Introduction
Drug-resistant epilepsy (DRE) can have devastating effects on children and imposes substantial morbidity and mortality on affected individuals, their families, and society. Despite the availability of more than 20 antiseizure medications (ASMs), approximately 30% of patients with epilepsy do not achieve adequate seizure control [1]. Although current ASMs help control seizures, they primarily address symptoms and do not directly target the mechanisms that generate seizures [2]. Long-term ASM therapy has also been associated with side effects, including cognitive impairment, fatigue, and behavioral disorders [3]. The molecular mechanisms underlying epileptogenesis remain incompletely understood. Therefore, a more comprehensive understanding of seizure-related molecular pathways is needed to support the development of targeted therapeutic approaches.
Inflammatory mediators secreted by brain cells during epileptic activity, including cytokines, complement factors, and prostaglandins, contribute to epileptogenesis. These mediators not only stimulate inflammation but also have neuromodulatory effects that influence neuronal function and excitability [4]. The Toll-like receptor (TLR) family plays a central role in immune activation and inflammatory responses [5]. Among these receptors, TLR4 recognizes lipopolysaccharides and initiates inflammatory cascades by stimulating gene expression. This receptor has been extensively studied as investigators continue to clarify its involvement in a wide range of pathological conditions. High-mobility group box 1 (HMGB1) is an endogenous activator of TLR4 and is classified as a damage-associated molecular pattern released from injured or disrupted tissues [6]. Studies using animal models and/or brain samples have demonstrated that both HMGB1 and TLR4 contribute to seizure development and persistence [7]. The HMGB1–TLR4 regulatory axis plays a key role in seizure generation and exacerbation and may represent a therapeutic target for interventions designed to inhibit this signaling pathway.
Several previous studies have examined inflammatory mediators, particularly HMGB1 and TLR4, in epilepsy. Research in adults has demonstrated elevated serum HMGB1 and TLR4 levels in epilepsy, with higher concentrations in drug-resistant cases and positive correlations between both markers and seizure frequency [8]. Pediatric studies have also reported increased HMGB1 levels, particularly among children with drug resistance, as well as significant associations with cognitive impairment [9]. In addition, upregulation of inflammatory cytokines such as interleukin (IL)-1β, IL-1R1, and HMGB1 has been observed in pediatric DRE, suggesting a link between neuroinflammation and seizure refractoriness [10]. Despite these contributions, several areas remain insufficiently explored. Previous studies have not jointly analyzed HMGB1 and TLR4 expression specifically in pediatric epilepsy or examined their associations with radiological abnormalities and age-related immune maturation. Moreover, prior research has provided limited clinical characterization, with insufficient attention to detailed electroencephalogram (EEG) findings, neuroimaging features, etiologic classifications, and epilepsy syndromes. The present study addresses these limitations by evaluating the combined role of HMGB1 and TLR4 as potential serum biomarkers in children with epilepsy and correlating them with clinical, electrophysiological, and radiological characteristics, thereby providing further insight into inflammatory processes underlying pediatric DRE.
Materials and Methods
1. Study design and participants
This cross-sectional observational study used consecutive sampling and was conducted among pediatric patients with epilepsy who were hospitalized or attended the Pediatric Neurology Outpatient Clinic at Sardjito Hospital, Yogyakarta, between April 1 and December 31, 2022. The study enrolled patients aged 6 months to 18 years who were diagnosed with DRE or drug-responsive epilepsy according to International League Against Epilepsy (ILAE) criteria and whose parents provided consent for participation. The criteria used to classify patients into the DRE and drug-responsive epilepsy groups followed ILAE guidelines, as shown in Supplementary Table 1 [11].
Patients were excluded if they had systemic, neurological, infectious, autoimmune, malignant, metabolic, renal, or hepatic conditions that could affect seizure activity or inflammatory biomarker levels. These conditions included hypoglycemia, degenerative diseases, traumatic brain injury, cerebral ischemic injury, cerebral hemorrhage, sepsis, meningoencephalitis, pneumonia, and other acute infections. Patients receiving immunosuppressive therapy or those with recent vaccination were also excluded. In addition, patients with modifiable seizure triggers, such as sleep deprivation or poor medication adherence, were excluded. Patients who did not meet the criteria for either DRE or drug-responsive epilepsy were also excluded, including those who had not experienced seizures but had less than 1 year of follow-up after ASM therapy and those with uncontrolled seizures due to inappropriate ASM selection or failure to achieve an effective therapeutic dose. All patients who met the inclusion and exclusion criteria were enrolled sequentially as they presented to the clinic or were admitted to the hospital, without randomization or prior stratification. The nearly equal distribution between the drug-resistant and drug-responsive epilepsy groups resulted naturally from this consecutive recruitment process.
2. Ethics approval and consent to participate
The Medical and Health Ethics Committee of the Faculty of Medicine, Universitas Gadjah Mada, approved this study (KE/FK/0264/EC/2022). Patients were included after their parents signed an informed consent form.
3. Procedures and data collection
Demographic and clinical data were collected, including age, sex, seizure type, disease course, average seizure duration, seizure frequency, and ASM type. History of status epilepticus, family history of epilepsy, presence of epilepsy syndromes, epilepsy etiology, EEG findings, neuroimaging results from magnetic resonance imaging and/or computed tomography, and other relevant clinical information were also documented. Seizure frequency was evaluated using standardized sources, such as family reports or physician records. Seizure duration was defined as the total observable ictal period, measured from the onset of clinical seizure activity to its cessation and excluding the postictal recovery phase. Epilepsy syndromes were classified according to the ‘ILAE position statement on the definition and classification of epilepsy syndromes with onset in neonates and infants’ (2022).
Based on these criteria, patients were categorized into three major groups: self-limited epilepsies; developmental and epileptic encephalopathies; and etiology-specific syndromes [12]. Etiologic classification was based on available clinical history, seizure semiology, neurological examination, EEG findings, neuroimaging results, and relevant clinical investigations. Genetic etiology was recorded when a documented clinical or laboratory diagnosis suggestive of genetic epilepsy was available; however, genetic testing was not systematically performed in all participants.
Serum HMGB1 and TLR4 levels were measured once for each participant. Blood samples were collected after a minimum fasting period of 8 hours. In the DRE group, samples were obtained within 24 hours after a seizure episode, whereas in the drug-responsive group, samples were collected during a clinically stable fasting condition. Samples were centrifuged for 20 minutes at approximately 1,000 ×g to obtain the supernatant for testing. The blood collection tubes were single-use, nonpyrogenic, and nonendotoxic. Samples intended for use within 5 days were stored at 2°C to 8°C to maintain integrity. Blood samples were processed and tested at the Integrated Research Laboratory of the Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, under special protection protocols. Qualified laboratory personnel blinded to patients’ clinical information measured serum HMGB1 and TLR4 concentrations using a sandwich enzyme-linked immunosorbent assay (ELISA). Concentrations were measured using a commercially available ELISA kit according to the manufacturer’s instructions (ABclonal Technology, Woburn, MA, USA). Each test was performed at least twice for calibration and validation. Specifically, 100 µL of the standard reagent or sample was added to each plate and incubated for 2 hours at 37°C. The liquid was then removed from each plate without washing. Subsequently, 100 µL of the biotin-labeled antibody working solution was added to each plate and incubated for 60 minutes at 37°C. The plates were then aspirated and washed three times. Next, 100 µL of the horseradish peroxidase-avidin solution was added to each plate and incubated for 30 minutes at 37°C. The plates were then aspirated and washed five times. Then, 90 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added and incubated for 15–30 minutes at 37°C while avoiding exposure to light. Finally, 50 µL of stop solution was added, and the plates were read at 450 nm within 5 minutes, followed by the necessary calculations.
4. Sample size calculation
The sample size was determined using a formula for comparing two independent means, with reference to previous research demonstrating differences in HMGB1 levels between patients with drug-resistant and drug-responsive epilepsy. Using an estimated effect size of 0.70, a two-sided α of 0.05, and 80% power, the minimum required sample size was 30 participants per group. To account for potential data loss or withdrawal, 66 participants were included in the study, with 33 participants per group.
5. Statistical analysis
Statistical analyses were performed using IBM SPSS Statistics for Windows, version 29 (IBM Corp., Armonk, NY, USA). Descriptive statistics were used to summarize demographic and clinical characteristics. Between-group differences were analyzed using the independent t-test for normally distributed variables and the Mann–Whitney U test for non-normally distributed data. Age was categorized using a cutoff of 5 years as an exploratory clinical subgroup to reflect potential developmental and immunological differences within the pediatric population. To address the potential limitation of dichotomization, an additional sensitivity analysis was performed by treating age as a continuous variable. Correlations between serum HMGB1 and TLR4 levels and clinical parameters, such as seizure frequency and disease duration, were assessed using Spearman rank correlation coefficient for nonparametric data and Pearson correlation coefficient for parametric data. Multivariable linear regression analyses were performed to evaluate factors associated with serum HMGB1 and TLR4 levels after adjustment for relevant covariates. Variables included in the model were drug responsivity, age, seizure frequency, and neuroimaging results, based on clinical relevance and significant findings in the univariate analysis. All estimates were reported with 95% confidence intervals (CIs). Diagnostic accuracy was evaluated using receiver operating characteristic (ROC) curve analysis to determine the area under the curve (AUC), optimal cutoff points, sensitivity, and specificity. ROC curves were generated for each biomarker individually and for their combined use through a linear composite model. ROC analysis was performed without covariate adjustment because the objective was to assess unadjusted discriminatory performance. Statistical significance was set at P<0.05.
Results
This cross-sectional study was conducted at the child neurology outpatient and inpatient clinics of Sardjito Hospital in Yogyakarta from April 1 to December 31, 2022. Initially, 285 pediatric patients with epilepsy of varying severity were identified. Of these, 66 met the inclusion and exclusion criteria, with 33 patients in the DRE group and 33 in the drug-responsive epilepsy group. Participant selection is illustrated in Fig. 1.
The mean ages were 5.65±3.78 years in the DRE group and 6.91±4.87 years in the drug-responsive epilepsy group, with no significant between-group difference. The two groups were comparable in patient age, sex, seizure type, disease duration, seizure duration, EEG features, history of status epilepticus, family history of epilepsy, and epilepsy syndrome course (P>0.05). However, compared with the drug-responsive group, the DRE group had a significantly different etiologic distribution and a significantly higher proportion of patients with seizure frequency >3 times/month. The demographic and clinical characteristics of each group are presented in Table 1.
As shown in Table 2, the mean HMGB1 level was significantly higher in the DRE group (298.92±52.30 pg/mL) than in the drug-responsive group (237.27±41.48 pg/mL). Similarly, the mean TLR4 level was significantly higher in the DRE group (139.55±22.56 pg/mL) than in the drug-responsive group (122.18±11.08 pg/mL). Higher HMGB1 and TLR4 levels were also observed in children aged >5 years, patients with seizure frequency >3 per month, and patients with abnormal radiological findings.
Multivariable linear regression analysis was performed to evaluate factors associated with serum HMGB1 and TLR4 levels after adjustment for drug responsivity, age, seizure frequency, and neuroimaging results. DRE remained significantly associated with higher HMGB1 levels (B=49.1, SE=14.7, β=0.442, P=0.001) and higher TLR4 levels (B=13.9, SE=5.8, β=0.355, P=0.020). Age >5 years was also independently associated with higher HMGB1 levels (B=39.7, SE=10.8, β=0.357, P=0.001) and higher TLR4 levels (B=8.7, SE=4.3, β=0.223, P=0.046). In contrast, seizure frequency and neuroimaging results were not independently associated with HMGB1 or TLR4 levels after adjustment. These findings are presented in Table 3. In an additional sensitivity analysis in which age was treated as a continuous variable, age remained significantly associated with HMGB1 and TLR4 levels after adjustment for drug responsivity, seizure frequency, and neuroimaging findings.
The performance of HMGB1, TLR4, and the combined HMGB1–TLR4 model in assessing epilepsy severity is described in Table 4. HMGB1 showed sensitivity of 81.8%, specificity of 60.6%, and an AUC of 0.805, with P<0.001. The AUC CI was 0.702–0.910, and the HMGB1 cutoff value was 247.5 pg/mL. In comparison, TLR4 showed sensitivity of 72.7%, specificity of 51.5%, and an AUC of 0.733, with P=0.001 at a cutoff value of 122.5 pg/mL. The AUC CI for TLR4 was 0.613–0.854. The combined HMGB1–TLR4 model showed an AUC of 0.819, with sensitivity of 81.8% and specificity of 66.7%, representing a modest improvement compared with HMGB1 or TLR4 alone. The discriminatory ability of HMGB1 and TLR4 as biomarkers for assessing epilepsy severity in children is further illustrated by the ROC curve shown in Fig. 2. In addition, the Pearson correlation coefficient was 0.587 (P=0.001), indicating a significant relationship between HMGB1 and TLR4 levels (Fig. 3).
Discussion
This cross-sectional study investigated serum HMGB1 and TLR4 levels in pediatric patients with epilepsy and examined their relationship with epilepsy severity. The results showed that (1) serum HMGB1 and TLR4 levels were higher in patients with DRE than in those with drug-responsive epilepsy; (2) higher HMGB1 and TLR4 levels were associated with more frequent seizures, older age, and radiological abnormalities; and (3) higher HMGB1 and TLR4 levels were associated with a greater likelihood of ASM resistance.
The mean serum HMGB1 level was significantly higher in the DRE group (298.92±52.30 pg/mL) than in the drug-responsive group (237.27±41.48 pg/mL). This finding is consistent with several previous studies that reported higher serum HMGB1 levels in patients with DRE, including children [13,14] and adults [5,8,15]. In a study comparing HMGB1 levels across neurological conditions, levels were higher in the encephalopathy and meningitis group than in the West syndrome and control groups [16]. Another study reported that HMGB1 expression in the cerebrospinal fluid (CSF) was significantly higher in the DRE group [17]. However, a study conducted in Iraq found no significant differences in serum HMGB1 levels between patients with epilepsy and healthy participants or between patients with controlled and refractory epilepsy [18].
The mean serum TLR4 level was significantly higher in the DRE group (139.55±22.56 pg/mL) than in the drug-responsive epilepsy group (122.18±11.08 pg/mL). Although research on serum TLR4 levels in patients with epilepsy remains limited, a preliminary study found significantly higher TLR4 levels in patients with epilepsy than in healthy controls [5]. These findings are consistent with studies reporting higher serum TLR4 levels in both children [14] and adults [8] with DRE. In addition, a study of TLR4 genotypes and epilepsy risk in children showed that the AA genotype of the TLR4 single-nucleotide polymorphism rs11536858 was associated with a higher risk of epilepsy than the GG genotype and was also associated with an increased risk of clonazepam resistance [19].
The role of HMGB1 in DRE is closely related to inflammatory mechanisms, blood–brain barrier disruption, and increased neuronal excitability. As a damage-associated molecular pattern, HMGB1 is released by damaged cells and triggers inflammatory responses, which may contribute to DRE development [20]. Chronic inflammation can impair blood–brain barrier permeability, reducing the ability of ASMs to reach the brain while allowing additional inflammatory molecules to enter the brain, thereby creating a self-perpetuating inflammatory cycle and worsening seizure control [21]. HMGB1 can also influence neuronal excitability by increasing the release of glutamate, the primary excitatory neurotransmitter in the brain. High glutamate levels contribute to neuronal hyperexcitability, which can trigger seizures. This process is exacerbated by changes in synaptic plasticity commonly observed in patients with DRE, in which HMGB1 is believed to play a key role [20,22,23]. HMGB1 also interacts with TLR4 on immune cells, including microglia in the brain. HMGB1-induced TLR4 activation triggers the release of additional inflammatory molecules, prolongs the inflammatory response, and contributes to a chronic inflammatory state. This process can amplify neuronal hyperexcitability, thereby worsening seizures and reducing ASM effectiveness [14,24,25]. Overall, inflammatory mechanisms mediated by HMGB1 and TLR4 may contribute to DRE development and complicate clinical management.
This study found significantly higher serum HMGB1 and TLR4 levels in patients with seizure frequency exceeding three times per month. Previous studies also demonstrated significant differences in HMGB1 and TLR4 levels according to seizure-frequency thresholds of more than two times [8] and three times per month [5]. This finding suggests a positive correlation between serum HMGB1/TLR4 expression and seizure frequency. Repeated seizures may create recurrent cycles of HMGB1 release, thereby exacerbating inflammation [25]. Through its interaction with TLR4, HMGB1 activates microglia and astrocytes, leading to the release of proinflammatory cytokines and reactive oxygen species. TLR4 activation in neurons can also directly influence synaptic plasticity, potentially contributing to the development of hyperexcitable brain networks [26]. In addition, chronic inflammation induced by HMGB1 and TLR4 can compromise the blood–brain barrier, which may inhibit ASM delivery to the brain and allow more peripheral inflammatory molecules to enter, further exacerbating seizures [27].
In this study, patients with abnormal radiological findings, such as lesions, tumors, or malformations, had significantly higher HMGB1 and TLR4 levels. Radiological abnormalities can cause cellular stress and damage, triggering the release of HMGB1 from damaged neurons and glial cells. HMGB1 then binds to TLR4 on immune cells, activating inflammatory pathways and promoting proinflammatory cytokine production. In addition, some radiological abnormalities can disrupt the blood–brain barrier, allowing peripheral immune cells and inflammatory molecules to enter the brain and further intensify inflammation and HMGB1/TLR4 signaling. Chronic brain inflammation associated with radiological abnormalities, such as scarring or gliosis, may result in persistent immune activation and sustained elevation of HMGB1 and TLR4 levels [28]. This process may establish a positive feedback loop in which HMGB1 activates TLR4, leading to cytokine production that stimulates further HMGB1 release and amplifies the inflammatory response.
This study found higher HMGB1 and TLR4 levels in children aged >5 years than in younger children. Previous research on the relationship between age and HMGB1/TLR4 expression has not specifically focused on pediatric populations. In adult samples, one study reported no significant differences in HMGB1 and TLR4 levels between patients aged <35 and >35 years [8]. However, the immune system undergoes substantial maturation throughout childhood. Children aged >5 years may have a more developed immune response to triggers that induce HMGB1 and TLR4 release, resulting in higher detectable levels of these molecules [29]. The blood–brain barrier also undergoes developmental changes; although conclusive evidence that it becomes more permeable with age is lacking, subtle functional shifts could contribute to the observed differences in HMGB1 and TLR4 levels in older children [27]. In addition, children aged >5 years may have experienced a greater cumulative seizure burden and associated inflammation, leading to higher HMGB1 and TLR4 levels. This mechanism may be particularly relevant in the context of DRE, in which prolonged seizure activity and inflammation may create a self-perpetuating cycle [5,23]. However, the 5-year cutoff should be interpreted as an exploratory clinical categorization rather than a definitive biological threshold. Further studies are needed to validate age-related differences in HMGB1 and TLR4 levels across pediatric developmental stages.
This study demonstrated a significant positive correlation between serum HMGB1 and TLR4 levels, with a Pearson correlation coefficient of 0.587 and P=0.001. This finding is consistent with previous studies in both adult and pediatric populations [5,8]. The correlation suggests that HMGB1 and TLR4 may be components of an interconnected pathway involved in DRE rather than acting independently. HMGB1 is a known ligand for TLR4. When HMGB1 is released after cellular stress or seizure-induced damage, it binds to TLR4 on microglia, astrocytes, and neurons, triggering a downstream inflammatory cascade. This direct interaction may explain the observed correlation, in which higher HMGB1 levels are associated with greater TLR4 activation [30]. HMGB1-induced TLR4 activation triggers the release of proinflammatory cytokines and other signaling molecules, which may further exacerbate neuronal damage and blood–brain barrier dysfunction and lead to additional HMGB1 release. This process creates a positive feedback loop, amplifying the inflammatory response and strengthening the association between HMGB1 and TLR4 levels [31].
In this study, serum HMGB1 showed sensitivity of 81.8%, specificity of 60.6%, and an AUC of 0.805, with P<0.001. TLR4 showed sensitivity of 72.7%, specificity of 51.5%, and an AUC of 0.733, with P=0.001. The combined HMGB1–TLR4 model slightly improved discriminatory performance compared with either biomarker alone, with an AUC of 0.819. However, this improvement was modest, and the combined model remains exploratory. External validation in larger cohorts is required before the model can be considered for clinical use. The AUC values, particularly for HMGB1, indicate moderate to good ability to differentiate between DRE and drug-responsive epilepsy. An AUC of 0.8 is often considered a reasonable starting point for biomarker development. The sensitivity and specificity values are promising, but they are not high enough to support the use of HMGB1 as the sole diagnostic marker for DRE. Similarly, TLR4 is not suitable for single-marker evaluation because of its lower AUC and specificity. This limitation reflects the involvement of HMGB1 and TLR4 in various inflammatory and immune responses, not only epilepsy-specific processes. Levels of these molecules are elevated in several other neurological conditions, infections, and systemic inflammatory states [5,14,23]. To address this limitation, strict inclusion and exclusion criteria were used during participant recruitment. Because HMGB1 and TLR4 were measured after clinical classification according to ILAE criteria, these biomarkers should not currently be interpreted as tools for establishing a diagnosis of DRE in routine clinical practice. Rather, they may serve as adjunctive exploratory biomarkers reflecting inflammatory burden, seizure activity, or treatment resistance when interpreted together with clinical history, seizure frequency, EEG findings, neuroimaging results, and treatment response. Their modest specificity and the cross-sectional design of this study preclude their use as standalone diagnostic markers. Longitudinal studies comparing clinical outcomes between patients with high and low HMGB1 or TLR4 levels are needed to clarify their prognostic value.
The absence of a healthy control group is an acknowledged limitation of this study. Because of the ethical limitations of obtaining blood samples from healthy children, recruitment of such a group was not feasible. Nevertheless, based on previous literature demonstrating elevated HMGB1 and TLR4 levels in epilepsy, we hypothesized that serum levels would be higher in our patient cohort than in healthy individuals. Another limitation is the lack of CSF measurements, which could have provided complementary insight into central inflammatory processes and improved diagnostic specificity. However, lumbar puncture is not routinely performed in pediatric patients with epilepsy without a clinical indication; therefore, CSF sampling was not ethically feasible in this population. To improve diagnostic accuracy and provide a more comprehensive understanding of the inflammatory processes underlying DRE, further studies should explore HMGB1 and TLR4 in combination with other potential biomarkers, such as inflammatory cytokines or microRNA profiles.
The heterogeneous etiology of epilepsy in this cohort should be considered when interpreting the results. Although etiology was categorized in relation to HMGB1 and TLR4 levels, epilepsy etiology may still have influenced the observed biomarker patterns. Structural brain abnormalities, malformations, gliosis, prior hypoxic or infectious injury, and other symptomatic etiologies may independently increase neuroinflammatory activity. Thus, residual confounding by epilepsy etiology cannot be excluded. The present findings should therefore be interpreted as exploratory associations rather than evidence of independent causality. In addition, genetic etiologies could not be comprehensively evaluated because genetic testing was not systematically available for all participants. Therefore, patients classified as having unknown, idiopathic, or nongenetic etiology may still have had undetected genetic epilepsy. This is important because some genetic epilepsies may intrinsically alter inflammatory signaling pathways, potentially contributing to variation in serum HMGB1 and TLR4 levels and affecting the diagnostic accuracy of these biomarkers. Future studies should incorporate standardized genetic testing to clarify the relationship between genetic etiology and inflammatory biomarkers in pediatric epilepsy.
Because HMGB1 and TLR4 were measured only once in each participant, this study could not evaluate intra-individual temporal changes in biomarker levels or determine whether these markers fluctuate according to seizure timing, seizure burden, or treatment response. This single-time-point design also limits the ability to assess prognostic value. Longitudinal studies tracking changes in HMGB1 and TLR4 levels over time in relation to treatment response and seizure control may therefore provide valuable insight into their potential role as prognostic biomarkers. The timing of blood sampling differed between groups. In the DRE group, samples were obtained within 24 hours after the most recent seizure episode, whereas in the drug-responsive group, samples were collected during a clinically stable fasting condition. This difference may have influenced the observed biomarker levels because HMGB1 and TLR4 may increase after seizure events. However, ongoing seizure activity is also part of the clinical phenotype of DRE, as these patients continue to have seizures despite adequate trials of two appropriately chosen and tolerated ASMs. Therefore, elevated HMGB1 and TLR4 levels may reflect the inflammatory burden associated with active DRE, including recent seizure-related inflammatory activation. Nevertheless, this study cannot distinguish persistent inflammation related to drug resistance from transient postictal inflammatory changes. Future longitudinal studies with standardized seizure-to-sampling intervals are needed to clarify whether HMGB1 and TLR4 are markers of drug resistance, recent seizure burden, or both. To further clarify inflammatory mechanisms underlying epilepsy, future studies should validate these findings through longitudinal designs and incorporate additional biomarkers such as IL-1β and tumor necrosis factor-α, which are known to contribute to seizure generation through inflammatory pathways. Future research should also assess the prognostic value of HMGB1 and TLR4 in predicting treatment response and long-term outcomes in pediatric epilepsy and explore the potential for targeted therapies that modulate HMGB1/TLR4 activity.
This study supports a potential role for HMGB1 and TLR4 in inflammatory processes associated with pediatric DRE. Significantly higher serum HMGB1 and TLR4 levels were observed in patients with DRE, seizure frequency exceeding three times per month, age >5 years, and radiological imaging abnormalities. Furthermore, serum HMGB1 and TLR4 levels were positively correlated. Although HMGB1 and TLR4 show promise as potential biomarkers, their individual diagnostic value is limited when they are used alone.
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