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. 2026 Feb 10;46:50. doi: 10.1007/s10571-026-01683-0

Early HMGB1 Inhibition Reduces Hippocampal Injury During Adolescence in a Young Mouse Model of Radiation-Induced Brain Injury

Yanyan Sun 1,#, Mingrui Shi 2,#, Leijie Ma 3, Huanhuan Xie 3, Mingyan Hei 2,✉, Wenli Zuo 1,✉
PMCID: PMC12946357  PMID: 41667899

Abstract

Radiotherapy is a primary treatment for childhood malignant brain tumors. However, it often leads to radiation-induced brain injury (RIBI), which significantly impairs neurodevelopment in pediatric patients. Effective treatment for this complication remains limited. Given the established link between RIBI-associated neuroinflammation and HMGB1, we investigated the neuroprotective potential of early HMGB1 inhibition in a young mouse model. Four-week-old male C57BL/6 mice received a single 10 Gy whole-brain irradiation and were divided into Sham + PBS, Rad + PBS, and Rad + glycyrrhizin (GL, 10 mg/kg for 14 days post-irradiation) groups. Immunofluorescence showed that irradiation significantly triggered nuclear-to-cytoplasmic translocation of HMGB1 in hippocampus 7 days post-irradiation. Preliminary data suggests that GL administration abrogates increases in HMGB1 expression and translocation as well as of the key inflammatory receptors TLRs and RAGE. Immunofluorescence and Nissl staining confirmed that GL treatment inhibited microglial activation and mitigated neuronal loss 14 days post-irradiation. Critically, behavioral assessment via the Morris water maze 8 weeks post-irradiation showed that this early intervention significantly improved spatial learning and memory deficits. Complementary preliminary in vitro experiments demonstrate irradiation-associated increases in microglia-derived inflammatory mediators and corresponding changes in neuronal MAP2 staining following exposure to microglia-conditioned media, with altered cytokine profiles observed in the presence of GL. Collectively, our results demonstrate that early pharmacological blockade of the HMGB1 pathway alleviates microglial activation and hippocampal damage, offering a potential new treatment target for pediatric RIBI.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10571-026-01683-0.

Keywords: Radiation-induced brain injury, HMGB1, Microglia, Neuroinflammation, Hippocampus, Children

Introduction

Malignant brain tumors are the second most common type of pediatric malignant tumor, and radiotherapy is the first-line treatment for primary and metastatic brain tumors (Cheng et al. 2023). However, the radiation field includes the normal brain tissue surrounding the tumor, which may lead to neuronal loss, abnormal gliosis, a loss of capillaries or a sparse microvasculature, and the involvement of the hippocampus in the temporal lobe may lead to cognitive dysfunction, such as learning and memory impairments, known as radiation-induced brain injury (RIBI) (Tang et al. 2017), posing a significant threat to the normal development of the pediatric nervous system. Moreover, children’s brains are not yet fully developed and are more susceptible to damage than are those of adults (Rübe et al. 2023).

In the acute phase and early delayed phase of RIBI, the main clinical manifestations include headache, nausea, vomiting, and mood changes such as lethargy and irritability (Wang et al. 2024), and most of these symptoms are reversed with treatment. However, more than 50%-90% of patients who survive more than 6 months after whole-brain radiotherapy develop irreversible cognitive impairments that affect their quality of life (Rübe et al. 2023; Zhou et al. 2021; Liu et al. 2022). Currently, an effective treatment to improve RIBI is unavailable (Zhou et al. 2021). Therefore, exploration of the pathophysiological mechanisms of pediatric RIBI is urgently needed to identify effective treatments.

The molecular and cellular mechanisms of RIBI are complex (Liu et al. 2022), and the main pathological characteristics of RIBI include overactivation of glial cells, neuroinflammation, immune cell infiltration, disruption of the blood‒brain barrier, and neuronal loss (Rübe et al. 2023; Wang et al. 2024). Increasing evidence has shown that microglia play a significant role in neuroinflammatory processes following exposure to ionizing radiation (He et al. 2020; Zhang et al. 2022). Microglia are resident immune cells of the central nervous system that play key roles in immune surveillance and maintaining homeostasis of the central nervous system (Salter and Stevens 2017; Woodburn et al. 2021). Studies have indicated that microglia are highly sensitive to radiation, and within hours after exposure, microglia rapidly become activated (Rübe et al. 2023; Wang et al. 2024; Zhang et al. 2022). Activated microglia are cytotoxic and can secrete proinflammatory cytokines such as IL-1β, IL-6, and TNF, impairing neurogenesis in the hippocampus (Liu et al. 2022). Research has shown that a radiation dose of 10 Gy can induce microglial activation and trigger the release of proinflammatory cytokines, resulting in the occurrence of RIBI (Dong et al. 2015).

High mobility group box protein 1 (HMGB1) is a nonhistone nuclear DNA-binding protein that is widely expressed in the nervous system and can promote brain development under physiological conditions (Sun et al. 2019; Le et al. 2020). HMGB1 is located in the nuclei of various cells and is expressed primarily in neurons. Ionizing radiation can upregulate the expression of HMGB1, promoting its translocation to the cytoplasm and extracellular space, where it binds to cell surface receptors such as receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs), inducing microglia to release proinflammatory cytokines and participating in the signaling pathways of neuroinflammatory responses (Wang et al. 2024; Fan et al. 2020). Studies have shown (Zhang et al. 2022) that knocking out the HMGB1 gene in neurons or the TLR2, TLR4, and RAGE genes in microglia can effectively suppress the vicious cycle of radiation-induced microglial activation, neuroinflammation, and neuronal injury. However, whether early inhibition of HMGB1 can improve hippocampal injury during adolescence in pediatric RIBI patients remains unclear. This study used in vivo and in vitro models to verify that early inhibition of HMGB1 after brain irradiation can reduce the activation of microglia, alleviate neuroinflammatory responses, and reduce hippocampal injury caused by RIBI during adolescence. Therefore, it holds promise as a potential therapeutic strategy for pediatric RIBI.

Materials and methods

Animals and Ethical Permission

Four-week-old male C57BL/6 mice (weighing 12–16 g) were purchased from SPF (Beijing, China) Biotechnology Co., Ltd. This specific age group was selected as it represents a key developmental stage corresponding to human childhood, making it a well-established model for RIBI. To minimize potential confounding effects arising from hormonal fluctuations associated with the female estrous cycle, only male mice were included in the current study. All the experiments were performed in accordance with the National Institute of Health guidelines. The protocol was approved by the Institutional Animal Care and Use Committee of Zhengzhou University (No. 2021042501). All the mice were housed in a facility with a 12-h light/dark cycle and free access to food and water with 5–7 mice per cage. Efforts were made to reduce the number of animals used and mitigate their suffering during the experiment.

Randomization and Blinding in Animal Study

In our animal study, we used computer-generated random numbers for group allocation to minimize bias and ensure comparability. Each animal was assigned a unique ID matched to the random sequence by a researcher not involved in the experiment, maintaining objectivity. We also implemented a double-blind design, with both the experimenter and data analysts unaware of group assignments until final analysis, ensuring unbiased data collection and enhancing result credibility.

RIBI Animal Model and Drug Administration

The mice were randomly assigned to three groups: Sham + PBS, Rad + PBS, and Rad + glycyrrhizin (GL). Glycyrrhizin was dissolved in DMSO as a stock solution (-20 °C) and freshly diluted before each injection in a sterile vehicle (2% DMSO, 30% PEG300, 2% Tween-80, 66% ddH₂O). For irradiation, mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and subjected to a single 10 Gy cranial dose using a 6 MeV photon beam (TrueBeam SN1403, Varian), targeting the area from the post-canthus to the post-aurem line at 3 Gy/min. The dosage and irradiation parameters were selected based on previously established protocols (Dong et al. 2015). Sham group mice were anesthetized and positioned but not irradiated. After irradiation, body temperature was maintained at 37 °C. Starting on the day of irradiation, the Rad + GL group received daily i.p. injections of glycyrrhizin (10 mg/kg) for 14 days. The dose and duration were selected based on its previously demonstrated neuroprotective efficacy via suppression of the HMGB1 pathway in neural injury models (Fan et al. 2020). The other two groups received an equal volume of 0.01 M PBS on the same schedule.

Morris Water Maze Test

The Morris water maze (MWM) test was used to assess the spatial learning and memory of the mice. It consisted of a labyrinth in a water pool (diameter, 160 cm; height, 50 cm) with a water temperature of 25.0 ± 1.0 °C; the water covered the platform to a depth of 1 cm. The pool was divided into four equivalent quadrants: north, west, south, and east. The platform was placed in a quadrant equidistant from the sidewall and the center of the pool. At the start of the experiment, the mice were placed in a quadrant facing the wall and allowed to swim for 90 s or until the platform was found. If the animal found the platform, it could remain on it for 20 s. If the platform was not found, the animal was directed to the platform and allowed to remain on the platform for 20 s. A camera hanging above the maze and connected to an animal behavior analysis system (SANS, China) was used to record the time taken and the distance traveled by the mouse to find the hidden platform. Beginning at 7 weeks after irradiation, each mouse was tested 4 times a day for 4 consecutive days. On the fifth day, the platform was removed for probe trials to record the percentage of the total time spent in the target quadrant, the percentage of the total distance traveled in the target quadrant, and the number of times the mice crossed the previous platform location.

Immunofluorescence Staining

The animals were anesthetized and transcardially perfused with 0.01 M PBS and 4% paraformaldehyde (PFA). The brains were then removed and postfixed with 4% PFA immediately. After dehydration with a sucrose gradient, 10 serial coronal sections were cut across the middle of the hemisphere. Slices were fixed, rinsed, washed three times with 0.01 M PBS, blocked with 5% bovine serum albumin (BSA), and used for active HMGB1, neuronal nuclei (NeuN), Iba1, and glial fibrillary acidic protein (GFAP) staining. The sections were then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-HMGB1 (1:1000 dilution, Abcam, ab18256, RRID: AB_444360), rabbit anti-NeuN (1:200 dilution, Cell Signaling Technology, 24307, RRID: AB_2651140), rabbit anti-Iba1 (1:100 dilution, Abcam, ab178847, RRID: AB_2832244), mouse anti-GFAP (1:300 dilution, Cell Signaling Technology, 3670T, RRID: AB_561049). After three washes with 0.01 M PBS, the sections were incubated with Cy3-conjugated goat anti-rabbit IgG (1:2000 dilution, Boster Biological Technology, BA1032, RRID: AB_2716305) or FITC-conjugated goat anti-mouse IgG (1:2000 dilution, Boster Biological Technology, BA1101, RRID: AB_10920052) for 1 h at room temperature. After three washes with 0.01 M PBS, the sections were covered with diamidino-2-phenylindole (DAPI, 1:1000, Beyotime, C1002, RRID: AB_3675433) for 5 min.

For primary cortical neurons, cells were fixed with 4% PFA for 24 h following treatment with BV2 cell-conditioned medium, then incubated overnight at 4 °C with a rabbit anti-MAP2 polyclonal antibody (1:200 dilution, Proteintech, 17490-1-AP, RRID: AB_2138153). After three washes in 0.01 M PBS, neurons were incubated with Cy3-conjugated goat anti-rabbit IgG (1:2000 dilution, Boster Biological Technology, BA1032, RRID: AB_2716305) and counterstained with DAPI.

For both tissue sections and cultured neurons, five nonoverlapping digital microscopy images of cortical areas (or MAP2-positive areas) stained with each antibody were randomly captured using a fluorescence microscope (IX71, OLYMPUS, Japan). The integrated optical density (IOD) and percentage of positive signal area was calculated using Image-Pro Plus 6.0 (Media Cybernetics, United States). HMGB1 translocation was quantified by calculating the percentage of HMGB1-positive cells exhibiting a clear extranuclear localization (i.e., signal present in the cytoplasm or extracellular space) relative to the total number of HMGB1-positive cells in the dentate gyrus.

Nissl Staining

The animals were euthanized and perfused with normal saline, followed by 4% paraformaldehyde (PFA). The mouse brains were removed and infiltrated with 4% PFA for 24 h. Xylene and ethanol were used for gradient dehydration, and the brains were subsequently embedded in paraffin. Coronal Sect. (4 μm thick) were prepared using a microtome (CM1950, Leica Biosystems, Wetzlar, Germany). Nissl staining was performed according to the manufacturer’s instructions and visualized using microscopy (LM80196S, LMAI Bio, Shanghai, China) to observe histopathological changes. The hippocampal CA1, CA3, and DG regions was analyzed, taking three images from each area. The number of Nissl bodies was counted in each image, and the average value was calculated for each region.

Western Blotting

Western blotting was performed to assess the expression of HMGB1, RAGE, TLR2, TLR4, β-tubulin and GAPDH in the hippocampus. Briefly, frozen hippocampal samples were completely homogenized in lysis buffer containing phenylmethanesulfonyl fluoride (PMSF, Solarbio, P0100) and radioimmunoprecipitation assay (RIPA, Beyotime, P0013B) buffer and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected and contained the total protein was extracted from the tissue. The quantity of protein in the samples was determined using a BCA protein assay kit (CWBIO, CW0014S) according to the manufacturer’s instructions. The samples were separated via sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) on 12% gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010). The membranes were blocked with 5% nonfat milk for 2 h at room temperature (temperature of 20–25 °C) and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-HMGB1 (1:1000 dilution, Abcam, ab18256, RRID: AB_444360), rabbit anti-RAGE (1:1000 dilution, Cell Signaling Technology, #6996), rabbit anti-TLR2 (1:1000 dilution, Abcam, ab209216, RRID: AB_3662636), rabbit anti-TLR4 (1:1000 dilution, Cell Signaling Technology, #14358, RRID: AB_10830221), rabbit anti-Tubulin (1:5000 dilution, Proteintech, 11224-1-AP, RRID: AB_2210206) and rabbit anti-GAPDH (1:20000 dilution, Proteintech, 10494-1-AP, RRID: AB_2263076). After three washes with PBST (0.01 M PBS containing 0.1% Tween-20), the membranes were incubated with secondary antibodies (goat anti-rabbit IgG, 1:5000 dilution, Licorbio, 926-32211, RRID AB_621843) at room temperature for 2 h. Finally, the protein bands were visualized using a Touch Imager System (Touch Imager XLi, E-Blot) and quantified by densitometry. The relative protein expression levels were normalized by calculating the ratio of the target protein (HMGB1, RAGE, TLR2 and TLR4) to Tubulin or GAPDH.

Primary Neuron Cultures and Microglia-Conditioned Medium Treatment

BV-2 microglial-like cells were seeded into 6-well plates at a density of 1 × 105 cells/mL and incubated overnight in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). BV-2 cells were then exposed to a radiation dose of 10 Gy, and the cells were divided into the following three groups: Ctrl + PBS, Rad + PBS, and Rad + GL. Briefly, PBS (0.01 M) and GL (55 mM) were added to the respective cell groups for 4 h. Finally, the culture supernatant was collected, one part was used for ELISA, and the other part was used as conditioned medium (CM) for primary neurons.

Primary cortical neurons were obtained from P1 mouse pups. Briefly, the cortices of P1 rats were isolated, digested with trypsin, and filtered through a 50 μm sterile nylon filter. The cells were then placed in 24-well plates precoated with poly-L-lysine in Neurobasal medium supplemented with 10% FBS and B27. The cells were placed in an incubator (37 °C, 5% CO2) and allowed to differentiate for 7 days. At this point, the neuronal medium was removed and replaced with the aforementioned CM from BV-2 cells.

ELISA

The culture supernatant from the BV-2 cells was collected for ELISA. The concentrations of HMGB1, IL-1β and IL-6 were determined using ELISA kits. Accurate concentrations of each protein were calculated from a standard curve. The ELISA kits used in this study were as follows: HMGB1 (JONLNBIO, China, JL13693-96T), IL-1β (R&D Systems, United States, DY501-05), and IL-6 (Abcam, United States, ab218796). All measurements were performed according to the manufacturers’ protocols.

Statistical Analysis

Normality of the data was assessed using the Shapiro-Wilk test, and homogeneity of variance was evaluated using Levene’s test. All the data are shown as the means ± SDs. Data that met the assumption of homogeneity of variances were analyzed using one-way ANOVA and Bonferroni test for post hoc comparisons. For data that did not meet the homogeneity assumption, Welch’s ANOVA was used followed by Games-Howell post-hoc tests. Longitudinal data in the same biological replicates were analyzed using repeated measures test following Bonferroni test for post hoc comparisons. Statistical Package for the Social Sciences 19.0 (SPSS, IBM, United States) and GraphPad Prism 10.0 (GraphPad, San Diego, CA, United States) were used for statistical analyses. A p value < 0.05 was considered statistically significant.

Results

GL Inhibited HMGB1 Translocation from the Nucleus To the Cytoplasm in the Hippocampi of Irradiated Mice

A radiation dose of 10 Gy was used in this study to establish a mouse model of RIBI and to investigate the effects of ionizing radiation on HMGB1. Radiation can cause HMGB1 to translocate from the nucleus to the cytoplasm (Zhang et al. 2022). Our immunofluorescence staining revealed that HMGB1 translocated from the nucleus in the hippocampal dentate gyrus (DG) region of mice 7 days after exposure to ionizing radiation (Fig. 1a), and the rate of nuclear translocation was significantly increased (M = 39.45%, SD = 15.17) compared with control group (M = 11.55%, SD = 4.14), F(2,11) = 17.50, p = 0.023 (Fig. 1b). After the application of GL, HMGB1 translocation, as determined by immunofluorescence staining, decreased, and the rate of nuclear translocation decreased compared to the Rad + PBS group (M = 16.74%, SD = 3.15), F(2,11) = 10.31, p = 0.044 (Fig. 1a, b).

Fig. 1.

Fig. 1

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GL reduced the rate of HMGB1 translocation from the cell nucleus after irradiation. a The translocation of HMGB1 from the nucleus in the hippocampal DG region of each group at 7 days after irradiation was labeled by immunofluorescence staining. HMGB1-positive cells are green, and DAPI-stained nuclei are blue. b Quantification of the HMGB1 translocation rate in each group. n = 5 mice per group. The data are presented as the means ± SDs, ∗p < 0.05

Preliminary Observations on HMGB1-TLR4/RAGE Pathway-Related Protein Levels in the Mouse Hippocampus After Irradiation and GL Administration

Emerging evidence implicates the alarmin HMGB1 in microglial priming via TLR2/TLR4 and RAGE, thereby amplifying post-irradiation neuroinflammation (Agalave et al. 2021; Han et al. 2016). To chart the temporal profile of this axis, hippocampal lysates were collected seven days after exposure and examined by quantitative Western blotting. Relative to the control group (HMGB1: M = 1.00, SD = 1.25; TLR2: M = 1.00, SD = 0.14; TLR4: M = 1.00, SD = 0.87; RAGE: M = 1.00, SD = 0.13), irradiated animals displayed elevated signals for all four targets (HMGB1: M = 1.67, SD = 0.80; TLR2: M = 1.57, SD = 0.33; TLR4: M = 2.14, SD = 0.27; RAGE: M = 2.03, SD = 0.15) (Fig. 2a, b) (The full Western blot images are presented in the supplementary material). Concomitant GL administration modified these levels (HMGB1: M = 0.86, SD = 0.81; TLR2: M = 1.53, SD = 0.28; TLR4: M = 1.45, SD = 0.16; RAGE: M = 1.34, SD = 0.17), suggesting a potential regulatory influence on the HMGB1–TLR4/RAGE pathway. We therefore present these descriptive observations without inferential statistics and regard them as preliminary.

Fig. 2.

Fig. 2

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Preliminary assessment of HMGB1-TLR4/RAGE signaling protein expression in the irradiated hippocampi. a Representative images of immunoblots showing the levels of HMGB1, TLR4, TLR2, and RAGE in the hippocampi of the mice from the different groups 7 days after irradiation. b Quantitative analysis of HMGB1, RAGE, TLR2, and TLR4 protein expression levels. n = 3 mice per group. The data are presented as the means ± SDs for all panels

Early Application of GL To Inhibit HMGB1 Ameliorated Cognitive Dysfunction in RIBI Mice During Adolescence

Cognitive dysfunction is a common side effect following radiotherapy for brain tumors. We further examined the behavioral performance of the mice 8 weeks after exposure to whole-brain ionizing radiation. The MWM results revealed that the swimming distance in d31 and d32 were significantly increased in the Rad + PBS group (M = 9079.84, SD = 2.59 and M = 9251.78, SD = 2.33) compared with the control group (M = 5107.25, SD = 1.51 and M = 5279.95, SD = 2.13), F(2, 18) = 12.27, p = 0.012 and F(2, 18) = 11.12, p = 0.013 (Fig. 3a). The escape latency in d32 were also increased in the Rad + PBS group (M = 76.01, SD = 18.30) compared with the control group (M = 39.09, SD = 26.11), F(2, 18) = 9.387, p = 0.027, (Fig. 3b), whereas the percentage of distance traveled in the target quadrant, the percentage of time spent in the target quadrant and the number of times the platform was crossed were significantly decreased in the Rad + PBS group (M = 23.05%, SD = 4.00, M = 27.74%, SD = 7.48 and M = 0.71, SD = 0.78) compared with the control group(M = 48.00%, SD = 8.54, M = 56.12%, SD = 11.23 and M = 2.29, SD = 1.60), F(2,18) = 48.91, p < 0.001, F(2,18) = 30.951, p < 0.001 and F(2,18) = 8.45, p = 0.048, respectively, (Fig. 3c, d, e). Compared with those in the Rad + PBS group, the swimming distances in d31 and d32, and escape latencies in d32 of the HMGB1 inhibitor GL group were reduced (M = 5706.18, SD = 2.52, M = 5454.21, SD = 2.40 and M = 38.88, SD = 25.62), F(2,18) = 6.10, p = 0.036, F(2,18) = 9.05, p = 0.018 and F(2,18) = 9.736, p = 0.026, respectively, (Fig. 3a, b), and the percentage of distance traveled in the target quadrant and the percentage of time spent in the target quadrant were increased (M = 38.27%, SD = 13.00 and M = 44.00%, SD = 10.66) compared with the Rad + PBS group F(2,18) = 8.8, p = 0.048 and F(2,18) = 10.894, p = 0.02, (Fig. 3c, d). However, no significant difference in the number of platform crossings was observed between the Rad + GL group (M = 1.57, SD = 0.57) and the Rad + PBS group F(2,18) = 1.8, p = 0.709, (Fig. 3e). These results suggest that the application of GL can ameliorate the learning and memory deficits caused by ionizing radiation in mice.

Fig. 3.

Fig. 3

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Spatial memory deficits induced by radiation were alleviated by GL treatment. a Escape path length in the MWM test plotted against training days. b Escape latency in the MWM test plotted against training days. c The percentage of distance traveled in the target quadrant during the probe test of the MWM. d The percentage of time spent in the target quadrant during the probe test of the MWM. e The number of platform crossings during a 90-s probe trial of the MWM test. f Representative swimming paths of the rats on d28 and d32. n = 7 mice per group. The data are presented as the means ± SDs for all panels: *p < 0.05 and ***p < 0.001 compared with the Con group; #p < 0.05 compared with the Rad + PBS group; ns, not significant

Early Application of the HMGB1 Inhibitor GL Reduced Neuronal Loss in the Hippocampi of RIBI Mice

Given the significant role of HMGB1 in the neuroinflammatory response in the RIBI model and the improvement in learning and memory deficits in mice through the inhibition of HMGB1, we further investigated whether inhibiting the expression of HMGB1 could ameliorate neuronal loss. Immunofluorescence staining was used to label neurons with NeuN, and the results revealed a reduction in the number of neurons in the hippocampal CA1 region of the mice 14 days after exposure to ionizing radiation (M = 101.4, SD = 10.29) compared with control group (M = 133.6, SD = 3.13), F(2,12) = 44.85, p < 0.001, (Fig. 4a, b), which was effectively mitigated by the application of GL (M = 118.6, SD = 5.32), F(2,12) = 11.03, p = 0.006, (Fig. 4a, b). The Nissl staining results also revealed a significant decrease in the number of neurons in the hippocampal DG, CA1, and CA3 regions of the mice after exposure to ionizing radiation (M = 136.2, SD = 10.29, M = 58.0, SD = 6.56 and M = 29.4, SD = 2.51, respectively) compared with control group (M = 229.2, SD = 13.28, M = 87.2, SD = 8.16 and M = 45.0, SD = 2.55, respectively), F(2,11) = 150.21, p < 0.001, F(2,12) = 38.86, p < 0.001 and F(2,12) = 95.06, p < 0.001, respectively, (Fig. 4c, d), and these changes were attenuated by GL treatment (M = 175.75, SD = 16.66, M = 71.0, SD = 3.67 and M = 37.2, SD = 0.83, respectively), F(2,11) = 24.94, p = 0.003, F(2,12) = 14.95, p = 0.023 and F(2,12) = 43.46, p < 0.001, respectively, (Fig. 4c, d).

Fig. 4.

Fig. 4

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GL attenuated radiation-induced neuronal loss. a Immunofluorescence labeling of neurons in the hippocampal CA1 region of different groups at 14 days after irradiation. The NeuN-positive cells are green, and the DAPI-stained nuclei are blue. Scale bar = 50 μm. b Quantification of neurons in the hippocampal CA1 region. n = 5 mice per group. c Nissl-stained neurons in the hippocampal CA1, CA3, and DG regions of different groups at 14 days after irradiation. Scale bar = 50 μm. d Quantification of Nissl-stained neurons. n = 7 mice per group. The data are presented as the means ± SDs for all panels: ***p < 0.001 compared with the Con group; #p < 0.05, ## p < 0.01 and ###p < 0.001 compared with the Rad + PBS group

Early Application of the HMGB1 Inhibitor GL Reduced the Activation of Hippocampal Glial Cells in RIBI Mice

According to previous studies, ionizing radiation can lead to the activation of microglia and astrocytes (Chew et al. 2020). We further investigated the activation of glial cells in RIBI mice. The immunofluorescence results revealed that the expression of Iba-1, a marker of microglia, was significantly increased in the hippocampus of the Rad + PBS group (M = 2.33, SD = 0.51; M = 7.20%, SD = 0.62) in IOD and percentage of positive signal area compared to the control group 7 days after whole-brain irradiation (M = 1.00, SD = 0.32; M = 3.53%, SD = 0.45), F(2, 12) = 24.51, p < 0.001, F(2, 12) = 114.107, p < 0.001, (Fig. 5). The expression of GFAP, a marker of astrocytes, was also significantly elevated in both IOD and percentage of positive signal area (M = 1.91, SD = 0.35; M = 9.15%, SD = 1.44) compared to the control group (M = 1.00, SD = 0.31; M = 4.96%, SD = 2.39), F(2, 12) = 18.97, p = 0.006; F(2, 12) = 13.302, p = 0.038, (Fig. 5). These changes confirmed the activation of both microglia and astrocytes after exposure to ionizing radiation. However, after the application of the HMGB1 inhibitor GL, the activation of microglia was significantly inhibited in both IOD and percentage of positive signal area (M = 1.32, SD = 0.12; M = 4.72%, SD = 0.44), F(2, 12) = 18.992, p = 0.002, F(2, 12) = 52.839, p < 0.001(Fig. 5), whereas the activation of astrocytes did not change significantly (M = 1.78, SD = 0.43; M = 8.07%, SD = 3.11), F(2, 12) = 0.321, p = 1.000, F(2, 12) = 0.734, p = 0.417 (Fig. 5), indicating that GL primarily targets microglia rather than astrocytes.

Fig. 5.

Fig. 5

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GL attenuated the activation of hippocampal microglia in RIBI mice. a Immunofluorescence labeling of microglia in the hippocampus 7 days after irradiation. Iba-1-positive cells are red, and DAPI-stained nuclei are blue. b Immunofluorescence labeling of astrocytes in the hippocampus 7 days after irradiation. GFAP-positive cells are red, and DAPI-stained nuclei are blue. c Relative IOD of Iba-1/ GFAP in each group. d Percentage of Iba-1/ GFAP positive area in each group. Scale bar = 50 μm, n = 5 mice per group. The data are presented as the means ± SDs for all panels: *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

Preliminary Observations Linking GL Treatment to Microglial inflammatory Markers and Neuronal MAP2 Staining In Vitro

We performed preliminary in vitro assays to investigate the potential of GL to attenuate neuronal structural damage by limiting microglial activation. BV-2 cells were exposed to 10 Gy, and supernatants were collected 4 h later. ELISA measurements indicated apparent increases in HMGB1 (M = 137.45, SD = 11.47), IL-6 (M = 142.89, SD = 22.97) and IL-1β (M = 158.23, SD = 3.47) compared with control groups (HMGB1: M = 48.89, SD = 2.66; IL-6: M = 38.67, SD = 1.30; IL-1β: M = 39.93, SD = 1.30) (Fig. 6a–c). GL-treated cultures showed lower levels of these inflammatory cytokines (HMGB1: M = 85.00, SD = 0.80; IL-6: M = 61.10, SD = 0.83; IL-1β: M = 115.40, SD = 11.30) (Fig. 6a–c). Primary neurons subsequently cultured with the supernatant of BV-2 cells from each group assessed using immunofluorescence staining for MAP2 as a neuronal marker. Neuronal integrity appeared reduced in the Rad + PBS group (M = 0.49, SD = 0.07) relative to controls (M = 1.00, SD = 0.88) and partially preserved after GL exposure (M = 0.65, SD = 0.04) (Fig. 6d, e).

Fig. 6.

Fig. 6

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Preliminary observations of GL treatment on inflammatory factor production and neuronal integrity in vitro. a–c ELISAs of HMGB1, IL-6, and IL-1β levels in each group at 4 h after irradiation. n = 3 mice per group. d Immunofluorescence labeling of neurons in different groups after irradiation. MAP2-positive cells are green, and DAPI-stained nuclei are blue. e Quantification of MAP2-positive cells in each group. All data are presented as the means ± SDs for all panels. n = 3 mice per group

Discussion

RIBI is primarily divided into three clinical stages: acute, early delayed, and late. In pediatric RIBI research, specific studies on the mechanisms by which microglia cause neuroinflammatory responses during the acute and early stages are lacking. In this study, we explored whether early application of an HMGB1 inhibitor in children could improve hippocampal tissue injury and cognitive deficits during adolescence following RIBI. We utilized GL, a specific inhibitor of HMGB1, to confirm the effect of inhibiting HMGB1 on RIBI. Previous research has indicated that GL can alleviate inflammatory cascades, protecting the brain tissue (Fan et al. 2020). By applying both in vivo and in vitro experimental models, we examined microglial activation, the expression of TLR4/RAGE, the production of inflammatory factors, neuronal injury, and the learning and memory functions of mice following early application of the HMGB1 inhibitor GL in the RIBI model. Early application of GL reduced microglial activation, decreased neuronal injury, and improved long-term learning and memory. Preliminary findings further indicated that these effects were characterized by alleviated neuroinflammation, which may be mediated through the TLR4/RAGE pathway. This study provides new insights into brain protection strategies after radiotherapy in children and has clinical significance.

Neuroinflammation plays a significant role in the pathogenesis and progression of RIBI. HMGB1 is a nuclear DNA-binding protein expressed in nucleated animal cells that is involved in physiological functions, including DNA replication, transcription, and repair (Jin et al. 2023). As a damage-associated molecular pattern (DAMP), it can translocate from the nucleus to the extracellular space during cranial tumor radiotherapy, where it is detected by pattern recognition receptors on cells, activating the innate immune system and damaging cancer cells (Ashrafizadeh et al. 2020). However, the excessive release of HMGB1 can initiate inflammatory response-related pathways by binding to cell surface receptors, leading to RIBI (Wang et al. 2024). Numerous studies have confirmed that HMGB1 is involved in the pathological processes of various central nervous system diseases, including neuroinflammatory responses, traumatic brain injury, and cognitive dysfunction (Paudel et al. 2018). HMGB1 has become a new focus of research because of its potential role in neuroinflammation (Lee et al. 2014), and therapy involving HMGB1 inhibitors has been shown to exert significant neuroprotective effects on several neuroinflammation models (Kim et al. 2006).

Our research group previously confirmed that the application of GL to inhibit the expression of HMGB1 could reduce neuroinflammatory responses in a rat model of hypoxic–ischemic encephalopathy (Zhu et al. 2022). In this study, observations from both in vivo and in vitro models suggested an increase in hippocampal HMGB1 levels following whole-brain ionizing radiation. Notably, a significant increase in HMGB1 nuclear translocation was statistically confirmed in vivo via immunofluorescence; similarly, preliminary in vitro data from ELISA assays suggested an apparent upward trend in HMGB1 levels, although these observations remain descriptive. This result is consistent with the findings of Zhang et al. (2022), who reported increased HMGB1 expression and nuclear translocation rates in the cerebral cortex of adult mice and cultured neurons after irradiation. These findings indicate that HMGB1 is involved in the pathological process of RIBI.

Overexpressed HMGB1 is primarily released from injured neurons, microglia, and activated astrocytes following exposure to ionizing radiation. It acts on receptors on the cell surface, such as TLRs (Agalave et al. 2021), RAGE (Han et al. 2016), the scavenger receptor Mac1 (Gao et al. 2011), and the chemokine receptor CXCR7 (Das et al. 2019), to activate microglia, induce inflammatory cytokine production and exacerbate brain damage (Markarian et al. 2021). In various central nervous system disease models, such as hypoxic injury (Zhu et al. 2022), traumatic brain injury (Gao et al. 2018), depression (Wu et al. 2022), and neurodegenerative disease models (Tian et al. 2020; Yang et al. 2022), HMGB1 has been reported to mediate neuroinflammatory responses by activating microglia. Microglia are indispensable resident mononuclear cells in the central nervous system with immune surveillance functions that maintain central nervous system homeostasis (Zhou et al. 2017; Zhang et al. 2022).

The neuroinflammatory response induced by ionizing radiation is intricately associated with the activation of microglia within the central nervous system. Microglia exhibit different morphological changes under various conditions and are categorized as ramified, hypertrophic, or ameboid (Wang et al. 2017; Sheu et al. 2023). Ramified microglia in a resting state have abundant branches, which aid in immune surveillance of the brain. However, exposure to ionizing radiation induces an amoeboid morphology in microglia, leading to changes in phagocytic activity and ultimately resulting in their activation (Wang et al. 2024). Activated microglia migrate to the site of injury, phagocytose apoptotic neurons and cellular debris, and simultaneously produce large amounts of cytokines, chemokines, and reactive oxygen species (Zhou et al. 2017; Boyd et al. 2021). The persistent activation of microglia increases susceptibility to subsequent injury (Zhou et al. 2017). In our study, the number of Iba-1-positive microglia was significantly increased after whole-brain irradiation, indicating that radiation could lead to the activation of microglia.

In vitro, we observed that BV-2 cells from irradiated cultures produced higher amounts of IL-6 and IL-1β than controls, whereas concurrent GL application appeared to attenuate these elevations. This finding is consistent with previous research findings (Zhou et al. 2017; Liu et al. 2022). These descriptive trends provide preliminary evidence for a potential anti-inflammatory effect of GL. Additionally, activated microglia and TNF remain at high levels for at least six months after exposure to a single high dose of radiation (Hong et al. 1995; Greene-Schloesser et al. 2014), with these continuously activated microglia constantly releasing proinflammatory factors, maintaining an inflammatory state in the brain microenvironment, and further leading to the death of neurons and precursor cells to create a vicious cycle (Jenrow et al. 2013).

In addition to the activation of microglia, other factors also play important roles in the process. In our study, the number of GFAP-positive astrocytes increased after exposure to ionizing radiation, which may be related to the interaction between microglia and astrocytes after irradiation, as well as the activation of astrocytes due to the disruption of the blood‒brain barrier constituted by astrocytes after irradiation (Chew et al. 2020). However, the application of GL did not significantly reduce the activation of astrocytes, indicating that the primary target cells of HMGB1 are microglia rather than astrocytes.

In the microglia-mediated neuroinflammatory response, TLRs and RAGE are recognized as key surface receptors. Previous work by Xu et al. (2020) and Li et al. (2023) indicates that interfering with the HMGB1/TLR4/NF-κB axis can dampen microglial activation and attenuate neuroinflammation in models of depression and ischemia–reperfusion injury, respectively. In our preliminary observations, whole-brain irradiation tended to raise hippocampal levels of TLR4 and TLR2, while GL administration appeared to lower TLR4 with a less evident effect on TLR2. Similarly, RAGE expression—reported to participate in neuroinflammatory cascades following ischemia, spinal cord injury, stress-induced hypertension, and RIBI (Liesz et al. 2015; Zhang et al. 2020, 2022; Fan et al. 2020)—was also elevated after irradiation and seemed to be reduced by GL. These descriptive trends, although not yet statistically confirmed, provide initial evidence that HMGB1 may modulate microglial activation through the TLR4/RAGE pathway and further studies are required to establish this mechanism.

Microglial activation is associated with a persistent immune‒inflammatory response in the late stages of brain injury and mediates cognitive functions in the hippocampus by modulating neurodevelopment and neuroplasticity (Chew et al. 2020). Cognitive dysfunction, which mainly involves learning and memory, is a common side effect of ionizing radiation therapy for brain tumors. Radiation therapy may lead to a reduction in the structural integrity of anatomical regions crucial for memory formation (Rübe et al. 2023), especially in children whose developing brains are more sensitive to ionizing radiation injury than are adults (Han et al. 2016). Adolescence is also a critical period for the development of cognition and signal integration functions, determining the function of the hippocampus in adulthood (Chew et al. 2020). When the irradiation field involves the temporal lobe where the hippocampus is located, it can lead to long-term neurocognitive effects throughout the life of children and adolescents (Rübe et al. 2023). In the MWM test, the learning and memory functions of the mice in the Rad + PBS group were significantly decreased, whereas early application of GL significantly improved the cognitive deficits of the adolescent mice. This result is consistent with previous research findings (Chew et al. 2020). This improvement in cognitive function is related mainly to the alleviation of neuronal loss. Our in vivo experiments revealed that the application of GL could alleviate neuronal loss. Therefore, the early application of an HMGB1 inhibitor can reduce the activation of microglia, alleviate neuronal injury, and improve learning and memory functions during adolescence. In vitro, treating primary neurons with the supernatant of irradiated BV-2 cells appeared to reduce MAP2 IOD levels in primary neurons, whereas GL treatment seemed to lessen this reduction, suggesting a potential role for GL in preserving neuronal morphology.

This study has several limitations. First, a priori sample size calculation was not performed. While our sample size was determined based on comparable published studies and consultation with institutional biostatisticians to ensure sufficient power for detecting meaningful effects while minimizing animal use, the lack of a prospective power analysis remains a constraint. Second, the model employed exclusively involved four-week-old mice receiving a single 10 Gy dose. According to previous research, the degree of brain injury caused by different radiation doses may vary at different ages (Rübe et al. 2023). Third, the investigation centered on neuroinflammatory mechanisms. Although neuroinflammation is a key pathogenic factor in RIBI (Cheng et al. 2023), complementary mechanisms such as neurogenesis impairment and blood-brain barrier disruption remain unexamined. Notably, the absence of Western blotting quantification for Iba-1 and GFAP, coupled with the reliance on immunofluorescence rather than subcellular fractionation for assessing HMGB1 translocation, represents a limitation of the current evidence. Additionally, due to limited biological replicates, the biochemical data for TLR2/TLR4/RAGE and the in vitro data are presented as descriptive and preliminary. Finally, our neuroinflammatory analysis maintained defined boundaries. Microglial polarization states (M1/M2) were not characterized (Jin et al. 2023; Ye et al. 2019). TLR4/RAGE assessments were conducted only at the protein level without transcriptomic data or functional validation using specific inhibitors. And intracellular mechanisms linking TLR4/RAGE activation to microglial effector functions such as mitochondrial autophagic flux (Wu et al. 2022), remain unexplored. These questions will be the main focus of our future work.

Conclusions

Our study revealed that in a pediatric RIBI model, the early application of an HMGB1 inhibitor can alleviate hippocampal neuronal injury and improve cognitive function during adolescence. This outcome may be attributed to the blockade of HMGB1 binding to TLR4/RAGE on microglial surfaces, consequently mitigating microglial activation and the subsequent production of inflammatory cytokines.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (2.1MB, pdf)
Supplementary Material 2 (14.9KB, xlsx)

Author Contributions

All authors contributed to the study conception and design. Y. S. designed the research and performed the experiments. L. M. established the animal model. H. X. collected and analyzed the data. M. H. and W. Z. conceptualized the research and directed the study. M. S. wrote the first draft of the manuscript. All authors read and approved the manuscript .

Funding

This work was supported by Science and Technology Project of Henan Province (222102310137).

Data Availability

All data generated or analyzed during this study are included in this published article. The data that support the findings of this study are available from the first author upon reasonable request.

Declarations

Competing Interests

The authors declare no competing interests.

Ethical Approval

All animal experiments were conducted in compliance with National Institutes of Health Guidelines and were approved by the Institutional Animal Care and Use Committee of Zhengzhou University (No. 2021042501).

Consent for Publication

All authors have approved the contents of this manuscript and provided consent for publication.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yanyan Sun and Mingrui Shi have been contributed equally to this work.

Contributor Information

Mingyan Hei, Email: heimingyan@bch.com.cn.

Wenli Zuo, Email: wenlizuoqq@aliyun.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (2.1MB, pdf)
Supplementary Material 2 (14.9KB, xlsx)

Data Availability Statement

All data generated or analyzed during this study are included in this published article. The data that support the findings of this study are available from the first author upon reasonable request.

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