2.1 Animals and Ethical Approval

Animal procedures were conducted in strict compliance with the guidelines set forth by the Animal Ethics Committee of Wenzhou Medical University and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57Bl/6J male and female mice (6 to 8 weeks old) were obtained from Vital River (Zhejiang, China) and bred under standard laboratory conditions with ad libitum access to water and fodder, a 12 h light-cycle, as well as temperature- and humidity-controlled environments. The pups were generated by crossing male and female mice. Male pups at postnatal day 5 (P5) were utilized, corresponding to 23–32 weeks of gestation in terms of OL development in the human brain [27]. The animal experiments strictly adhered to the principles of replacement, reduction, and refinement (3Rs).

2.2 Neonatal Mice WMI Model and Treatment

The neonatal WMI model was established following previously described methods [27]. Briefly, P5 pups underwent anesthesia using isoflurane inhalation. The left common carotid artery was carefully ligated between double ligatures after separation before being severed; subsequently, the wound was sutured. The total operation time did not exceed 4 min. Following this procedure, mice were allowed to recover for 1.5 h with their mother before being placed in a temperature-controlled hypoxic container (36.0°C) with humidified mixed gas (10% oxygen and 90% nitrogen at a flow rate of 2 L/min) for 60 min. Sham-operated mice without exposure to hypoxia constituted the Sham group. Animals that succumbed during these procedures (mortality rate: 5%) were excluded from further experimentation.

The mice were allocated into four groups using a randomized block design: sham group, WMI group, WMI + rosiglitazone (WMI + RSGI) group, and WMI + GW9662 (WMI + GW) group. Rosiglitazone (BRL 49653, MCE, Shanghai, China) is a widely utilized PPAR-γ agonist while GW9662 (HY-16578, MCE, Shanghai, China) serves as an efficient PPAR-γ antagonist. Both compounds were dissolved in DMSO, with the final concentration of DMSO being 1% in the solutions diluted with phosphate-buffered saline (PBS). The drug delivery method and dosages used in this study were based on preliminary investigations [28-30]. In the WMI + RSGI group, mice received intraperitoneal injections of Rosiglitazone at a dose of 6 mg/kg 1 h before establishing the WMI model and once daily for 28 days. For the WMI + GW group, mice were administered GW9662 (1 mg/kg) via intraperitoneal injection 1 h before modeling daily for 28 days. The sham and WMI groups of mice received equivalent volumes of 1% DMSO through intraperitoneal injections performed daily for 28 days. The diagram of the in vivo experimental design is shown in Figure 1A,B.

2.3 Cell Culture and Oxygen–Glucose Deprivation (OGD) Model

The human microglial clone three (HMC3) cell line was procured from Shanghai FuHeng Biology located in Shanghai, China. The cells were cultured optimally using Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S) (Gibco, United States). They underwent incubation within a humidified environment at 37°C supplied with a 5% CO₂. OGD induction followed previous protocols but included modifications. HMC3 cells were seeded in 6-well plates and allowed to adhere and grow for 24 h. The next day, the cells were rinsed twice with PBS and subsequently placed under serum/glucose-free DMEM condition within an anoxic incubator (Thermo Fisher Scientific) set at 1% oxygen concentration alongside 5% CO₂ to stimulate HI conditions in vitro. Control group cells underwent similar treatment without exposure to OGD.

2.4 Cell Counting Kit-8 Assay

To determine the appropriate duration of OGD for the in vitro experiments, we performed a cell viability assay on HMC3 cells utilizing Cell Counting Kit-8 (CCK-8, 40203ES88, Yeasen Biotechnology, Shanghai, China). Cells were seeded at a density of 1 × 10⁴ cells/mL in 100 μL culture medium per well into 96-well plates and incubated for 24 h. Subsequently, cells were exposed to OGD for varying durations including 6 h, 12 h, 18 h, and 24 h. After OGD, the culture medium was removed, and CCK-8 solution (1:10) diluted in fresh medium was added into each well at an equivalent volume of 100 μL per well. Following a 2-h incubation in darkness at 37°C, the absorbance was then measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, United States).

2.5 Short Interfering (Si) RNA and Plasmid Transfection

The PPAR-γ siRNA (si-PPAR-γ) and PPAR-γ overexpression plasmid vector (pcDNA3.1-PPAR-γ) utilized in the experiments were synthesized by RiboBio Co. Ltd. (Guangzhou, China). HMC3 cells were seeded in 6-well plates and allowed to stabilize for 24 h. Subsequently, transfection with si-PPAR-γ or pcDNA3.1-PPAR-γ was carried out using Lipofectamine 3000 Transfection Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. At 48 h after transfection, the cells were exposed to OGD for 18 h and then collected for subsequent experiments.

2.6 Reactive Oxygen Species (ROS) Detection

An ROS Assay Kit (S0033S, Beyotime, Shanghai, China) was employed to detect intracellular ROS as per established protocols. Following various treatments, HMC3 cells were washed with fresh medium and incubated with DCFH-DA (10 μM) in fresh medium for 30 min in the dark. The cells were then rinsed thrice with fresh medium and counterstained with Hoechst at 37°C for 10 min. Images were captured using a fluorescence inverted microscope (Ni-U, Nikon, Japan), and the fluorescence intensities of DCFH-DA were quantified using Image J software.

2.7 Open Field Test (OFT)

Anxiety-related behavior was assessed through an open field test conducted 28 days after HI injury. Mice were individually placed in an acrylic glass box measuring 40 × 40 cm, and their movement trajectories were automatically tracked for a duration of 5 min [31]. The central area of the box was defined as a field measuring dimensions of 20 × 20 cm located at its center. The behaviors exhibited by mice including total distance traveled, the percentage of time spent in the central area, and the percentage of distance traveled in the central area were analyzed using animal behavioral analysis system (Shanghai JiLiang SoftwareTechnology Co. Ltd., Shanghai, China).

2.8 Novel Object Recognition (NOR) Test

As previously described [32], the task was conducted from 30 to 32 days after WMI establishment. The NOR test consisted of three stages: habituation, object familiarization, and NOR testing. During the habituation stage on the first day, mice were acclimated in an open field box (40 cm in height, 25 cm in width, 25 cm in length) for 5 min. On the second day during object familiarization, mice had 5 min to explore two identical objects in a familiar box before being returned to their cage for 1 h. In the testing phase, one of the previously presented objects was replaced with a novel object and mice were given 5 min to explore both objects. Exploration was recorded when mice directed their nose within a distance < 1 cm from the object and/or touched it with their nose. The movement trajectory and exploration time toward each object were analyzed using ANYmaze 7.0 (IL, USA). The Recognition Index (RI) is determined by the formula (exploration time of novel object/exploration time of both objects) × 100, while the Discrimination Index (DI) is calculated as [(exploration time of novel object—exploration time of old object)/exploration time of both objects] × 100.

2.9 Beam-Walking Test

The beam-walking task was conducted 33 days after WMI establishment as previously reported [32], with modification. A 0.5 cm-wide beam was positioned 50 cm above the ground, with a black box placed at the beam's end as a target for the mouse to reach. During training, padding material from their own cages was placed in the box to attract them to walk through the beam. Mice received three trials per day for 3 days prior to assessment, and performance was observed by recording videos while assessing motor coordination and balance based on hind limb slippage frequency and time taken to cross the beam.

2.10 Brain Water Content

Mice underwent deep anesthesia followed by decapitation. The left hemisphere was rapidly harvested and weighed to acquire the wet weight. The brain tissue was placed into an electrothermal oven at 70°C for 72 h to obtain the dry weight. The percentage of moisture content was calculated as [(Wet weight—Dry weight)/Wet weight] × 100%.

2.11 Measurement of GSH and MDA Levels

Mice underwent deep anesthesia followed by decapitation. White matter (WM) was promptly extracted, quantified, and homogenized in extraction buffer. HMC3 cells were collected and ultrasonically lysed. Malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) levels were then quantified using commercially available kits from Beyotime Technology (Shanghai, China) in accordance with the manufacturer's guidelines.

2.12 RT-qPCR Analysis

The total RNA extracted from WM tissues and HMC3 cells at a specific time point using the TRIzol/chloroform method was subjected to RT–qPCR analysis. Equal amounts of RNA (1 μg) were reverse-transcribed into cDNA utilizing the PrimeScript RT Master Mix kit (Takara Bio Inc., Japan) according to the manufacturer's protocol. PCR assays were carried out on the CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) employing the TB Green Premix Ex Taq II kit (Takara Bio Inc., Japan). The mRNA expression level of the target gene was quantified using the equation $$$ {2}^{-\Delta \Delta {C}_t} $$$ and normalized to β-actin control. The primers utilized in this investigation were designed with the NCBI Primer-Blast Tool and are detailed in Tables S1 and S2.

2.13 Western Blot Analysis

Mice were euthanized by intraperitoneal injection of an lethal dose of pentobarbital sodium at a specific time point. Brains were harvested on the ice-cold medium, and WM tissues were isolated under a dissecting microscope as previously described. Samples of WM or HMC3 cells were lysed in ice-cold RIPA lysis buffer supplemented with phenylmethane-sulfonyl fluoride and phosphatase inhibitors (all from Solarbio, Beijing, China). The extraction of nuclear proteins followed standard procedures utilizing a Nuclear Proteins Extraction Kit (R0050; Solarbio Biotechnology). Protein concentrations were determined using the BCA protein assay kit (ZJ102L, Epizyme Biotech, Shanghai, China). Equal amounts of protein (30 μg for in vitro studies and 60 μg for in vivo studies) underwent separation via 10%–12% SDS-PAGE gels before being transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using the Bio-Rad wet-blot system along with blot transfer buffer (25 mM Tris and 190 mM glycine) containing 20% methanol.

Following blocking with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) for 2 h at room temperature, the membranes underwent three washes in TBST before being immunoblotted with specified primary antibodies (listed in Table S3) overnight at 4°C on a rotating shaker. After further washing steps, membranes were incubated with the appropriate HRP-conjugated secondary antibodies: Anti-Rabbit IgG (1:10,000, Proteintech, SA00001-2), Anti-Mouse IgG (1:10,000, Proteintech, SA00001-1), or Anti-goat IgG (1:5000, Proteintech, SA00001-4) secondary antibodies at room temperature for 2 h. Proteins were visualized with the enhanced chemiluminescence (ECL) substrate (P2300, NCM Biotech, Suzhou, China) and imaged utilizing the ChemiDoc XRS Biorad Imager. Finally, densitometric measurements concluded through Image Lab Software5.1 (Bio-Rad, CA, USA).

2.14 Luxol Fast Blue (LFB) Staining

Mice were humanely euthanized with a lethal dose of pentobarbital sodium via intraperitoneal injection and subsequently underwent transcardial perfusion with cold normal saline followed by 4% paraformaldehyde (PFA) in PBS at 28 days post-induction of the WMI model. The brains were meticulously dissected, fixed in 4% PFA, dehydrated, and embedded in paraffin. Paraffin-embedded brain samples were sectioned coronally into 5 μm-thick slices using a HistoCore Biocut (Leica, Weztlar, Germany) and mounted on slides. The paraffin sections were stained with Luxol Fast Blue (LFB) staining (Solarbio Biotechnology) according to the manufacturer's instructions. Bright-field images were acquired using an optical microscope (Nikon, Tokyo, Japan) and analyzed using image analysis software (ImageJ).

2.15 Immunohistochemistry

The coronal tissue sections were prepared as previously described. Following deparaffinization in xylene and rehydration in an ethanol gradient, the sections were treated with 3% hydrogen peroxide at room temperature for 15 min. Antigen retrieval was achieved by heating the sections in preheated sodium citrate buffer for 25 min. After cooling and three washes in PBS, the sections were blocked with a solution containing 10% normal goat serum and 0.3% Triton X-100 for 1 h at room temperature. Incubation with primary antibodies (listed in Table S3) was carried out overnight at 4°C. Subsequently, the sections were incubated with a goat anti-rabbit IgG horseradish peroxidase-labeled secondary antibody (1:200) for 2 h at room temperature, followed by PBS washes. Visualization was performed using a DAB kit (ZLI-9018, ZSGB-BIO) according to the manufacturer's instructions. The slides were coverslipped with Cytoseal 60 and imaged using a digital scanner (Leica HS6, Germany). The images were analyzed utilizing Image J software.

2.16 Immunofluorescence Staining

Paraffin-embedded sections were dewaxed, rehydrated, and subjected to microwave-based antigen retrieval. HMC3 cells cultured on coverslips in a 24-well plate were fixed with 4% PFA for 20 min and permeabilized with 1× PBS + 0.3% Triton X-100 for 15 min. For immunofluorescence analysis, brain tissue sections and cells were incubated with primary antibodies (listed in Table S3) overnight at 4°C, followed by fluorescently labeled secondary antibodies for 2 h at room temperature. Fluorescently labeled secondary antibodies included Donkey Anti-Goat IgG H&L (Alexa Fluor 488) at a dilution of 1:1000 (Abcam), CoraLite488-conjugated Goat Anti-Rabbit IgG H&L, and CoraLite594-conjugated Goat Anti-Rabbit/mouse IgG H&L at a dilution of 1:200 (Proteintech, Wuhan, China). Subsequently, brain slices and cells were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime Biotech) for 5 min and mounted with Mowiol before observation under a fluorescence microscope (Ni-U, Nikon, Japan).

2.17 Electron Microscopy

At 28 days after HI insult, mice were humanely euthanized and underwent transcardial perfusion with a solution of 4% PFA and 2.5% glutaraldehyde in PBS following an initial flush with PBS. The brains were then carefully dissected and trimmed to obtain 1-2 mm³ sections containing the corpus callosum. These sections were subsequently fixed in the same solution at 4°C overnight. Following dehydration with acetone, the tissues were embedded in epoxy 812 and sectioned into ultrathin slices (60–80 nm) using a Leica ultrathin microtome (Weztlar, Germany). The sections were stained with uranyl acetate and lead citrate before being examined under a transmission electron microscope (Tokyo, Japan). G-ratios were determined by calculating the ratio of the inner axonal diameter to the total outer diameter (d/D) of myelinated axons using Image J software.

2.18 Statistical Analysis and Reproducibility

All measurements were conducted with a minimum of three independent replicates obtained from distinct experiments. Statistical evaluations for each experiment are detailed in the corresponding figure legends. The data presented are expressed as the mean ± SD. Shapiro–Wilk test was used to analyze the normal distribution. Nonparametric tests were employed for data that did not follow a normal distribution. For experiments involving multiple groups, one-way ANOVA with Tukey's post hoc test was applied. Student's t-test was utilized for comparisons between two groups. Statistical significance was determined using GraphPad Prism 8.1 (GraphPad Software, Inc., CA, USA). A p value < 0.05 was considered statistically significant across all types of statistical analyses.

3.1 Effects of PPAR-γ on Persistent Neurological Deficits in a Neonatal Mouse Model of HI-Induced WMI

In this study, we examined the effects of intraperitoneal delivery of either vehicle, RSGI, or GW9662 over a period of 28 days on the neurofunctional deficits in the neonatal WMI model. The OFT was employed to assess the impact of PPAR-γ on anxiety-like behaviors in the WMI mice induced by HI insult. The results depicted in Figure 1C,D revealed no significant differences in total travel distance and average speed among animals in the four experimental groups. Notably, compared with the Sham group, mice in the WMI group showed significantly less time spent and shorter distance traveled in the central area of the open field, indicative of anxiety-like behaviors. And mice in the WMI + RSGI group conferred a better performance with increasing time spent and longer distance traveled in the central area of the open field compared with that in the WMI group. Conversely, administration of GW9662 led to poorer performance in the OFT than of mice in the WMI group (Figure 1C,E).

To investigate the potential of PPAR-γ activation by RSGI in preserving recognition memory in neonatal mice with WMI, NOR tests were conducted (Figure 1F). In the NOR testing period, mice from the WMI + RSGI group spent more time exploring the new object than mice from the WMI group, suggesting the preservation of recognition memory by RSGI treatment. Conversely, mice in the WMI + GW group showed reduced exploration time toward the novel object and a significant decrease in DI and RI compared to the WMI mice (Figure 1G–I), indicating an exacerbation of cognitive impairment.

Next, a beam-walking test was conducted to assess motor coordination in mice following the exposure to HI injury. The results depicted in Figure 1J demonstrated that HI-induced WMI mice exhibited prolonged traversal time and increased frequency of foot slippage on an 80-cm long beam as compared to the Sham mice. Treatment with RSGI significantly improved the performance in beam-walking test than that of mice in the WMI group, while mice from the WMI + GW group displayed exacerbated performance with longer transmit time and increasing frequency of foot slippage relative to the WMI group (Figure 1J).

Collectively, these findings suggest that PPAR-γ activation facilitates neurofunctional recovery, as evidenced by the alleviation in anxiety-like behaviors, enhancement of motor coordination, and mitigation of cognitive impairments induced by neonatal WMI. Conversely, while blockade of PPAR-γ aggravated poor performances in these neurobehavior tests.

3.2 Effects of PPAR-γ on Blood–Brain Barrier (BBB) Disruption and OLs Differentiation in the Neonatal WMI Model

To determine the effects of PPAR-γ on BBB disruption caused by HI in neonatal mice, brain water content and western blot were performed 24 h after WMI model establishment. The results of western blot illustrated in Figure 2A–D demonstrate that HI significantly induced the downregulation of tight junction proteins (ZO-1, Occludin, and Claudin-5) and adherens junction proteins (β-Catenin and P120 Catenin), but this effect was effectively mitigated by treatment with RSGI. Notably, compared with the WMI group, GW9662 treatment further downregulated the expression levels of these proteins. The quantitative results of brain water content showed that RSGI treatment significantly ameliorated brain edema induced by HI insult, but edema extent was further aggravated after GW9662 treatment (Figure 2E). Overall, these findings suggest that PPAR-γ protects BBB integrity against HI insult in a neonatal mouse model of WMI.

To study the effects of PPAR-γ on OLs differentiation after neonatal HI, we performed western blot and immunofluorescence staining. Western blot analysis demonstrated downregulated levels of platelet-derived growth factor receptor alpha (PDGFRα, an OL progenitor marker), neuron glia antigen-2 (NG2, oligodendrocyte precursor cells (OPCs) marker), and oligodendrocyte transcription factor 2 (Olig2) 72 h after HI insult in the WMI group compared with the Sham group, while activation of PPAR-γ with RSGI significantly increased the levels of these proteins (Figure 2F–H). Consistently, immunofluorescence staining further showed that HI resulted in obvious reduction in the density of Olig2-positive cells in the corpus callosum and striatum compared with the Sham group, while RSGI treatment dramatically restored the density of Olig2-positive cells (Figure 2K,L). Next, we conducted immunofluorescent staining for CC1 (a mature OL marker). Quantification of CC1-positive cells in the corpus callosum 28 days post-modeling showed a reduction in cell density following HI, while the forced activation of PPAR-γ using RSGI was sufficient to upregulate the density of CC1-positive cells (Figure 2I,J). Notably, GW9662 further exacerbated the reduction in protein levels of PDGFRα, Olig2, and NG2 and the density of Olig2-positive or CC1-positive cells at indicated time when compared with those in the WMI group (Figure 2). Overall, these findings demonstrate the protective impact of PPAR-γ in mitigating OLs loss and promoting OLs differentiation in a neonatal mouse model of HI-induced WMI.

3.3 Effects of PPAR-γ on OLs Maturation, Myelination, and Synaptic Deficits in Neonatal WMI Mice Induced by HI

Given the protective effect of PPAR-γ on OLs differentiation, we proceeded to investigate whether PPAR-γ activation could expedite OLs maturation, promote remyelination, and improved synaptic deficits in a mouse model of neoantal WMI. We initially performed immunostaining assay and western blot analysis to determine the expression level of myelin proteins expressed in mature OLs 28 days after model establishment, respectively. As expected, western blot analysis revealed that the expression levels of Proteolipid Protein (PLP), 2′, 3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase), myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) were greatly diminished in the WMI group compared with the Sham group, while RSGI treatment demonstrated higher levels of these proteins than those of the WMI group (Figure 3A,B). Furthermore, immunostaining results exhibited a similar tendency as compared with western blot analysis of the same marker (Figure 3C–G). LFB staining revealed an improvement in the arrangement of nerve fibers and an increase in the average optical density (AOD) values in the WMI mice following RSGI treatment (Figure 4A). Similar to the results of LFB staining, analyses of G-ratio from transmission electronic microscopy (TEM) revealed significantly fewer myelinated axons in the corpus callosum of HI brains compared to the Sham animals, while this trend was substantially mitigated by RSGI treatment (Figure 4B–D).

To assess the impact of PPAR-γ activation on transcriptional activity in OLs and subsequent enhancement of OLs differentiation, qRT-PCR analysis was performed to quantify the mRNA levels of differentiation-associated transcriptional factors including homeobox gene Nkx2.2 and the SRY-related HMG-box gene SOX10 at 28 days after modeling. As depicted in Figure 4E, we found that PPAR-γ activation with RSGI treatment significantly upregulated the mRNA levels of both Nkx2.2 and SOX10 in HI-induced mice, suggesting a promotion of OL transcription. Additionally, western blots analysis showed RSGI treatment substantially reversed the HI-induced downregulation in the protein level of SOX10, thereby providing further validation for the role of PPAR-γ activation in promoting transcriptional activity in OLs (Figure 4F,G).

Next, western blot was utilized to assess protein levels of the presynaptic protein (synaptophysin) and postsynaptic protein (PSD-95), both crucial for synapse formation and remodeling. The results indicated decreased protein levels of synaptophysin and PSD-95 in the WMI group as compared to the Sham group, suggesting synaptic deficits. However, this downregulation greatly was prevented by RSGI treatment (Figure 4H,I). In contrast, GW9662 treatment exacerbated impaired myelination and deteriorated synaptic damage as evidenced by reduced levels of myelin proteins and transcription factors of OLs, further decrease in AOD values from LFB staining, increased G-ratio, and decreased levels of synaptic protein (Figure 4).

Taken together, these results indicate that PPAR-γ activation promotes OLs maturation, myelination, and attenuates synaptic deficits in neonatal WMI mice induced by HI.

3.4 Effects of PPAR-γ on the Activation of Astrocyte and Microglial Polarization Induced by HI in Neonatal Mice

The involvement of activation of microglia and astrocyte is widely acknowledged for its substantial contribution to the regulation of the neuroinflammation, which is crucial in the pathogenesis of WMI in premature infants [11]. To validate an inhibitory effect of PPAR-γ on the activation of astrocyte and microglia, western blot was initially conducted in the WMI mice 24 h after HI injury. Notably, results showed that protein levels of Iba1 (a classical bio-marker of microglia activation) and GFAP (a classical bio-marker of reactive astrogliosis) were upregulated in the WMI group compared with the Sham group. RSGI treatment reversed this effect in HI-induced mice (Figure 5A,D,E). Moreover, GFAP-positive cells (Figure 5B,C) and Iba1-positive cells (Figure 5F,G) in each group as detected by immunofluorescent staining showed the same expression trend as the protein expression of GFAP and Iba1. However, inhibiting PPAR-γ using GW9662 upregulated the protein levels of Iba1 and GFAP and increased the density of both Iba1-positive cells and GFAP-positive cells, suggesting a heightened activation of microglia and astrocyte.

A microglial transition from M2 phenotype to M1 phenotype plays a pivotal role in the microglial activation and the subsequent neuroinflammatory response [11, 33]. To investigate the effect of PPAR-γ on microglial polarization, we performed western blot to detect protein levels of M1-specific markers (CD86) and M2-specific markers (CD206, Arginase-1). Upregulated levels of both M1-specific and M2-specific markers were observed in the WMI group; however, RSGI treatment reduced levels of M1-specific markers, while remarkably upregulating those of M2-specific markers, whereas GW9662 treatment increased the levels of M1-specific markers and decreased M2-specific markers in HI-induced mice, indicating microglial polarization toward the M1 phenotype (Figure 5D,E).

Overall, these data indicate that PPAR-γ activation exhibits an inhibitory effect on activation of microglia and astrocyte and promotes microglial polarization to the M2 phenotype in the neonatal HI WMI model.

3.5 HMGB1/NF-κB Pathway Is Involved in the Amelioration of Neuroinflammatory Response After Activation of PPAR-γ in the Neonatal WMI Model

To determine the protein expression of PPAR-γ in the neonatal WMI model, western blot was performed at 12 h, 24 h, and 48 h after HI injury. HI led to a reduction in protein expression of PPAR-γ at 24 h and 48 h after HI injury (Figure 6A). Notably, the decreased level of PPAR-γ protein induced by HI insult was reversed by treatment with RSGI. Conversely, treatment with GW9662 exacerbated the downregulation of PPAR-γ protein levels (Figure 6B). We further elucidate the regulatory function of PPAR-γ in neuroinflammatory response through western blot and qRT-PCR of inflammatory cytokines. The WMI group exhibited significantly higher levels of pro-inflammatory cytokines (iNOS, TNF-α, IL-1β, and IL6) at both protein (Figure 6C,D) and mRNA levels (Figure 6E) compared with the sham group, while RSGI treatment decreased HI-induced expression of these cytokines. Conversely, blocking PPAR-γ with GW9662 significantly worsened inflammatory response in HI-induced mice as revealed by the upregulated expression levels of pro-inflammatory cytokines (Figure 6C–E).

The translocation and release of High Mobility Group Protein 1 (HMGB1) play important roles in inflammatory response in neonatal HI brain injury [34]. Assessing cytosol protein via western blot showed that HI injury increased the cytosol transfer of HMGB1 at 24 h after HI. Treatment with RSGI led to a reduction in cytosol HMGB1 levels (Figure 6F,G). Activation of the NF-κB pathway is associated with HMGB1 expression and the post-WMI neuroinflammation [35, 36]. To further investigate the specific mechanisms of PPAR-γ-mediated alleviation of inflammation under WMI condition, NF-κB activation was assessed at 24 h after HI in neonatal mice. The results showed an upregulation in the protein levels of p-NF-κB p65, NF-κB p65, and p-IκBα and a downregulation in the protein levels of IκBα in the WMI mice, indicating activation of NF-κB pathway (Figure 6F–H). Moreover, qRT-PCR analysis (Figure 6I) mirrored the protein expression trends of HMGB1 and NF-κB p65. However, GW9662 treatment remarkably exacerbated the activation of HMGB1/NF-κB pathway as revealed by elevated levels of cytosol HMGB1, p-NF-κB p65, p-IκBα, and NF-κB p65 proteins, and lower protein levels of IκBα (Figure 6F–I).

Therefore, based on the above evidence, we speculate that PPAR-γ activation might exert inhibitory effects on the excessive neuroinflammation in the WMI mice via suppressing the HMGB1/NF-κB pathway.

3.6 NRF-2/KEAP1 Pathway Is Involved in the Amelioration of Excessive Oxidative Stress After Activation of PPAR-γ in the Neonatal WMI Model

In addition to the excessive neuroinflammatory response, microglial activation also induced excessive oxidative stress in cerebral WMI. Given that activation of PPAR-γ with RSGI inhibited the microglial activation and subsequent inflammation, we further investigated the inhibitory effects and mechanism of PPAR-γ on the oxidative stress induced by HI in neonatal mice. We initially quantified the levels of MDA, antioxidant activity (superoxide dismutase (SOD)2 and GSH-Px), and found that HI induced higher levels of MDA and lower levels of GSH-Px and SOD2 as compared with the Sham group, while RSGI treatment remarkably downregulated the levels of MDA with concomitant upregulation in the levels of GSH-Px and SOD2 as compared with the WMI group (Figure 7A,B).

NRF2 is a major regulator against oxidative stress. Several scientific studies have demonstrated the therapeutic potential of targeting the NRF2/Kelch-like ECH-associated protein 1 (KEAP1) pathway in HI-induced brain injury [37-39]. Western blot was further utilized to detect NRF2/KEAP1 pathway, and analyses showed that RSGI treatment significantly increased the expression levels of T-NRF2, heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1 (NQO-1), and decreased the expression levels of KEAP1 and facilitated the nuclear translocation of NRF2 as compared with the Sham group (Figure 7C–E). qRT-PCR analysis showed upregulation in mRNA levels of NRF2 and HO-1 in the RSGI-treated WMI group compared with the WMI group (Figure 7F), confirming the antioxidant effects of PPAR-γ activation against HI-induced WMI. However, GW9662 treatment significantly suppressed the activation of NRF2, increased expression levels of KEAP1, downregulated levels of antioxidant proteins (HO-1, NQO-1, SOD2, and GSH-Px), and elevated the levels of MDA compared with the WMI group (Figure 7C–F).

Therefore, based on the above evidence, we speculate that PPAR-γ activation might exert inhibitory effects on the excessive oxidative stress in WMI mice via activating the NRF2/KEAP1 pathway.

3.7 HMGB1/NF-κB Pathway Is Involved in the Regulation of Microglial Polarization After Activation of PPAR-γ in HMC3 Cells After OGD

Considering the constraints in systemic administration layout, it is imperative to ascertain the role of PPAR-γ in microglia through further advancements. OGD was performed to simulate HI conditions, and the effect of OGD on HMC3 cell viability in different time periods was examined through the CCK-8 assay. Results showed that the viability of cells exposed to OGD for 18 h was reduced to 60.17% ± 3.01% (Figure S2A); thus, OGD for 18 h was chosen in subsequent experiments. Subsequent in vitro experiments revealed that the protein expression level of PPAR-γ was downregulated in HMC3 cells exposure to OGD injury (Figure S2B). Furthermore, utilizing small interfering RNA and plasmid techniques, we successfully manipulated PPAR-γ expression levels and confirmed the transfection efficiency. The map and sequencing information of recombinant expression vector of pcDNA 3.1-PPAR-γ, and sequencing information of si-PPAR-γ were shown in Figure S1. Results from qRT-PCR and western blot showed that the administration of pcDNA 3.1-PPAR-γ effectively increased PPAR-γ expression at both mRNA and protein levels (Figure S2C,D), while si-PPAR-γ led to its downregulation in HMC3 cells (Figure S2E,F). In OGD-treated HMC3 cells, the decrease in the mRNA and protein levels of PPAR-γ was significantly counteracted upon PPAR-γ overexpressing (Figure 8A–C), but was aggravated upon knockdown of PPAR-γ (Figure 9A–C).

Consistent with in vivo findings, western blot analysis showed that overexpressing PPAR-γ significantly decreased the protein expression levels of M1-specific markers (CD16), and increased the protein expression levels of M2-specific markers (CD206, Arginase-1) in HMC3 following OGD injury (Figure 8D,E). However, silencing PPAR-γ showed higher expression levels of M1-specific markers, and lower expression levels of M2-specific markers compared with that in the OGD-preconditioned HMC3 cells (Figure 9D,E). qRT-PCR analysis of CD86 and CD206 further confirmed the effect of PPAR-γ activation on microglial transition from M1 to M2 phenotype (Figures 8F, 9F). Moreover, we assessed inflammatory response in OGD-preconditioned HMC3 cells. Compared with the OGD-treated HMC3 cells, overexpressing PPAR-γ obviously downregulated pro-inflammatory cytokines, such as iNOS, TNF-α, IL-6, and IL-1β both at protein and mRNA levels (Figure 8G–I), whereas silencing PPAR-γ showed higher expression of pro-inflammatory cytokines induced by OGD injury both at protein and mRNA levels than that in OGD-treated HMC3 cells (Figure 9G–I). Quantitative immunofluorescence showed a similar trend in the expression of iNOS and IL-1β in each group as observed in western blot analysis (Figures 8J,K and 9J,K).

Based on the aforementioned data, we evaluated the effects of PPAR-γ on HMGB1/NF-κB pathway. Compared with the control group, OGD induced cytosol transfer of HMGB1, promoted the phosphorylation of NF-κB p65 and IκBα, upregulated the protein levels of NF-κB p65, and downregulated the protein level of IκBα. However, overexpressing PPAR-γ reversed this effect (Figure 8L,M). qRT-PCR analysis showed that mRNA levels of NF-κB p65 were decreased in PPAR-γ-overexpressing HMC3 cells after OGD injury (Figure 8N). Notably, knockdown of PPAR-γ elevated expression levels of cytosol HMGB1, p-NF-κB p65, NF-κB p65, p-IκBα, and elevated mRNA levels of NF-κB p65, as well as decreased expression levels of IκBα compared with OGD-treated HMC3 cells (Figure 9L–N). Thus, our findings suggest that the involvement of PPAR-γ is pivotal in modulating the HMGB1/NF-κB pathway and inflammatory response in HMC3 cells after OGD.

3.8 NRF-2/KEAP1 Pathway Is Involved in the Reduction in Excessive Oxidative Stress After Overexpression of PPAR-γ in HMC3 Cells After OGD

Given that activation of PPAR-γ attenuated the microglial activation both in vivo and in vitro, we further evaluated the inhibitory effects and mechanism of PPAR-γ on the oxidative stress induced by OGD in HMC3 cells. To measure intracellular ROS produced by oxidative stress, the ROS analysis kit was used in OGD-treated HMC3 cells. Our results indicated that ROS fluorescence intensity in OGD-treated HMC3 cells was reduced upon administration of pcDNA 3.1-PPAR-γ (Figure 10A,B), while it was elevated with si-PPAR-γ administration (Figure 11A,B). These findings suggest that PPAR-γ activation effectively prevents the upregulation of ROS produced by oxidative stress in OGD-treated HMC3 cells, while inhibition of PPAR-γ fails to scavenge ROS. Further investigation into the molecular mechanism of PPAR-γ revealed increased expression levels of total NRF2 (T-NRF2), nuclear NRF2 (N-NRF2), and elevated amounts of antioxidant proteins including HO-1, NQO-1, SOD2, and GSH-Px, as well as decreased levels of KEAP1 and MDA in PPAR-γ-overexpressing HMC3 cells after OGD injury (Figure 10C–E). However, silencing PPAR-γ decreased the expression of T-NRF2, N-NRF2, antioxidant proteins (HO-1, NQO-1, SOD2, and GSH-Px), and increased the levels of KEAP1 and MDA (Figure 11C–E) in OGD-treated HMC3 cells. Consistent trends were observed in fluorescence intensity (Figures 10F,G and 11F,G) and mRNA levels of NRF2 and HO-1 compared to western blot analysis (Figures 10H and 11H).

Overall, our findings suggest that overexpression of PPAR-γ activates the NRF-2/KEAP1 antioxidant pathway, while knockdown of PPAR-γ inhibits this pathway in OGD-treated HMC3 cells.