apart from those disclosed.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Beijing Municipal Natural Science Foundation - Haidian Original Innovation Joint Fund Sponsored Projects (L222132), National Natural Science Foundation of China (82204914) and Fundamental Research Funds for the Central Universities (China) (2024-JYB-JBZD-0400, 2024-JYB-JBZD-023, 2024-JYB-JBZD-045).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eExperiments were designed by YZ, HK and YZ, and conducted by JH, XKW, KC, YFZ and XRT. YZ, HK and YZ support the funding. Data was analyzed by JH, KC, YH, CXH, XWZ, PZ and XHQ. Manuscript was prepared by JH and KC, and was revised by KC and KH. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBeghi E, Giussani G, Sander JW. The natural history and prognosis of epilepsy. Epileptic Disord. 2015;17(3):243\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTHIJS RD, SURGES R, O'BRIEN T J, et al. Epilepsy in adults[J]. Lancet. 2019;393(10172):689\u0026ndash;701.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu N, Lin XJ, Zhang SG, et al. Analysis of the reasons and costs of hospitalization for epilepsy patients in East China. Seizure. 2019;72:40\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalco-Walter J, Epilepsy-Definition. Classification, Pathophysiology, and Epidemiology. 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Biotechnol Appl Biochem. 2022;69(4):1633\u0026ndash;45.\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":"Epilepsy, Carbon Dots, Apoptosis, Neuroprotection","lastPublishedDoi":"10.21203/rs.3.rs-6202061/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6202061/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEpilepsy is a brain neurological disease with a high incidence and recurrent attacks. Currently, there is still a lack of simple, long-term prevention and control measures. Crinis Carbonisatus (named \u0026ldquo;Xue-yu-tan\u0026rdquo; in Chinese) is forged from healthy human hair and is widely used in traditional Chinese medicine to treat epilepsy, hemostasis, stroke and other diseases. Previous studies have successfully isolated and characterized carbon dots derived from Crinis Carbonisatus (CrCi-CDs), confirming their pharmacological activity in treating ischemic stroke and demonstrating neuroprotective effects against neural injury. Building on these findings, this study aims to explore the potential therapeutic effects of CrCi-CDs on acute epilepsy.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eClean, healthy human hair was calcined in a muffle furnace at 350\u0026deg;C for 1 hour and then decocted in deionized water and filtered to obtain a solution of CrCi-CDs. We used Pentylenetetrazole (PTZ), Pilocarpine (PILO) and Penicillin (PNC) to simulate clinical epileptogenic factors to establish three acute epilepsy models in mice and investigate the anti-epileptic effect of CrCi-CDs. We explored whether CrCi-CDs can reduce nerve excitability, improve nerve tissue inflammation, and oxidative stress levels, thereby reducing nervous system damage and improving epileptic symptoms. Based on the classic neuronal apoptosis pathway, we preliminarily explored the anti-epileptic mechanism of CrCi-CDs.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this study, we successfully isolated CrCi-CDs by referring to the previous method. CrCi-CDs is spherical in shape, well dispersed in aqueous solution, with uniform and consistent particle size distribution, and contains a large number of hydroxyl, amino and carbonyl/carboxy groups on the surface. The antiepileptic effects of CrCi-CDs were evaluated using Pentylenetetrazole (PTZ), Pilocarpine (PILO) and Penicillin (PNC)-induced epileptic mouse models. After CrCi-CDs intervention, the latency period of epileptic mice in each group was prolonged, and their spatial learning and memory abilities were improved. In addition, nerve damage in the hippocampus of epileptic mice was reduced by the CrCi-CDs intervention, the imbalance of neurotransmitters such as Glutamic acid (GLU) and Gamma-Aminobutyric acid (GABA) was regulated, the levels of inflammatory factors such as Interleukin-1β(IL-1β), Interleukin-6 (IL-6), Tumor Necrosis Factor-α(TNF-α) and Interleukin-18 (IL-18), and oxidative stress such as malondialdehyde (MDA) and superoxide dismutase (SOD) was improved. The above results showed that the improvement effect of high-dose CrCi-CDs was the most significant. Initial mechanistic investigations suggest that CrCi-CDs may ameliorate epileptic damage by suppressing neuronal apoptosis in brain tissue through modulation of the Bax/Bcl-2/Caspase-3 signaling pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCrCi-CDs show significant anti-epileptic potential, which may be achieved through multiple pathways including regulating neurotransmitter balance, inhibiting neuroinflammation and oxidative stress. This study lays the foundation for the clinical application of CrCi-CDs and further drug development.\u003c/p\u003e","manuscriptTitle":"The neuroprotective effect of carbon dots from Crinis Carbonisatus (carbonized human hair) against epilepsy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-14 10:03:55","doi":"10.21203/rs.3.rs-6202061/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":"842be061-5137-46d7-836d-7f6e1950c7a0","owner":[],"postedDate":"March 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-24T04:23:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-14 10:03:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6202061","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6202061","identity":"rs-6202061","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}