Abstract Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by diverse clinical presentations, often associated with dysregulated oxidative stress mechanisms leading to heightened production of reactive oxygen species (ROS) in the brain. Due to its multifactorial etiology, effective therapeutic approaches for ASD remain challenging to ascertain. This work engineers Prussian blue nanoparticles (PB NPs) designed to mimic the enzymatic functions of key antioxidants such as superoxide dismutase, glutathione peroxidase, catalase, and peroxidase. PB NPs effectively scavenge ROS and restore cellular redox homeostasis. These nanoparticles attenuate neuronal apoptosis by reducing activation of apoptotic markers like cleaved caspase-3 and B-cell lymphoma-2 associated X protein, while enhancing the expression of anti-apoptotic protein B-cell lymphoma-2. Furthermore, PB NPs mitigate neuroinflammation by downregulating pro-inflammatory cytokines and upregulating anti-inflammatory cytokines, thereby alleviating glial cell hyperactivity. In preclinical ASD models, PB NPs significantly improve social interaction deficits, diminish anxiety-like behaviors, and enhance cognitive functions. The therapeutic application of PB NPs represents a notable advancement in ASD treatment, offering a novel approach for clinical intervention aimed at enhancing the quality of life for individuals affected by ASD. Keywords: Nanoenzymes, Oxidative stress, Redox equilibrium, Anti-inflammation, Autism spectrum disorder Graphical abstract [39]Image 1 [40]Open in a new tab Highlights * • PB NPs mimic key antioxidant enzymatic functions. * • PB NPs scavenge reactive oxygen species and stabilize mitochondrial function. * • PB NPs reduce neuronal apoptosis and mitigate neuroinflammation in vitro. * • PB NPs improve social interaction deficits and cognitive functions in ASD models. * • PB NPs offer a novel therapeutic strategy for enhanced ASD clinical intervention. 1. Introduction Autism spectrum disorder (ASD) is a complex neuropsychiatric condition that typically manifests in early childhood, characterized by deficits in social communication, restricted interests, and repetitive behaviors [[41][1], [42][2], [43][3], [44][4]]. The etiology of ASD is multifaceted, involving genetic predispositions, environmental factors, structural brain anomalies, and immune system dysregulation [[45][5], [46][6], [47][7]]. The prevalence of ASD has been increasing annually, a trend attributed to evolving diagnostic criteria and heightened awareness [[48]8,[49]9]. Individuals with ASD face substantial challenges in societal integration, which severely impacts their physical and mental health as well as their overall quality of life [[50]4,[51]10]. Given the multifaceted nature of ASD, which results from a complex interplay of factors, no definitive and effective treatment currently exists for the core symptoms of the disorder. Treatment strategies primarily involve comprehensive, individualized intervention approaches. Currently, the U.S. Food and Drug Administration (FDA) has approved only two medications, i.e., risperidone and aripiprazole, for the management of ASD. These medications do not specifically target the core symptoms of ASD and are frequently associated with adverse effects in clinical practice, including drowsiness, upper respiratory infections, and tremors [[52]11]. This underscores the urgent need for innovative therapeutic strategies to enhance the survival and well-being of individuals with ASD individuals. Despite extensive research, the molecular mechanisms underlying ASD remain incompletely understood. Emerging evidence suggests that oxidative stress plays a critical role in ASD pathophysiology [[53][12], [54][13], [55][14], [56][15]]. Reactive oxygen species (ROS) such as peroxide, superoxide radical (O[2]^−), hydroxyl radical (·OH), singlet oxygen, and hydrogen peroxide (H[2]O[2]) are natural by-products of oxygen metabolism and are essential for cell signaling and homeostasis [[57]16,[58]17]. However, in ASD, there is a significant imbalance between antioxidant defenses and ROS production, exacerbated by reduced glutathione levels, leading to elevated ROS and subsequent oxidative damage to biomolecules, cells, and tissues [[59]18,[60]19]. Such oxidative stress can result in apoptosis, necrosis, inflammation, neurodegeneration, and other pathological responses [[61]14,[62]20,[63]21]. Under physiological conditions, the redox balance is maintained by endogenous enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and peroxidase (POD) [[64]22,[65]23]. However, these enzymes can become ineffective under harsh conditions, such as high temperature and acidity. In recent years, artificial nanozymes such as Fe[3]O[4], Pt, Au, CeO[2], and MnO[2] have garnered significant attention for their ability to combine the unique properties of nanomaterials with enzymatic functions, showing promise in treating a variety of diseases including infectious diseases, cancer, neurodegenerative disorders, gastrointestinal ailments, and cardiovascular diseases [[66][24], [67][25], [68][26], [69][27]]. For example, Yang et al. developed a single-atom nanozyme that mimics natural antioxidant enzymes, significantly reducing pro-inflammatory cytokines, mitigating multi-organ damage, and lowering mortality rates in sepsis animal models [[70]28]. Du et al. reported a 2D niobium carbide MXene-based nano-chelator capable of chelating copper ions and scavenging ROS, demonstrating excellent therapeutic efficacy in treating Alzheimer's disease by facilitating blood-brain barrier penetration through the photothermal effect in the second near-infrared region [[71]29]. Furthermore, Liao et al. introduced a ceria nanoenzyme synergistic drug-carrying nanosystem targeting mitochondria, which alleviates oxidative stress and modulates the mitochondrial microenvironment, thereby restoring brain damage induced by ischemia [[72]30]. These studies underscore the significant potential of nanoenzymes in addressing a range of diseases, particularly neurological disorders. However, to date, there are very few reports on the applications of nanoenzymes for the treatment of ASD. Prussian blue (PB) is a biocompatible dye with the remarkable ability to traverse the human body harmlessly, and it has found extensive applications in diverse domains including magnetics, optics, electrochemistry, and biomedicine [[73]31]. In 2010, FDA approved PB as an antidote for thallium poisoning. PB nanoparticles (NPs) can effectively scavenge ROS, attributed to their affinity for ·OH and their ability to mimic antioxidant enzymes such as SOD, CAT, POD, and GPx [[74]32]. Recent evidence indicates that the enzymatic activity and therapeutic efficacy of PB NPs are highly dependent on their physicochemical properties. Specifically, smaller PB NPs with reduced crystallinity exhibit enhanced POD and CAT activities due to increased surface defects and active sites, enabling superior ROS scavenging capacity [[75]33]. Moreover, cysteine-assisted synthesis strategies can optimize the size and defect density of PB NP, which not only amplify their antioxidant enzyme-mimicking functions but also improve anti-inflammatory effects by suppressing pro-inflammatory cytokines in macrophage models [[76]34]. Emerging studies have also highlighted the role of PB NPs in modulating oxidative stress and reactive nitrogen species in ischemic stroke models, where they have been shown to alleviate oxidative damage, suppress apoptosis and inflammation, and enhance cerebral tolerance to ischemic insults [[77]35]. In Parkinson's disease, they have demonstrated potential as inhibitors of pyroptosis, thereby mitigating neuroinflammation and dopaminergic neuronal degeneration, which contributes to the attenuation of neurodegenerative processes [[78]36]. These compelling findings underscore the significant promise of PB NPs for advancing clinical applications in both neuroprotection and beyond. In this study, we design and fabricate an engineered nanocatalyst composed of PB NPs, demonstrating excellent biocompatibility and efficacy in ameliorating symptoms of ASD by effectively scavenging excessive ROS to maintain redox balance ([79]Scheme 1). PB NPs possess versatile enzyme-mimicking capabilities, encompassing activities akin to SOD, CAT, POD, and GPx. At the cellular level, these functions enable PB NPs to restore cellular redox homeostasis and inhibit cellular apoptosis and neuroinflammation. These cellular benefits translate into significant improvements in social and cognitive functions observed in ASD animal models. Our findings underscore the therapeutic potential of multifunctional catalytic nanoenzymes with ROS-regulating properties for treating ASD, establishing a promising approach for clinical management and offering new prospects for individuals affected by this condition. Scheme 1. [80]Scheme 1 [81]Open in a new tab Schematic illustration of PB NPs in the treatment of ASD. PB NPs, with diverse enzyme-mimetic activities, regulate ROS and mitigate oxidative stress, thereby reducing cellular apoptosis and fostering an anti-inflammatory neural environment, which significantly alleviates autism-like behaviors in animal models of ASD. 2. Experimental section 2.1. Materials and reagents All chemical agents and solvents were purchased from commercial sources and used according to operation instruction. Potassium hexacyanoferrate (Ⅲ) (K[3] [Fe (CN)[6]]), lipopolysaccharide (LPS), valproic acid (VPA), and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich. Ferric chloride (FeCl[3]) and polyvinylpyrrolidone (PVP) were purchased from Greagent (Shanghai, China) and Adamas-beta Inc (Shanghai, China). TAC assay kit, GPx assay kit, SOD assay kit, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), and bicinchoninic acid (BCA) protein assay kit were obtained from Beyotime Biotechnology (Shanghai, China). Calcein acetoxymethyl ester/propidium iodide (Calcein-AM/PI) cell double staining kit and cell counting kit-8 (CCK-8) assay were bought from Dojindo (Kumamoto, Japan). Cell apoptosis analysis kits were obtained from Yeasen (Shanghai, China). Caspase-3 antibody, B-cell lymphoma-2 (Bcl-2) antibody and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody were purchased from ABclonal (Wuhan, China). B-cell lymphoma-2 associated X protein (Bax) antibody was obtained from HUABIO (Hangzhou, China). The secondary anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP) was purchased from ABclonal (Wuhan, China). 2.2. Synthesis of PB NPs The preparation of PB NPs was achieved using PVP as a stabilizer through simple colloidal chemical methods. K[4] [Fe (CN)[6]] solution was prepared by adding K[4] [Fe (CN)[6]]·3H[2]O (0.042339 g, 0.1 mM) to ultra-pure water (80 mL). FeCl[3] solution was prepared by adding FeCl[3]·6H[2]O (0.0162204 g, 0.1 mM) and PVP (K-30, MW 38–40 k, 10 mM in terms of monomer) to ultra-pure water (80 mL). Then K[4] [Fe (CN)[6]] solution was added to FeCl[3] solution with vigorously stirring at 60 °C. The reaction was continued for 30 min and then cooled to room temperature under ambient condition. Then ultrafiltration was carried out using a tangential flow filtration system (MWCO: 50 kDa, Millipore, USA) to exclude the impurities. The ultra-filtered solution was then filtrated with a 220 nm filter (Merck Millipore, Germany) to remove the aggregates. 2.3. Characterization of PB NPs Transmission electron microscopy (TEM) image and mapping were obtained by a Tecnai G2 F30 field emission transmission electron microscope at 300 Kv (FEI Company, USA). X-ray photoelectron spectroscopy (XPS) was performed by an ESCALAB 250Xi (Thermo Scientific, USA). X-ray diffraction (XRD) measurement was carried out on an X-ray diffractometer (Panalytical Empyrean, Netherlands). The ultraviolet and visible spectrophotometry (UV–vis) absorption spectra was determined by a DU800 spectrophotometer (Beckman Coulter Inc, USA). Raman spectra was determined by a DER2xi spectrophotometer (Thermo Scientific, USA). Fourier-transform infrared spectroscopy (FTIR) spectra was conducted by a spectrometer (Nicolet AVATAR 370, Thermo Scientific, USA). The ζ-potential and size distribution were performed by ZetaSizer (Nano-ZS, Malvern Instruments, UK). 2.4. SOD-like activity of PB NPs The SOD-like activity of PB NPs was evaluated using a commercially available colorimetric SOD assay kit (Beyotime Biotechnology, Shanghai, China), based on the xanthine–xanthine oxidase system. In this system, superoxide anions generated from the enzymatic reaction reduce nitro blue tetrazolium (NBT) to form a water-soluble formazan, which is quantitatively measured at 560 nm. The presence of SOD or SOD mimetics inhibits this reduction, leading to a decreased absorbance. PB NPs dispersions were prepared in phosphate-buffered saline (PBS) at various concentrations (0, 30, 60, 120, 240, 480, and 960 μg/mL) to assess the dose-dependent activity of PB NPs. The reaction system included 20 μL of PB NPs dispersion, 160 μL of NBT/enzyme working solution (comprising 158 μL of SOD detection buffer, 1 μL of 2.5 mM NBT, and 1 μL of xanthine oxidase), and 20 μL of reaction initiator. Samples were incubated at 37 °C for 30 min, followed by absorbance measurement at 560 nm using a microplate reader (SpectraMax M5, Molecular Devices). To ensure accurate assessment, four experimental groups were established: (i) Test group (Sample): 20 μL of PB NPs + 160 μL of NBT/enzyme working solution + 20 μL of reaction initiator; (ii) Maximal reaction control (Blank 1): 20 μL of SOD detection buffer + 160 μL of NBT/enzyme working solution + 20 μL of reaction initiator; (iii) Background control (Blank 2): 40 μL of SOD detection buffer + 160 μL of NBT/enzyme working solution; (iv) NP interference control (Blank 3): 20 μL of PB NPs + 20 μL of SOD detection buffer + 160 μL of NBT/enzyme working solution. Formazan production was quantified spectrophotometrically at 560 nm (defined as A). The superoxide scavenging rate was calculated using the following formula: [MATH: Inhibition(%)=(ABlank1−ABlank2)−(ASample−ABlank2)ABlank1−ABlank2×100% :MATH] 2.5. POD-like activity of PB NPs The POD-like activity assay was performed by using TMB as the POD substrate with the assistance of H[2]O[2]. In brief, TMB (10 mM), H[2]O[2] (10 mM), and PB NPs were mixed at room temperature in an acetate buffer solution. As the reaction proceeded, the blue color was developed, then the absorbance changes of solution were immediately measured in time-scanning mode at 652 nm using multifunction microplate reader to assess the POD-like activity. 2.6. CAT-like activity of PB NPs The CAT-like ability assay was monitored by using dissolved oxygen meter to obtain concentration of O[2] solution. H[2]O[2] solution (5 mL, 10 mM) was mixed with PB NPs (500 μL) in the glass bottle and the concentration of O[2] solution was monitored during 300 s. The kinetic assays of PB NPs were performed by adding 500 μL PB NPs to different amounts (0.1, 0.2, 0.4, 0.8, and 1.6 M) of H[2]O[2] solution (n = 3 for each group). 2.7. GPx-like activity of PB NPs The GPx-like activity assay was studied spectrophotometrically using a GPx assay kit as the manufacturer's instructions. In brief, GPx can catalyze glutathione (GSH) to produce glutathione disulfide (GSSG), while glutathione reductase subsequently uses nicotinamide adenine dinucleotide phosphate (NADPH) to catalyze GSSG back to GSH. The GPx activity is quantified by measuring the reduction in NADPH, as the decrease in NADPH absorbance is directly proportional to GPx activity. Test working solution was prepare according to the instructions. Subsequently, this solution was incubated with either the GPx detection buffer or PB NPs for 15 min at 37 °C. The absorbance changes of the reaction mixture at 340 nm were immediately measured in time-scanning mode to assess the GPx-like activity (n = 3 for each group). 2.8. ·OH scavenging activity The scavenging ability of ·OH was investigated by the electron spin resonance (ESR) spectrum. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) (1 mg) was added in H[2]O[2] (1 mM) or PBS buffer (200 μL, 10 mM) for radical capture. Fenton reagent was then introduced to generate ·OH. The scavenging capability was evaluated by comparing the ESR signal intensity with/without PB NPs (n = 3 for each group). 2.9. ·O[2]^− scavenging activity The scavenging ability of ·O[2]^− was measured by the ESR spectrum with DMPO as a trapping agent. KO[2] (35.56 μg) was mixed with the 18-crown-6 in Dimethyl Sulfoxide (DMSO) solution (200 μL, 0.35 mM) and further added with DMPO (1 mg) under ultrasonic treatment. Then ESR signals were collected in the absence and presence of PB NPs to estimate ·O[2]^− scavenging ability. 2.10. Cell culture Human neuroblastoma (SH-SY5Y), rat microglia (GMI-R1), rat neuroblastoma (B104), and mouse brain microvascular endothelial cells (bEnd.3) were originally obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). All cells were regularly maintained in complete Dulbecco's Modified Eagle Medium (DMEM) medium supplemented 10 % fetal bovine serum (FBS), 1 % penicillin, and 1 % streptomycin. All cultures were kept at 37 °C in a humidified atmosphere of 5 % CO[2] and 95 % air. The medium was changed every two days, and the cells were routinely harvested by using 0.25 % trypsin solution before approached 80 % confluence. 2.11. Cell viability study The cytotoxicity of PB NPs was evaluated by Cell Counting Kit-8 (CCK-8) assay. Briefly, SH-SY5Y, GMI-R1, B104, and bEnd.3 cells in growth medium were placed into each well of 96-well plates at 37 °C (100 μL, 5 × 10^4 cells/mL). After incubation for 24 h, the culture medium was replaced fresh one containing PB NPs at various concentrations (0, 30, 60, 120, 240, 480, and 960 μg/mL). After another 24 h culture, the cells were rinsed twice lightly with prewarm PBS. CCK-8 work solution (100 μL total, including 10 μL CCK-8) was then added and treated for further 1.5 h. Subsequently, the relative cell viability was calculated by measuring the absorbance at 450 nm using a Spectra M2 microplate reader (BIO-RAD, USA) (n = 3 for each group). 2.12. Hemolytic test Red blood cells from rat donors were seeded into anticoagulant tubes and added with PB NPs (30, 60, 120, 240, 480, and 960 μg/mL) for 4 h of treatment at room temperature. PBS and Triton were used as negative and positive controls, respectively. Then, the culture solution was collected and centrifuged (4 °C, 2000 rpm, 10 min). The absorbance of the supernatant was measured at 540 nm by using a microplate reader (n = 3 for each group). The hemolysis rates were relative to the average absorbance values. 2.13. Construction of H[2]O[2] and VPA induced cell models SH-SY5Y cells were seeded in 96-well plates (5 × 10^4 cells/well). After incubation for 24 h, the medium was discarded, and the cells were exposed to fresh medium containing H[2]O[2] (500, 550, 600, 650, 700, 750, and 800 μmol/L) and VPA (1, 2, 4, 8, 16, 32, and 40 mM) for another 24 h. Finally, the cell viability was detected using CCK-8 assay, respectively (n = 3 for each group). The optimal stimulation concentration is around 50 % of cell viability, which is used for the construction of an oxidative stress and autism cell model in vitro. 2.14. Evaluation in cellular protecting from oxidative damage SH-SY5Y cells were seeded in 96-well plates (5 × 10^4 cells/well). Following a 24-h incubation, the medium was replaced with fresh medium containing either H[2]O[2] (650 μmol/L) or VPA (16 mM), supplemented with varying concentrations of PB NPs (0, 30, 60, 120, 240, and 480 μg/mL). The cells were then incubated for an additional 24 h. Cell viability was subsequently quantified using the CCK-8 assay (n = 3 for each group). 2.15. Live/dead assay of cell Calcein-AM solution (2.5 μL) and PI solution (5 μL) were added to PBS (1 mL) for the preparation of fluorescent dyes. Subsequently, the fluorescent dyes were co-cultured with the cells for 15 min and then washed by PBS for three times. Finally, the fluorescence images were conducted using confocal laser scanning microscopy (CLSM) (CarlZeiss LSM710). 2.16. Apoptosis detection Annexin-V and PI staining assays were used to quantify the apoptotic count of SH-SY5Y cells after different treatments. The medium was removed, and the cells were collected and washed three times with PBS. After that, PI (5 μL, 10 mg/mL) and Annexin-V (5 μL) were added and stained for 5 min. Finally, cells were washed three times with PBS and tested on a flow cytometer. 2.17. Oxidative stress assessment The intracellular redox status was determined using a fluorometric oxidative stress assay kit based on the oxidant-sensitive fluorescent probe dichlorofluorescein diacetate (DCFH-DA) as per the manufacturer's instructions. DCFH-DA, a non-fluorescent cell-permeable compound, diffuses across cell membranes and is hydrolyzed by intracellular esterases to form 2′,7′-dichlorodihydrofluorescein (DCFH). Subsequent oxidation of DCFH under pro-oxidant conditions produces the highly fluorescent 2′,7′-dichlorofluorescein (DCF), with fluorescence intensity correlating with cellular oxidative burden. To conduct the experiment, cells with different treatments were incubated with serum-free culture medium (1 mL) containing DCFH-DA (10 μM). After 30 min of incubation, the cells were washed three times with PBS to remove excess dye. Fluorescence intensity was then measured using flow cytometry (FCM), and fluorescence visualization was performed using CLSM. The excitation wavelength for DCF detection was 488 nm, and the emission was measured at 525 nm. 2.18. Western blot analysis To detect changes in Bcl-2, Bax, and Caspase-3 expression, cells were treated with Western & IP lysate containing phenylmethylsulfonyl fluoride (PMSF). After incubation in an ice-water bath for 30 min, proteins were extracted and quantified using a BCA protein assay kit. Based on the protein concentration, samples were heated at 100 °C for 20 min with 5 × SDS-PAGE loading buffer. Proteins were then separated by gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane, which was blocked at room temperature. The membrane was incubated with the primary antibody consisted of Bcl-2 antibody (1:1000, ABclonal, A0208), Bax antibody (1:30000, HUABIO, ET1603-34), Caspase-3 antibody (1:1000, ABclonal, A25309) and GAPDH antibody (1:10000, ABclonal, AC001) overnight. The following day, membranes were incubated with corresponding secondary antibodies (1:5000, ABclonal, AS014) at room temperature for 1.5 h and visualized using Enhanced Chemiluminescence (ECL) detection reagent (Yeasen, Shanghai, Cat No. 36208ES60). 2.19. Total RNA extraction and quantitative polymerase chain reaction (qPCR) For detection of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) and anti-inflammatory cytokine interleukin-10 (IL-10), TRIzol® reagent (TransGen Biotech, Beijing) was used to extract total RNA from GMI-R1 cells according to the manufacturer's instructions. RNA purity was examined utilizing a Nanophotometer-NP80 (IMPLEN, Cat No.CA10770-492). Reverse transcription-qPCR (RT-qPCR) was performed by using the Hieff® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China, Cat No. 11201ES08) and CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The reaction mixture contained each primer (0.4 μL, 10 μM), H[2]O (3.6 μL), cDNA (1 μL), and Hieff® qPCR SYBR Green Master Mix (5 μL). The relative mRNA expression of the target gene was normalized to that of β-actin. 2.20. ASD models construction and treatment Sprague-Dawley (SD) rats (male, 300 g; female, 250 g) were obtained from the Experimental Animal Center of the Chinese Academy of Sciences in Shanghai, China. All rats were housed under standard conditions (23 ± 2 °C; 55 % ± 5 % humidity) with a 12 h light-dark cycle. All experimental procedures were carried out in accordance with the guidelines in “the Animal Management Regulations” of the Ministry of Health of the People's Republic of China and were approved by the Animal Ethics Committee of Shanghai University (ECSHU-2020-030). Adult male and female rats were allowed to mate overnight. The day of detection of a vaginal embolus was recorded as day 0.5 of pregnancy. At gestational day 12.5, the pregnant females were randomly allocated into two groups. One group received a single intraperitoneal injection of VPA (600 mg/kg in 0.85 % saline), while the control group received an equivalent volume of sterile saline. The postnatal day (PN 0) was recorded on the day of parturition. Offspring were weaned at PN 23. Early physical and neurological development assessment were assessed during this period (n = 8 per group). Subsequently, male rats were selected as experimental subjects and divided into VPA-induced (VPA) and PB NPs-treated (VPA + PB NPs) group. Rats in the VPA + PB NPs group received a single stereotactic injection of PB NPs solution (4 μL) into the hippocampus, while rats in the VPA group and the age-matched healthy rat (CON) group were injected with an equivalent volume of sterile saline. Subsequently, behavioral tests of rats were conducted (n = 6 for each group). All experimental rats were humanely euthanized at the end of the study in accordance with institutional ethical guidelines. 2.21. Hematoxylin and eosin (H&E) staining Major organs (heart, liver, spleen, lung, and kidney) were harvested from each group. The specimens were then fixed in 4 % paraformaldehyde (PFA), embedded in paraffin, sectioned at 4 μm thickness, subjected to H&E staining, and examined under an inverted phase-contrast microscope. 2.22. Blood routine test Approximately blood (1 mL) per sample was collected via orbital puncture from each rat. Routine blood tests were conducted using an automated hematology analyzer. 2.23. Early physical and neurological development assessment in rat offspring Newborn rats underwent the following tests to evaluate early physical and neurological development parameters: righting reflex test, cliff avoidance test, negative geotaxis test, swimming test, eye-opening score, and weight gain (n = 8 for each group). All behavioral tests were conducted under controlled temperature and humidity conditions. For the righting reflex test, rats were placed ventral side up on a table, and the number of days from PN 2 to PN 7 when the rat completed a 180° turn and landed on all fours within 3 s was recorded. For the cliff avoidance test, the number of days from PN 2 to PN 7 when the rat exhibited withdrawal behavior within 15 s of being placed at the edge of a table was recorded. For the negative geotaxis test, the time required for the rat to perform a 180° turn when placed head-down on a 25° inclined slope from PN 7 to PN 10 was recorded, with a 60-s time threshold. For the eye-opening score, a score of 0 was given if both eyes were closed, 1 if one eye was open, and 2 if both eyes were open. For the swimming test, a tank with water maintained at 27–28 °C was used. The swimming behavior of rats for 5–10 s on PN 8, PN 10, PN 12, and PN 16 was recorded and scored. The scoring criteria were as follows: 0 for the head and nose submerged, 1 for the head raised but the nose submerged, 2 for the head and nose above water but the ears submerged, 3 for water at the middle of the ears, and 4 for the head, nose, and ears fully raised. After observation, the rats were dried with a towel to prevent cooling effects. To assess weight gain, the weight measurements of the rats were recorded on PN 7, PN 14, PN 21, PN 28, and PN 35. 2.24. Behavioral test Rats in each experimental group underwent a series of tests, including the three-chamber social test, Y-maze test, elevated plus maze test, open field test and Morris water maze test (n = 6 for each group). All behavioral tests were conducted in a controlled environment with regulated temperature and humidity. The light intensity used in each test was adjusted according to the specific experimental setup. All tests were conducted with the support of Shanghai Yuyan Instruments. To minimize potential biases associated with olfactory cues, the equipment used in the tests was cleaned with 75 % ethanol after each trial. The three-chamber social test is used to assess the social abilities of rodents. The test consists of four phases. Initially, the subject rat was acclimated in the central chamber for 5 min. Subsequently, the rat was allowed to freely explore all three chambers for 5 min, with transparent cylinders present in both the left and right chambers. In the third phase (social phase), an unfamiliar, age-matched rat (stranger 1) was placed within the transparent cylinder in the left chamber, while the right chamber contained an empty transparent cylinder. During the fourth phase (Social novelty phase), stranger 1 remained in the left chamber's cylinder, and a second unfamiliar rat (stranger 2) was introduced into the transparent cylinder in the right chamber. The time spent of rats in the three chambers during the third and fourth stages was analyzed separately. The Y-maze test was used to assess exploration and stereotypic behaviors. The apparatus consists of three identical arms. The spontaneous alternation rate of the rats was recorded over a 10-min period. The elevated plus Maze test is used to assess the exploratory behavior of rodents. It consists of two open arms, two closed arms, and a common central platform. The time spent by rats in the open arms during a 10-min period is recorded. The open field test is used to evaluate the locomotor activity and anxiety levels of rodents. A black open box is used, with the floor divided into 16 equal squares, and the central area contains 4 smaller squares, marked as the central squares. The total distance travelled and the time spent in the central area by rats within a 10-min period in the open field are recorded. The Morris water maze test was used to evaluate cognition and working memory in rodents. The apparatus consists of a circular pool filled with black water, facilitating the tracking of the rats, with the water temperature maintained at 26–27 °C. The entire experiment spanned 6 d. For the first 5 d, rats were placed into the maze from four different starting points and allowed to swim freely until they found a hidden platform. During the 5 d of training, the latency for the rats to find the hidden platform was recorded. On the final day, the platform was removed, and the trajectory of the rats through the memory area was recorded for 60 s. 2.25. Detection of apoptosis and inflammation of neurons Following the completion of behavioral assessments, rats from various experimental groups were euthanized, and their brains were promptly excised and fixed in a 4 % PFA solution for a period of 24 h. Subsequently, the fixed brain tissue underwent a series of dehydration steps and was embedded in paraffin, from which 10 μm thick sections were then cut. To assess neuronal apoptosis, the TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was employed. Neuronal integrity was evaluated using Nissl staining, which stains the rough endoplasmic reticulum and the cytoplasmic basophilia of neurons, thus providing a clear visualization of neuronal cell bodies. Furthermore, to investigate the inflammatory response within the brain tissue, immunofluorescence staining was conducted using antibodies against ionized calcium binding adaptor molecule-1(Iba-1), a marker for microglia, and glial fibrillary acidic protein (GFAP), a marker for astrocytes. 2.26. RNA sequencing Rats in different treatment groups were euthanized, and their hippocampus were collected for transcriptome sequencing (n = 3 for each group). Hippocampal tissue was promptly placed into pre-cooled RNase-free freeze-storage tubes and quickly frozen in liquid nitrogen for 30 min. The samples were then transferred to a refrigerator for storage at −80 °C. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Bio-pharm Biotechnology (Shanghai, China) according to the manufacturer's instructions (Illumina, San Diego, CA). The transcriptome library was prepared using the TruSeq™ RNA Sample Preparation Kit from Illumina (San Diego, CA) with 1 μg of total RNA. 2.27. Statistical analysis All results in this study were recorded as means ± standard deviation (Mean ± SD). Statistical analysis was performed by Two-tailed Student's t-test or one-way analysis of variance (ANOVA) to identify significant differences. Differences were considered significant when p < 0.05. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. All statistical analyses were performed with GraphPad Prism 8.0 software. 3. Results and discussion 3.1. Synthesis and characterization of PB NPs Uniformly sized PB NPs were synthesized via a straightforward colloidal chemistry approach, utilizing PVP as a stabilizer ([82]Fig. 1a). TEM images reveal that the PB NPs are cubic in shape with an average dimension of approximately 30 nm ([83]Fig. 1b). Elemental mapping images confirm a homogeneous distribution of Fe, K, C, N, and O within the PB NPs ([84]Fig. 1c). XPS survey spectra further validate the successful synthesis of PB NPs by displaying characteristic elemental signals ([85]Fig. 1d). The high-resolution Fe 2p spectra exhibit distinct contributions from Fe^2+ and Fe^3+ species, with the Fe^2+ 2p[3/2] and Fe^3+ 2p[3/2] peaks observed at binding energies of 709.3 eV and 713.2 eV, respectively ([86]Fig. 1e) [[87]37]. These findings align well with the mixed-valent Fe^2+(low-spin)–CN–Fe^3+(high-spin) structure typical of PB NPs [[88]36]. FTIR analyses display an absorption peak at 1670 cm^−1, attributed to C Created by potrace 1.16, written by Peter Selinger 2001-2019 O stretching vibrations in the PVP amide groups, consistent with the spectrum of pure PVP ([89]Fig. 1f). Raman spectroscopy of the PB NPs show a singular, sharp band at 2150 cm^−1, indicative of the C Created by potrace 1.16, written by Peter Selinger 2001-2019 N stretching mode ([90]Fig. 1g). A comparison with pure PVP reveals no significant peak shift, suggesting that PVP primarily acts as a steric stabilizer, with its amide groups weakly coordinating to Fe ions on the surface of PB NPs. XRD pattern is consistent with the crystal structure of PB NPs ([91]Fig. 1h). The PB NPs exhibit benign dispersity in FBS, DMEM, PBS, and water, with a pronounced absorbance around 700 nm, aligning with the characteristic absorption peak of PB NPs ([92]Fig. 1i). Besides, the PB NPs dispersion exhibits a uniform hydrodynamic diameter of 32.7 nm with a polydispersity index (PDI) of 0.167 ([93]Fig. 1j), collectively indicating excellent colloidal stability, which is an essential attribute for in vivo injectable applications. The zeta potential measurements of the PB NPs in water, PBS, and DMEM are approximately −11.1, −27.8, and −11.6 mV, respectively ([94]Fig. 1k), further affirming the synthesis fidelity and stability of the PB NPs. Furthermore, PB NPs exhibit excellent colloidal and structural stability in PBS and DMEM over an extended period, confirming their suitability for biological applications ([95]Fig. S1). Fig. 1. [96]Fig. 1 [97]Open in a new tab Synthesis and characterization of PB NPs. (a) Schematic illustration of the procedure for the preparation of PB NPs. (b) TEM image of PB NPs. (c) Elemental mapping images of PB NPs. (d) Full-survey XPS spectrum of PB NPs. (e) High-resolution Fe 2p XPS of PB NPs. (f) FTIR spectra of PB NPs and PVP. (g) Raman spectrum of PB NPs. (h) XRD spectrum of PB NPs. (i) Absorption spectrum of PB NPs dispersed in water and tyndall effect of PB NPs in different solutions including FBS, DMEM, PBS, and H[2]O. (j) Size distribution of PB NPs. (k) Zeta potentials of PB NPs in different solutions. Error bars represent the standard deviation. Data expressed as Mean ± SD. 3.2. Multienzyme-mimicking activities of PB NPs The ability of PB NPs to functionally mimic antioxidant enzymes was investigated through comprehensive characterization ([98]Fig. 2a). SOD, a crucial antioxidant metalloenzyme, catalyzes the conversion of superoxide radicals into oxygen and H[2]O[2], thus playing a key role in maintaining oxidative equilibrium. The SOD-like activity of PB NPs was quantified using a NBT colorimetric assay, where the activity of SOD is inferred from the inhibition of formazan formation, which demonstrates that formazan suppression increases with PB NPs concentration ([99]Fig. 2b), denoting concentration-dependent SOD-like activity. GPx is crucial for peroxide detoxification, catalyzing the conversion of GSH to its oxidized form GSSG while reducing NADPH. The GPx-like activity of PB NPs was evaluated by measuring the consumption of NADPH, revealing that the presence of PB NPs leads to a decline in NADPH absorbance over time ([100]Fig. 2c), indicative of robust GPx-like activity. CAT catalyze the decomposition of H[2]O[2] to produce O[2] and H[2]O, protecting cells from H[2]O[2] damage. A dissolved oxygen analyzer was used to measure the rate of H[2]O[2] decomposition in the presence of PB NPs to evaluate CAT-like performance. The results illustrate that PB NPs enhance the decomposition rate of H[2]O[2], with this increase being proportional to H[2]O[2] concentration ([101]Fig. 2d, e and [102]Fig. S2), which follows the representative Michaelis-Menten kinetics ([103]Fig. 2f and g). POD-like activity was assessed using TMB as the substrate, in which TMB oxidation by H[2]O[2] is enhanced by POD, yielding a detectable absorption at 652 nm. PB NPs in the presence of H[2]O[2] and TMB exhibit this absorption, confirming POD-like activity ([104]Fig. 2h and [105]Fig. S3), which is also H[2]O[2] concentration-dependent ([106]Fig. 2i). Finally, the antioxidant efficacy of PB NPs against ·OH and ·O[2]^− radicals was confirmed via ESR spectroscopy. ESR signals are strong in both Fenton reaction and H[2]O[2] solutions, but are significantly weakened after introducing PB NPs ([107]Fig. 2j), suggesting the removal of ·OH radicals. Similarly, ESR peaks in solutions of KO[2], 18-crown-6, and DMSO are also markedly reduced upon treatment with PB NPs ([108]Fig. 2k), signifying the effective elimination of ·O[2]^−. In contrast to nanozymes with more restricted activity profiles, PB NPs exhibit robust multi-enzyme mimetic activity and superior physiological compatibility, establishing them as a promising platform for oxidative stress-related therapies, particularly in the treatment of ASD [[109]38,[110]39]. Fig. 2. [111]Fig. 2 [112]Open in a new tab Multiple enzyme-like and ROS-scavenging activities of PB NPs. (a) Schematic illustration of enzyme-mimicking activities of PB NPs. (b) SOD-like activity of PB NPs. (c) GPx-like activity of PB NPs. (d) CAT-like activity of PB NPs (n = 3 for each group). (e) The solubility of O2 in the presence of PB NPs and varying concentrations of H[2]O[2] (n = 3 for each group). (f) Michaelis-Menten curve and (g) Lineweaver-Burk plots of the CAT-like activity of PB NPs. The Km value of the PB NPs was 1.185 mM, and the Vmax value was 0.07027 mM/s (h) POD-like activity of PB NPs (n = 3 for each group). (i) Absorbance in the presence of PB NPs and different concentrations of H[2]O[2] (n = 3 for each group). ESR spectra showing the elimination of (j) ·OH and (k) ·O[2]^− by PB NPs. Error bars represent the standard deviation. Data expressed as Mean ± SD. 3.3. Cytoprotective effect of PB NPs in vitro Motivated by the catalytic capabilities of PB NPs, we further investigated their potential neuroprotective abilities across various cellular contexts. Initial assessments were conducted to evaluate the cytotoxic impact of PB NPs on a range of cell lines, including SH-SY5Y, GMI-R1, B104, and bEnd.3. Administered at concentrations of 30, 60, 120, 240, 480, and 960 μg/mL, PB NPs exhibit negligible cytotoxicity across all cell lines, maintaining cellular viability even at the maximal concentration of 960 μg/mL ([113]Fig. S4). Given that both H[2]O[2] and VPA are known to induce oxidative stress in vivo, we employed H[2]O[2]-treated SH-SY5Y cells to model oxidative stress and VPA-treated SH-SY5Y cells to simulate a pathological model of ASD in vitro [[114]40,[115]41]. The half-maximal inhibitory concentration (IC50) of H[2]O[2] and VPA was determined using the CCK-8 assay ([116]Fig. 3a and b), and this concentration was used for subsequent experiments evaluating the efficacy of PB NPs in mitigating oxidative damage induced by H[2]O[2] and VPA. Notably, cell viability is increased in a concentration-dependent manner upon treatment with PB NPs, reaching levels comparable to the untreated control group at a concentration of 480 μg/mL ([117]Fig. 3c and d). CLSM images, following live/dead staining, further corroborate the capacity of PB NPs to effectively preserve cellular viability ([118]Fig. 3e and g). Moreover, treatment with PB NPs substantially reduce the rate of apoptosis triggered by H[2]O[2] and VPA, as quantified through FCM results; apoptosis rates drop from 26.3 % to 10.9 % and from 24.5 % to 7.3 %, respectively ([119]Fig. 3f and h). Western blot assays further validate that PB NPs treatment not only curtails the activation of apoptotic promoters cleaved caspase-3 and Bax, but also increases the expression of apoptotic inhibitor Bcl-2, underscoring their therapeutic potential in counteracting apoptosis ([120]Fig. 3i and j). Fig. 3. [121]Fig. 3 [122]Open in a new tab Cytoprotective performance of PB NPs in vitro. (a) Viability of SH-SY5Y cells treated with H[2]O[2] (n = 3 for each group). (b) Viability of SH-SY5Y cells treated with VPA (n = 3 for each group). (c) Protective effect of PB NPs on SH-SY5Y cells under oxidative stress induced by H[2]O[2] (n = 3 for each group). (d) Protective effect of PB NPs on SH-SY5Y cells under oxidative stress induced by VPA (n = 3 for each group). (e, g) CLSM images and quantitative analysis of SH-SY5Y cells after different treatments stained with Calcein-AM/PI (n = 3 for each group). (f, h) FCM and corresponding quantitative analysis of SH-SY5Y cells after different treatments stained by Annexin V-Alexa Fluor 647/PI (n = 3 for each group). (i) Western blot analysis of cleaved caspase-3, Bax, Bcl-2, and GAPDH expressions in SH-SY5Y cells after different treatments. (j) Quantitative protein expression levels in SH-SY5Y cells after different treatments (n = 3 for each group). Error bars represent the standard deviation. Data expressed as Mean ± SD. Significance was assessed using Two-tailed Student's t-test (for a, b, c, d, h) and one-way ANOVA (for j), ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. 3.4. Antioxidant capacity and anti-inflammatory action of PB NPs in vitro Growing evidence suggests that dysregulated oxidative homeostasis plays a pivotal role in the pathogenesis of ASD, with oxidative stress biomarkers emerging as potential therapeutic monitoring targets [[123]14,[124]15,[125]42]. To evaluate the antioxidative capacity of PB NPs, the intracellular oxidative stress status was quantified through DCFH-DA-based FCM. Fluorometric analysis reveal that SH-SY5Y cells exposed to H[2]O[2] and VPA exhibit elevated oxidative stress markers, as evidenced by significantly increased DCF fluorescence intensity ([126]Fig. 4a–d). Notably, PB NPs treatment effectively attenuate this oxidative challenge, demonstrating dose-dependent reactive species scavenging activity. Consistent with these findings, CLSM imaging confirm that PB NPs counteract the pro-oxidant effects induced by VPA and H[2]O[2] ([127]Fig. 4e and f). Furthermore, the impact of PB NPs on the inflammatory response induced by oxidative stress was investigated using a classical inflammation cell model induced by LPS. qPCR results demonstrate that, following LPS treatment for 36 h, GMI-R1 cells exhibit a significant upregulation of pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α, while the expression of the anti-inflammatory cytokine IL-10 was downregulated ([128]Fig. 4g–j). Upon treatment with PB NPs, the expression levels of these cytokines were markedly reversed. These findings suggest that PB NPs can effectively alleviate the inflammatory response induced by oxidative stress. Fig. 4. [129]Fig. 4 [130]Open in a new tab Antioxidant capacity and anti-inflammation action of PB NPs in vitro. (a, b) FCM and corresponding quantitative analysis of SH-SY5Y cells after different treatments, stained by DCFH-DA (n = 3 for each group). (c, d) FCM images and corresponding quantitative analysis of SH-SY5Y cells after different treatments, stained by DCFH-DA (n = 3 for each group). (e, f) CLSM images and quantitative analysis of SH-SY5Y cells after different treatments, stained with DCFH-DA (n = 3 for each group). (g–j) Quantification of the mRNA expression levels of pro-inflammatory factors including IL-1β, IL-6, and TNF-α and anti-inflammatory factor IL-10 in GMI-R1 cells following different treatments (n = 3 for each group). Error bars represent the standard deviation. Data expressed as Mean ± SD. Significance was assessed using Two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. 3.5. Therapeutic effect of PB NPs on ASD rats The cytocompatibility of PB NPs was examined using rat red blood cells (RBCs), revealing that PB NPs do not compromise the structural integrity of RBCs, even at a concentration of 960 μg/mL ([131]Fig. 5a and b). To further assess the toxicity of PB NPs treatment, H&E staining was performed on key organs, including the brain, heart, liver, spleen, lung, and kidney. The results showed no significant differences between control rats and those treated with PB NPs, as both groups exhibited intact and clear tissue structures ([132]Fig. 5c). Additionally, routine blood tests indicated that all parameters remained within normal ranges following PB NPs treatment ([133]Fig. 5d–o), underscoring the excellent biocompatibility of PB NPs. Besides, biodistribution analysis following stereotactic administration indicates that the PB NPs predominantly enrich in the brain within 24 h, with partial accumulation observed in the liver and kidneys, suggesting hepatic and renal clearance pathways ([134]Fig. S5 and S6). Overall, the remarkable in vivo biocompatibility of PB NPs establishes a foundation for the potential application of PB NPs in ASD treatment. Fig. 5. [135]Fig. 5 [136]Open in a new tab Biocompatibility of PB NPs. (a) The ratio of hemolysis at different concentrations of PB NPs treatments (n = 3 for each group). (b) Hemolysis image for PB NPs at different concentrations. (c) H&E-stained sections of major organs including heart, liver, spleen, lung and kidney from rats with different treatments. (d) White blood cells (WBC), (e) lymphocytes (LYM), (f) monocytes (MON), (g) neutrophils (GRAN), (h) red blood cells (RBC), (i) hemoglobin (HGB), (j) mean corpuscular volume (MCV), (k) mean corpuscular hemoglobin (MCH), (l) platelets (PLT), (m) mean platelet volume (MPV), (n) platelet distribution width (PDW), and (o) plateletcrit (PCT) in healthy rats before and after stereotactic technique of PB NPs (n = 3 for each group). The shaded portion represents the numerical reference range. Error bars represent the standard deviation. Data expressed as Mean ± SD. Encouraged by these promising results, the efficacy of PB NPs was validated in vivo using an ASD rat model, with administration to the hippocampal region performed through stereotaxic injection ([137]Fig. 6a). Exposure of rodents to VPA on gestational day 12.5 has been reported to induce behavioral characteristics of ASD in the offspring, along with neurobiological features analogous to those observed in human patients [[138]43,[139]44]. This approach was employed to establish a VPA-induced ASD rat model. Given the higher prevalence of autism patient in males, subsequent experiments were conducted on male offspring rats [[140]45]. VPA rats exhibit slower completion times for righting reflex and cliff avoidance, longer negative geotaxis times, lower scores in the swimming test and eye-opening and lighter body weight ([141]Figs. S7 and S8), indicating significant delays in early neurological and physiological development. Next, the therapeutic effect of PB NPs on VPA-induced ASD rats was evaluated using a series of behavioral tests. In the three-chamber social test, compared to VPA rats, the PB NPs treatment group significantly increase the interaction time with stranger 1 during the third phase of the social phase ([142]Fig. 6b and c). Similarly, during the fourth phase of the social novelty test, the PB NPs treatment group increase the interaction time with the less familiar stranger 2 compared to VPA rats ([143]Fig. 6b and d). These findings demonstrate that PB NPs enhance social interaction and social novelty in ASD rats. Additionally, in the Y-maze test, the PB NPs treatment group exhibits a higher spontaneous alternation rate compared to the VPA group, indicating an improvement in repetitive behaviors of ASD rats ([144]Fig. 6e and f). In the elevated plus maze test, the PB NPs treated group spend more time in the open arms relative to the VPA rats ([145]Fig. 6g and h), suggesting that PB NPs treatment alleviates exploration deficits in ASD rats. In the open field test, VPA rats exhibit shorter total distances travelled and reluctance to explore the central area ([146]Fig. 6i–k), both of which are improved in the PB NPs-treated group, indicating enhanced locomotor activity and reduced anxiety in ASD rats. Finally, in the Morris water maze test, the VPA rats spend more time searching for the hidden platform during the five days of training ([147]Fig. 6l–n). However, following PB NPs treatment, rats exhibit shorter escape latencies, suggesting improved learning capabilities in VPA rats. Additionally, the PB NPs-treated group spend more time swimming in the target quadrant compared to the VPA group, indicating an enhancement in spatial memory. Fig. 6. [148]Fig. 6 [149]Open in a new tab Evaluation of therapeutic efficacy of PB NPs in vivo. (a) Timeline illustrating the prenatal VPA-induced autism rat model, behavioral tests, and histopathological analysis following PB NPs treatment. (b–d) Schematic and quantification of time spent in each chamber during the third and fourth phases of the three-chamber social test (n = 6 for each group). (e, f) Representative traces and quantification of spontaneous alternation rates of the Y-maze test (n = 6 for each group). (g, h) Representative traces and quantification of time spent in open arms of the elevated plus maze test (n = 6 for each group). (i–k) Representative traces and quantification of total distance travelled and time spent in center of the open field test (n = 6 for each group). (l–n) Representative traces and quantification of latency to find the platform and distance in the target quadrant of the Morris water maze test (n = 6 for each group). (o) Representative imaging of Nissl staining, TUNEL staining, iba1 immunofluorescence, and GFAP immunofluorescence (n = 3 for each group). Error bars represent the standard deviation. Data expressed as Mean ± SD. Significance was assessed using one-way ANOVA (for c, d) and Two-tailed Student's t-test (for g, h, j, k, m, n), ∗∗∗p < 0.001. The pathology of the hippocampus is closely associated with the onset and progression of ASD, where the accumulation of ROS in the brain can trigger apoptosis, leading to the demise of hippocampal neurons [[150]6,[151]46,[152]47]. To assess neuronal loss and apoptosis in the hippocampus across different groups of rats, Nissl staining and TUNEL staining were employed. In VPA-treated rats, the hippocampus exhibits a loss of Nissl bodies, irregular neuronal morphology, and disorganized arrangement, indicative of severe neuronal damage ([153]Fig. 6o). The treatment with PB NPs ameliorates this condition, demonstrating robust neuroprotective effects. In the TUNEL staining, VPA-treated rats display a significant increase in green fluorescence, indicating a marked rise in apoptotic cells. Following PB NPs treatment, the green fluorescence is notably reduced ([154]Fig. 6o), highlighting superior anti-apoptotic properties. Furthermore, the levels of Iba-1 (a biomarker for microglia) and GFAP (a biomarker for astrocytes) in the hippocampus of the VPA group are decreased after PB NPs treatment ([155]Fig. 6o), confirming that PB NPs can attenuate the activation of microglia and astrocytes, thereby alleviating neuroinflammation and protecting neuronal cells. 3.6. Therapeutic mechanism of PB NPs on ASD rats To elucidate the mechanisms underlying the therapeutic effects of PB NPs in ASD, transcriptomic analyses were performed on rats subjected to VPA and VPA + PB NPs. The Venn diagram indicates that 16,943 genes are co-expressed across both treatment groups, while 553 genes are uniquely expressed in the VPA + PB NPs group ([156]Fig. 7a). Volcano plot analysis identified 91 differentially expressed genes (DEGs), with 56 genes upregulated and 35 downregulated ([157]Fig. 7b and [158]Table S1). A heatmap of DEGs post-PB NPs treatment is presented ([159]Fig. 7c). Pathway enrichment analysis utilizing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) reveal significantly altered pathways following PN NPs treatment ([160]Fig. 7d and e). Pathways related to oxidative phosphorylation, oxygen binding, oxygen transport, oxygen carrier activity, haptoglobin binding, and hydrogen peroxide catabolic process are notably affected, highlighting their roles in redox balance. Additionally, immune response pathways such as NF-kappa B signaling, adaptive immune response, and mononuclear cell proliferation are significantly altered, underscoring their importance in coordinating immune cell activation and proliferation, and mediating inflammatory responses to maintain homeostasis. The chord diagram emphasizes the top 50 pathways from the GO analysis, pinpointing key genes and pathways linked to these processes, including members of the Bcl2 family associated with apoptosis ([161]Fig. 7f and [162]Table S2). Notably, Bcl2l10, an anti-apoptotic protein that inhibits cell apoptosis and influences cell survival and fate, is significantly upregulated following PB NPs treatment, indicating the involvement of PB NPs in anti-apoptotic process, aligning with the aforementioned findings [[163]48]. Collectively, these findings imply that the therapeutic effects of PB NPs in ASD are highly related to antioxidant, anti-inflammatory, and anti-apoptotic pathways. Fig. 7. [164]Fig. 7 [165]Open in a new tab Therapeutic mechanisms of PB NPs on ASD rats. (a) Venn diagram of the transcriptomic profiles between the VPA and the VPA + PB NPs group. (b) Volcano plot of the significantly upregulated and downregulated genes after PB NPs treatment. (|log2FoldChange| > 1, p-value <0.05) (c) Heatmap diagram of transcriptomic profiles between VPA and VPA + PB NPs groups. (d) The KEGG pathways enrichment and GO enrichment analysis of the 91 DEGs between VPA and VPA + PB NPs groups with the 20 most significantly enriched pathways. (f) The chord diagram of genes and pathways related to oxidative stress, inflammation, and apoptosis within the top 50 pathways from the GO enrichment analysis of the VPA and VPA + PB NPs groups. 4. Conclusions In summary, we designed and engineered PB NPs as artificial nanozymes with broad-spectrum antioxidant enzyme properties and systematically evaluated their therapeutic performance in ASD. PB NPs feature robust total antioxidant capacity and effectively mimic the enzymatic activities of naturally-occurring enzymes involving SOD, CAT, POD, and GPx. In vitro efficacy assessments demonstrate that PB NPs significantly mitigate oxidative damage and suppress cell apoptosis and inflammatory responses in both H[2]O[2]-induced oxidative stress and VPA-induced autism cell models. For extending these observations to in vivo settings, PB NPs ameliorate social deficits and cognitive impairments in ASD rats, reduce hippocampal tissue apoptosis, neuronal damage, and neuroinflammation, and protect neural cells in the canonical VPA-exposed ASD rat model. Overall, our study underscores the therapeutic promise of PB NPs as an efficient approach for treating autism and potentially other neurological conditions. CRediT authorship contribution statement Yan Gong: Writing – original draft, Visualization, Validation, Software, Project administration, Methodology, Formal analysis. Lele Yu: Writing – original draft, Visualization, Validation, Software, Methodology, Formal analysis, Data curation. Lili Xia: Writing – original draft, Visualization, Validation, Resources, Investigation, Formal analysis, Data curation. Jilu Jin: Visualization, Validation, Methodology, Data curation. Yue Lang: Visualization, Validation, Formal analysis, Data curation. Shini Feng: Writing – review & editing, Supervision, Resources, Investigation, Funding acquisition, Formal analysis, Conceptualization. Wei Feng: Writing – review & editing, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Fuxue Chen: Writing – review & editing, Supervision, Investigation, Data curation, Conceptualization. Yu Chen: Writing – review & editing, Supervision, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements