The experiment was conducted using Kunming mice pups (n = 111). Approval for all animal procedures was obtained from the Ethics Committee on Animal Experiments of the Medical School of Shandong University. The dams and pups, with 9–11 pups per litter, were housed in animal facilities maintained at a constant temperature of 22°C ± 2°C, and subjected to a 12-h light/dark cycle. They were provided unlimited access to food and water. The day of birth was designated as postnatal day 1 (P1). In order to establish a white matter injury model, the mice were intraperitoneally injected with lipopolysaccharide (LPS) for three consecutive days, starting from P3.

The experiment was conducted following previous studies (Pang et al., 2012; Fan et al., 2013; Xie et al., 2016). The experimental design and drug treatment schedule are presented in Figure 2A. The experimental animals were divided into three groups: the Control group (treated with vehicle solution), the LPS group (treated with LPS at a dosage of 1 mg/kg/24 h), and the LPS + ISL group (treated with LPS at a dosage of 1 mg/kg/24 h, and ISL at a dosage of 4 mg/kg/24 h). At the end of the experiment, the mice were euthanized in accordance with institutional guidelines and approved protocols. The euthanasia was carried out using carbon dioxide (CO2) inhalation, followed by cervical dislocation to ensure death. CO2 was introduced into the chamber at a rate that displaced 10%–30% of the chamber volume per minute. The animals were monitored for cessation of respiration and lack of a heartbeat before proceeding with the subsequent procedures. All efforts were made to minimize animal suffering and to ensure a humane endpoint. The brain was removed, and the corpus callosum was collected and quickly stored at −80°C until further use. Some of the mice were used in the behavioral experiments. The LPS (Escherichia coli strain O111:B4) used in the experiments was purchased from Sigma-Aldrich (St. Louis, MO, United States) and prepared in saline solution (0.9% NaCl). ISL (98.07% purity) was purchased from MCE (shanghai, China) and dissolved in sterile saline with 1% dimethyl sulfoxide (Solarbio, Beijing, China). The chosen dosage of the drug was based on the results of our study (Supplementary Figure 1A, B) and consistent with previous literature demonstrating the neuroprotective effects of the agent (Liu et al., 2019; Wang et al., 2020).

Behavioral tests of mice were conducted between P42 and P56. Every behavioral experiment was conducted in an individual, dark chamber, devoid of unwanted external light and sound. During the experiment, the operator was blinded to the mice in the different treatment groups. Mice were moved there at least 30 min in advance before the experiment to adapt to the environment. After each mouse experiment, the behavioral device was cleaned with 75% ethanol. After each experiment, the mice rested for 24 h, waiting for the next experiment. A tracking system (TopScan 3.0) was used to record, measure and analyze all behavioral experiments.

Cryosections selected from each group were rinsed with distilled water for 5 min and dehydrated using an ascending ethanol series. The sections were then stained with preheated (55°C) luxol fast blue stain (LFB; 0.1 g LFB in 100 mL of 90% ethanol with 0.5 mL of 10% glacial acetic acid) for an extended period. Finally, the sections were sealed using neutral balsam and examined under a microscope.

The brain tissue of mice 21 days after birth was cut into 1 mm³ and fixed with 3% glutaraldehyde at 4°C for 2 h. The fixed tissue was washed 3 times with phosphate-buffered saline (PBS). The tissue was then fixed in 1% OsO4 at 4°C for 2 h. The tissues were dehydrated with ethanol gradient and infiltrated with 100% epoxy resin: acetone (1:3, 1:1, 3:1) for 1 h, 4 h, and 12 h, respectively. Finally, the tissues were embedded in fresh epoxy resin and polymerized at 37°C for 12 h, 45°C for 12 h, and 60°C for 1 h. The ultra-thin slices were supported on a grid (150 nm) and stained at room temperature for 20 min with uranyl acetate and 20 min with lead citrate. Images were captured using a Talos F200C transmission electron microscope (FEI Company).

The mice were anesthetized and then perfused with a 4% paraformaldehyde solution. Subsequently, the brain tissue was removed and fixed for frozen sectioning. The brain tissue sections were washed three times with PBS. Next, they were blocked with a mixture of 10% goat serum and 0.3% Triton X-100 for 3 h, followed by incubation with the primary antibody overnight at 4°C. The primary antibodies used were anti-Iba1 (1:200, Abcam), anti-PDGFR-α (1:20, CST), and anti-APC (1:50, Beyotime Biotechnology). Then, the cells or sections were incubated with fluorescently conjugated secondary antibodies (AbBkine-488 and AbBkine-594) for 1 h. The nuclei were stained with DAPI (Sigma-Aldrich Corp). The images were acquired using an IX71 Olympus fluorescence microscope.

The tissues were fixed using a 4% paraformaldehyde solution, dehydrated, embedded in paraffin, and then sectioned. Following dewaxing and hydration, the sections were immersed in a 3% hydrogen peroxide solution for 10 min. Subsequently, they were blocked with either 10% normal goat serum or 10% normal donkey serum, and incubated overnight at 4°C with the primary antibody (Anti-MBP antibody was used at a dilution of 1:600, CST). After washing with PBS, the samples were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Following that, they were stained with 3,3′-diaminobenzidine (DAB) and photographed.

Neonatal mice were sacrificed on postnatal day 3 (P3), and the brain tissue was removed, and the cerebellum, olfactory bulb, hippocampus and meninges were stripped. The rest of the brain tissue was then digested using 0.25% trypsin. The mixed glial single-cell suspension was further resuspended in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin/Amphotericin B (P/S/A) (100X). The cells were seeded into poly-lysine coated culture flasks at a density of approximately 5 × 10^6 cells/mL. On day 7, floating microglia were collected by gently tapping the culture flask. The isolated primary microglia were then seeded in either 6-well plates or 96-well plates for subsequent experiments. The cells were cultured in a humidified chamber at 37°C with 5% CO2.

The Cell Counting Kit-8 (CCK-8) assay (K1018, APExBIO) was performed to assess the impact of ISL on cell viability. Microglia were cultured in 96-well plates and exposed to varying concentrations of ISL for 24 h. Prior to testing, 100 μL of CCK-8 solution was added to each well of the culture plate and incubated in a CO2 incubator at 37°C for 2 h. The optical density at a wavelength of 450 nm was measured using a Microplate Reader (BIO-RAD iMark).

Mouse brain tissues were used to extract total RNA with TRIZOL reagent (Invitrogen). The purity and concentration of the total RNA were determined using a spectrophotometer (Thermo NanoDrop 2000). Subsequently, the cDNA was synthesized using the RevertAidTM First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR was performed with the SYBR Green Real-Time PCR Master Mix (TOYOBO CO., Ltd., Japan). β-actin expression served as the normalized control, and changes in gene expression were calculated using the 2^−ΔΔCT method. The primer sequences are provided in Table 1.

The corpus callosum tissue was lysed in RIPA buffer at 4°C for 30 min. Following centrifugation, the supernatant was collected, and the total protein concentration was measured using the BCA method. Equal amounts of protein from each sample were then separated using 8%–15% SDS-PAGE gel electrophoresis and transferred onto PVDF membranes with pore sizes of either 0.22 or 0.45 μm (Millipore). Subsequently, the PVDF membrane was blocked using TBST buffer containing 5% milk and incubated overnight at 4°C with the primary antibody. The secondary antibody, either goat anti-mouse IgG or rabbit IgG conjugated to HRP, was used. Finally, the bands were visualized using the enhanced chemiluminescence (ECL) detection kit (Merck Millipore), and the analysis was performed using ImageJ software (Version 1.46r). Primary antibodies were used at the following dilutions: rabbit anti-HDAC1 (1:500, Proteintech), rabbit anti-HDAC2 (1:500, Proteintech), rabbit anti-HDAC3 (1:500, Proteintech), rabbit anti-HDAC8 (1:500, Proteintech), rabbit anti-H3 (1:500, CST), rabbit anti-H3K18ac (1:500, CST), rabbit anti-H3K9/14ac (1:500, CST), rabbit anti-H3K27ac (1:500, CST), rabbit anti-AcH3 (1:1000, CST), mouse anti-β-actin (1:2000, CST).

Enzymatic chromatin immunoprecipitation (ChIP) was conducted using the SimpleChIP^® Enzymatic Chromatin IP Kit (CST). Formaldehyde fixation was performed on the tissue, followed by lysis and partial digestion of chromatin using Micrococcal Nuclease provided in the kit. This digestion process resulted in fragments ranging from 200–500 base pairs. The chromatin was immunoprecipitated overnight at 4°C using the specific antibody anti-HDAC3 or the control antibody (anti-IgG). The DNA was subsequently purified using a DNA purification centrifuge column, and after protein-DNA uncross-linking according to the manufacturer’s instructions, it was analyzed using real-time PCR. The primer sequences used for the PCR analysis can be found in Table 2.

The chemical structure and specification of ISL smile was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Importing the information into the PharmMapper server (https://www.lilab-ecust.cn/pharmmapper/) and Swiss Target Prediction (http://www.swisstargetprediction.ch/) allowed identification of ISL related targets. Potential targets for white matter injury were identified using “white matter injury” as a search term in the Genetic Society database (https://www.genecards.org/). Subsequently, the intersection of ISL and WMI targets was identified by analyzing the two sets of targets using Venny software (https://bioinfogp.cnb.csic.es/tools/venny/). The STRING database (https://cn.string-db.org/cgi/input.pl) can provide network visualization. Based on the STRING database, the interaction genes between ISL and WMI targets were analyzed (confidence data >0.4).

Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analyses were performed on an online bioinformatics platform (https://www.genedenovo.com/) to elucidate key signaling pathways and identify biological functions associated with potential therapeutic targets for ISL.

The data are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s test. Statistically significant differences were defined as p values <0.05.

A total of 362 potential targets for ISL were predicted using the Swiss Target Prediction and PharmMapper databases (Figures 1A, B; Supplementary Table 1). Of these, 336 therapeutic targets were identified by intersecting with 7,895 WMI-related targets (Figures 1C–E; Supplementary Tables 2, 3). Results from STRING analysis show that there are 321 targets in the PPI network, including 113 nodes and 3,445 edges, with an average node degree of 21.5 (Figure 1F). To analyze the role of these targets, GO enrichment analyses were performed to assess the biological functions of ISL targets (Figure 1G). The results indicated significant associations with inflammation, microglia cell activation, and myelination, which are important mechanisms of ISL treatment for WMI. Additionally, KEGG enrichment analysis suggested that ISL may affect WMI through multiple signaling pathways, including the MAPK signaling pathway, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, and NF-κB signaling pathway (Figure 1H).

To investigate the potential of ISL in improving behavioral abnormalities in mice with white matter injury, we initially assessed body weight and early neural reflexes (Figure 2A). The righting reflex and negative geotaxis are commonly used neurobehavioral tests in rodent research, which we utilized to assess early motor coordination function in mice development. Compared to the control group, mice subjected to LPS-induction exhibited significant reductions in body weight and early neurobehavioral deficits. ISL treatment significantly improved the body weight loss (Figure 2B). The righting reflex and negative geotaxis experiments showed that ISL treatment shortened the latency (Figures 2C, D). Furthermore, we conducted the rotary rod and grip tests in mice at P42 to evaluate the impact of ISL treatment on mice motor function. The findings demonstrated that ISL treatment significantly enhanced motor coordination impaired by LPS induction (Figure 2E) and grip reduction in mice (Figure 2F).

To further explore the enduring impacts of ISL treatment, we analyzed the effect on emotional behavior in mice. We conducted the open field test (OFT) to assess anxiety-like behavior. Mice in the LPS group spent less time in the center, indicating increased anxiety. ISL treatment increased center time, suggesting reduced anxiety (Figures 3A, B). Additionally, the elevated plus maze (EPM) confirmed higher anxiety levels in the LPS group, with reduced time in open arms. ISL-treated mice spent more time in open arms, indicating decreased anxiety (Figures 3C, D). We evaluated depressive-like behavior using the forced swim test (FST) and tail suspension test (TST). LPS group mice showed longer immobility periods, indicative of increased depressive-like behavior. ISL treatment significantly reduced immobility time in both tests, suggesting an antidepressant effect (Figures 3E, F). These findings collectively demonstrate the potential of ISL treatment to mitigate emotional deficits in mice.

The abnormal structure of white matter may lead to impaired or disrupted neural signal transmission, thereby affecting emotional regulation and motor coordination. Therefore, we conducted a pathological examination of the brain tissue. HE staining revealed that the LPS group exhibited subcortical and periventricular white matter lesions compared to the control group. The ISL group showed a noticeable improvement in the periventricular white matter looseness and nerve fiber disorder, with some white matter structures returning to normal (Figure 4A). Neonatal white matter injury can result in impaired myelination. Myelin basic protein (MBP) is a critical component of the myelin sheath, playing an essential role in the process of myelination. MBP is crucial for maintaining the structural integrity and function of myelin, and its expression levels are often used as an indicator of myelination status in the central nervous system. Immunohistochemical staining of MBP revealed a decrease in MBP expression within the corpus callosum of the LPS group compared to the control group. The ISL group exhibited increased expression of MBP compared to the LPS group (Figure 4B). Furthermore, Western blot analysis indicated that ISL treatment was able to reverse the decreased expression of MBP induced by LPS (Figures 4C, D). The density of LFB-staining myelin in the callosum was significantly increased upon ISL treatment, as observed in Figure 4E. The results of myelin transmission electron microscopy scanning showed that there were fewer myelinated axons in the corpus callosum of the LPS group, with irregular myelin shape and loose structure, while the myelinated axons in ISL group were significantly improved (Figure 4F). Collectively, our data strongly suggest that ISL has the potential to ameliorate neonatal white matter injury and prevent myelination defects.

GO analysis showed that ISL treatment was associated with microglial activation and inflammatory response. Microglia-mediated neuroinflammation plays a crucial role in white matter injury. We aimed to assess the potential of ISL in regulating the inflammatory response in a model of LPS-induced white matter injury. We examined the quantity and morphology of microglia in the corpus callosum region, as well as the mRNA expression levels of classical inflammatory factors secreted by microglia. In Figures 5A, B, the number of Iba1+ microglia (green) in the LPS group was higher compared to the control group, indicating that systemic LPS exposure activated microglia. Conversely, the number of Iba1+ microglia (green) in LPS + ISL treated mice was significantly reduced (Figure 5B). The mRNA expression analysis also confirmed that ISL can reduce the upregulation of LPS-induced inflammatory cytokines IL-1β (Figure 5C) and TNF-α (Figure 5D). We additionally assessed the impact of ISL on LPS-induced microglia activation in vitro. When microglia were treated with different concentrations of ISL, it was found that 40 μM of ISL did not significantly affect microglia viability (Supplementary Figure 2A). Increasing the dose of ISL significantly decreased microglia viability. Based on the results of CCK8, a dose of 40 μM was chosen for subsequent experiments. Immunofluorescence staining of microglia revealed that ISL treatment significantly improved their activation state (Supplementary Figure 2B). Furthermore, qPCR results demonstrated that the expressions of IL-1β and TNF-α in microglia were significantly downregulated in the ISL treatment group compared to the LPS group (Supplementary Figures 2C, D). These findings suggest that ISL may inhibit microglia activation and suppresses proinflammatory gene expression.

According to the biological process results of GO enrichment analysis, ISL treatment is related to myelination process. White matter injury primarily arises from a developmental disorder in oligodendrocyte precursor cells, resulting in defects in myelination and subsequent behavioral changes. In this study, we sought to investigate how ISL affects oligodendrocyte precursor cells. Immunofluorescence staining targeting PDGFR-α revealed an increase in the number of PDGFR-α⁺ cells in the LPS group. However, ISL treatment was found to restore the number of PDGFR-α+ cells reduced by LPS exposure (Figures 6A, B). Additionally, compared to the control group, the LPS group exhibited a decrease in the number of mature oligodendrocyte APC⁺ cells. However, in the LPS + ISL group, more APC⁺ cells were observed compared to the LPS group (Figures 6C, D). The development of oligodendrocytes involves numerous transcription factors and epigenetic regulation. To further investigate this, we conducted qPCR to examine changes in transcription factors known to play a role in oligodendrocyte maturation and differentiation. Our findings indicate an increase in the expression of SOX10 and a decrease in the expression of Olig1, Olig2, and NKX2.2 in the LPS group. However, treatment with ISL reversed these changes (Figure 6E). These results suggest that ISL may exert a beneficial effect on oligodendrocyte maturation by reducing the population of immature oligodendrocytes and increasing the population of mature oligodendrocytes. It is plausible that this effect is mediated through the influence of ISL on transcription factors involved in oligodendrocyte development.

Class I HDACs play a crucial role in neuroinflammation and the development of oligodendrocytes. Several studies have reported that ISL is capable of inhibiting class I HDACs. Therefore, we investigated the potential of ISL to ameliorate white matter injury through the regulation of class I HDACs. Expression levels of HDAC1, HDAC2, HDAC3, and HDAC8 were measured, demonstrating that ISL could suppress the upregulation of HDAC3 mRNA and protein expression following LPS induction (Figures 7A–C). HDACs are known to regulate histone acetylation; therefore, we assessed changes in histone acetylation. Western blot analysis revealed a decrease in total acetylation of histone H3 following LPS treatment, whereas ISL treatment significantly increased its acetylation (Figures 7D, E). Additionally, we assessed histone acetylation at key sites. Our findings demonstrated a significant decrease in H3K27ac levels in the LPS group, while no significant changes were observed in H3K9/14ac and H3K18ac. Treatment with ISL significantly increased H3K27ac levels (Figures 7F–I). Furthermore, chromatin immunoprecipitation (ChIP) results demonstrated that ISL effectively inhibited the LPS-induced upregulation of HDAC3 binding to the promoter regions of essential transcription factors involved in oligodendrocyte pro-differentiation, such as Olig1, Olig2, SOX17, and NKX2.2 (Figure 7J). Our findings indicate that ISL is capable of modulating histone acetylation levels and the expression of transcription factors involved in oligodendrocyte development through its interaction with HDAC3.