Mn₃O₄ nanozymes as potential therapeutic agents for autism spectrum disorder: insights from behavioral and molecular studies

Minghui Li† ^abd, Song Wang†^cd, Lingling Chang†^c, Ruiting Chen^c, Yuhang Liu^c, Zhengjie Ye^a, Yuhang Zhao^a, YiFan Ma^c, Jing Yang^a, Xinyu Gan^cd, Yongzhi Zhuang*^ed and Peng Wang*^cd
aDepartment of Pharmaceutics, Harbin Medical University, Heilongjiang 163319, China
^bExperimental Center of Traditional Chinese Medicine, Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning, China
^cDepartment of Exercise and Health Medicine, Harbin Medical University, Heilongjiang 163319, China. E-mail: wangpeng@hmudq.edu.cn
^dKey Laboratory of Frigid Zone Exercise Health Research and Translation in Heilongjiang Province, China
^eDaqing People's Hospital, Daqing, Heilongjiang 163319, China. E-mail: zhuangyongzhi@sina.com; Fax: +86-459-2796781; Tel: +86-459-2796781

First published on 12th August 2025

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1. Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by difficulties in social interaction, verbal communication, narrow-range interests or activities, and repetitive stereotypical behaviors.¹ In addition to these core features, individuals with ASD may also have significant deficits in learning, memory, and cognitive function.² The incidence of ASD has been increasing in recent years, with current estimates suggesting that 1 in 36 children in the United States has ASD. It is more common in boys than girls, with a ratio of 3.8:1.³ The exact pathophysiology of ASD is unknown, but it is believed to be a combination of genetic and environmental factors.⁴

A large number of studies have shown that patients with ASD experience oxidative stress.⁵ Oxidative stress refers to a state in which the body's oxidation capacity is stronger than its own antioxidant capacity and produces a large number of free radicals. Free radical induced oxidative damage has been shown to be a key factor in the onset, development, and severity of autism.⁶ ASD is associated with elevated levels of oxidative stress and decreased antioxidant capacity.⁷ Natural antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) play a key role in fighting reactive oxygen species (ROS).⁸ Some studies have shown that the activity of antioxidant enzymes is altered in autism.⁹ The level of SOD in autistic children was significantly lower than that in normal children, and the lower the level of SOD, the more serious the symptoms of autism, indicating the increase of oxidative damage in autistic patients.¹⁰ Taken together, these studies suggest that increased ROS levels may contribute to the progression of autism.

Manganese superoxide dismutase (MnSOD) is mainly responsible for the clearance of ROS under mitochondrial oxidative stress, and manganese (Mn) is one of the essential components of MnSOD.¹¹ As an essential trace element, Mn deficiency and intoxication are associated with adverse neuropsychiatric effects.¹² Although natural enzymes that remove ROS are effective at combating oxidation, they are sensitive to environmental conditions, such as temperature and pH, and are difficult to produce on a large scale.¹³ Therefore, in order to overcome the inherent defects of natural enzymes, a variety of artificial enzymes with ROS scavenging activity have been developed.¹⁴ In recent decades, nanozymes with natural enzyme-like activity have been developed as new artificial enzymes.¹⁵ Nanozymes have the advantages of excellent thermal and biological stability, versatility and easy large-scale production.¹⁶ Among these developed nanozymes, a variety of nanomaterials with ROS scavenging activity have been reported.^17–19 For example, ROS scavenging nanoparticles (nanozymes) made from manganese dioxide (MnO₂) have been explored to remove ROS from inflammatory diseases such as atherosclerosis and diabetes.²⁰ However, Mn-based nanozymes have not been explored in autism research yet.

In order to fill the research field gap, Mn₃O₄ NZs synthesized via a solvothermal method have demonstrated significant SOD mimicking activity. The Mn₃O₄ NZs can remove ROS in vivo and in vitro, regulate neurotransmitter levels, relieve oxidative stress effectively, reduce neuroinflammation and rescue nerve cell damage. ROS scavenging significantly improved the social function, repetitive stereotyped behavior and cognitive function in BTBR mice. Finally, this strategy suggests that biomimetic nanotechnology offers a more effective method for the treatment of autism.

2. Materials and methods

2.1 Chemicals and materials

Manganese acetate (Mn(OAc)₂) was sourced from Shanghai Meixing Chemical Reagent Co., Ltd. Hydrogen peroxide (H₂O₂, 30%) was acquired from Sinopharm Chemical Reagent Co., Ltd. Xanthine, xanthine oxidase, superoxide dismutase (SOD) derived from bovine erythrocytes, and phorbol 12-myristate 13-acetate (PMA) were provided by Sigma-Aldrich. 20,70-Dichlorofluorescein diacetate (DCFH-DA) and Rosup were obtained from Beyotime Chemical Reagent Co., Ltd. 5,5-Dimethyl-1-pyridine N-oxide (DMPO) was supplied by Nanjing Tongquan Chemical Reagent Co., Ltd. Hydroethidine (HE), salicylic acid (SA), and terephthalic acid (TA) were purchased from Aladdin Chemical Reagent Co., Ltd. All chemicals were utilized directly without additional purification. Deionized water (18.2 MΩ cm, Millipore) was used to prepare all aqueous solutions.

2.2 Preparation of Mn₃O₄ nanozymes

Mn₃O₄ nanozymes (NZs) were synthesized following established protocols.²¹ In brief, 1.225 g of manganese(II) acetate tetrahydrate was dissolved in 60 mL of anhydrous ethanol under vigorous stirring until complete dissolution. The resulting solution was transferred into a 100 mL Teflon-lined stainless steel autoclave and heated at 120 °C for 24 hours. After cooling to room temperature, the products were collected by centrifugation and washed three times with ultrapure water. The final dark brown Mn₃O₄ NZs were obtained after air-drying.

2.3 Characterization

The morphology of Mn₃O₄ NZs was observed using a Transmission Electron Microscope (TEM, Tecnai-F30, U.S.). A Zetasizer Nano-ZS90 (Malvern, UK) was used to measure the size of Mn₃O₄ NZs. An X-ray diffractometer (XRD, DX-2700, Bruker, Germany) was used to evaluate the chemical composition of the samples. Photoluminescence spectra were recorded on a Hitachi F-4600 spectrometer (Hitachi Co. Ltd, Japan) and confocal images were obtained by confocal laser scanning microscopy (CLSM) (Olympus, Japan).

2.4 Catalase-like activity assay

The H₂O₂ scavenging capacity of Mn₃O₄ nanozymes (NZs) was quantitatively assessed using fluorescence spectrophotometry by monitoring the characteristic emission of 2-hydroxyterephthalic acid (TAOH) at 425 nm. In a standard assay, reaction mixtures containing H₂O₂ (10 mM, pH 4.5) and Mn₃O₄ NZs in phosphate buffer (25 mM, pH 7.4) were homogenized by vortex mixing and subsequently incubated at 25 °C for 6 h. The fluorescence intensity corresponding to TAOH formation, resulting from hydroxyl radical-mediated terephthalic acid (TA) oxidation, was measured to determine catalytic H₂O₂ decomposition efficiency. Appropriate control experiments including TA solution alone and TA/H₂O₂ mixtures were performed.

2.5 Total superoxide dismutase activity assay

The superoxide anion radicals (O₂˙⁻), generated as reaction products of xanthine and xanthine oxidase, oxidize hydroxylamine to form nitrite, which exhibits distinct absorbance changes at 550 nm after a chromogenic reaction. This experiment was performed following the protocol of the Total Superoxide Dismutase Assay Kit (Nanjing JianCheng, China), where the absorbance changes before and after adding Mn₃O₄ NZs were measured using a UV-visible spectrophotometer.

2.6 Animals and drug administration

The BTBR mice used in this study were housed at the Animal Experiment Center of the Daqing Campus of Harbin Medical University. A one-week acclimation period was provided prior to the formal commencement of the experiments. All mice used in the study were male. All drug administration and behavioral testing were conducted between 9:00 AM and 2:00 PM. The experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH Publication no. 8023, revision 1978) and approved by the Ethics Committee of the Daqing Campus of Harbin Medical University. The mice were randomly divided into two groups: the BTBR group and the BTBR + Mn₃O₄ NZs group. Intracerebroventricular cannulas were implanted in each mouse for the administration of Mn₃O₄ NZs (1.0 μg kg⁻¹) and 0.9% saline, starting from postnatal day 14 (P14) and continuing for 15 days with injections given once every three days. After five injections, social function and cognitive behavior tests were conducted for each group of mice.

2.7 Open field test

The open field test is used to evaluate general motor activity and exploratory behavior. As mentioned before, general exploratory movement is carried out in a new open field environment.²² A CCTV camera is placed on top of the box (Security Camera Direct, Luling, TX, USA). The mice were first allowed to acclimate to the test box for 5 minutes and then their accumulated movement time and distance were recorded for 10 minutes.

2.8 Three-chambered social test

The three-chambered social test is a widely utilized method for evaluating social behavior in mice. The apparatus, constructed from transparent polycarbonate (60 cm long × 40 cm wide × 20 cm high), includes an empty wire cage (5.5 cm in radius, 20 cm in height) to confine a stranger mouse. The chamber is divided into three sections by removable partitions, each featuring a gate (5 cm wide × 7 cm high) in the central area to facilitate movement between compartments. A CCTV camera (Security Cameras Direct, Luling, TX, USA) mounted above the apparatus automatically recorded the time spent in each section and tracked the mouse's position during interactions with stranger mice or objects.²³

The test consisted of a 5 minute habituation phase, followed by two 10 minute experimental phases. In the first phase, an empty metal cage was positioned in the right chamber, while a cage housing a stranger mouse (Stranger 1) was placed in the left chamber. The test mouse was then introduced into the central chamber and allowed to explore freely for 10 minutes to evaluate its social behavior. In the second phase, the empty cage was replaced with the cage containing stranger 1, and a new stranger mouse (stranger 2) was introduced into the previously empty cage. The test mouse's interactions were observed for another 10 minutes. Between trials, the apparatus was cleaned using 70% ethanol and water to eliminate residual odors.

2.9 Marble burying assay

The marble burying test was conducted to evaluate repetitive and stereotyped behaviors in mice, as outlined in prior studies.²⁴ A standard mouse cage (27 × 16.5 × 12.5 cm) was filled with wood bedding to a depth of 5 cm. Twenty black glass marbles, each 1 cm in diameter, were arranged in a 4 × 5 grid on the bedding surface. Each mouse was placed in the cage for 30 minutes, after which the number of marbles buried was recorded. A marble was considered buried if it was more than 50% covered by the bedding material.

2.10 Morris water maze test

As previously described, a Morris Water Maze test system (WMT-100, Chengdu Techman Technology Co., Ltd), which included a place navigation test and a probe test, was used to assess spatial learning and memory of mice.²⁵ The apparatus, which is placed in the laboratory, consists of a white circular pool with a diameter of 100 cm and a height of 45 cm, along with a hidden platform 1.5 cm underneath the water surface. In brief, the place navigation test was conducted twice a day, once in the morning and once in the afternoon, for four consecutive days. The time it took the mouse to reach the hidden platform at 1.5 cm underneath the water surface was recorded. The mice underwent a probe test to assess spatial memory on day 9, during which the platform was removed from the pool. The number of times the mouse crossed the original platform area within 60 s was recorded and analyzed. A video analysis system was used to record and analyze the time, frequency and trajectory during the platform search.

2.11 Self-grooming test

The self-grooming test was employed to assess repetitive and stereotyped behaviors in mice. Following established protocols,²⁶ each mouse was placed individually in an empty standard cage under 40 lx illumination to prevent digging behavior. After a 5 minute acclimatization period, a trained observer recorded the total time spent grooming all body parts over a 10 minute interval.

2.12 Histology studies

2.13 Cytotoxicity assay and oxidative stress model

SH-SY5Y cells were seeded in 96-well plates at 30000 cells per well and incubated overnight. To assess cell viability, the culture medium was replaced, and Mn₃O₄ NZs were added at varying concentrations (0–80 μg ml⁻¹) for 24 hours. To establish the oxidative stress model, the culture medium was replaced with serum-free DMEM containing 500 μM H₂O₂, with or without co-treatment of Mn₃O₄ NZs (5, 10, and 15 μg ml⁻¹). After 24 hours of incubation, cell viability was evaluated using the MTT assay, which measures the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide to formazan.

2.14 Intracellular ROS scavenging detection

To measure intracellular reactive oxygen species (ROS) levels, the fluorescent probe dichlorodihydrofluorescein diacetate (DCFH-DA) was employed.²⁸ SH-SY5Y cells were cultured in 24-well plates for 24 hours, after which Rosup (0.5 mg ml⁻¹) was introduced to induce ROS generation. Following a 30 minute incubation, the medium was replaced three times, and the cells were treated with varying concentrations of Mn₃O₄ NZs for 1 hour. After washing the cells three times with PBS (pH 7.4), DCFH-DA (0.01 mM) dissolved in phenol red-free, and a serum-free medium was added. Finally, nuclei were stained with Hoechst, and images were captured using confocal laser scanning microscopy.

2.15 Oxidative stress measurement

To evaluate the impact of Mn₃O₄ NZs on hippocampal oxidative stress, we evaluate reactive oxygen species (ROS) and malondialdehyde (MDA) contents, the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and the level of glutathione (GSH) in hippocampal tissue. Hippocampal tissues were immediately dissected from euthanized gerbils and tissues were homogenized in ice-cold 0.9% physiological saline (1:10 w/v) using a homogenizer. Homogenates were centrifuged at 4 °C and supernatants were collected for analysis. All biomarkers were analyzed using standardized commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's specifications.

2.16 Inflammatory cytokine detection

Using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturers’ protocols, serum samples and the hippocampal tissues from mice in each group were used to measure the levels of the proinflammatory cytokines interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α).

2.17 Laser speckle contrast analysis (LSCA) of brain tissue

A moorFLPI-2 real-time blood flow zoom laser speckle imaging system was used for LSCA evaluation of brain areas, to assess the blood flow velocity of brain sites.

2.18 Statistical analysis

Statistical analysis was reported using SPSS 22.0 software (SPSS Inc., USA). All data are published as mean ± standard error of the mean (SEM). The results were statistically assessed by two way ANOVA with status (B6 vs. BTBR) and treatment (Mn₃O₄ NZs vs. saline) as between-subject factors. Comparisons among multiple groups were made using unpaired Student's t-test. The Newman–Keuls test was used for post hoc comparisons when the F value was significant and differences with a p-value below 0.05 were considered statistically significant.

3. Results and discussion

3.1 Preparation and characterization of Mn₃O₄ nanozymes

Mn₃O₄ NZs were synthesized by a solvothermal method using ethanol as solvent. First, TEM was carried out to observe the morphology of the as-prepared Mn₃O₄ NZs, displaying uniform polyhedrons (Fig. 1A). The results of dynamic light scattering measurement showed that the average hydration diameter of Mn₃O₄ NZs was 10.59 nm, with an exceptionally low polydispersity index (PDI) of 0.039, which was consistent with the results of TEM (Fig. 1B). The crystalline structure of the as-prepared samples was investigated using powder X-ray diffraction. In the XRD pattern of the as-prepared Mn₃O₄ NZs (Fig. 1C), many diffraction peaks were observed, which was consistent with the standard pattern of Mn₃O₄ (JCPDS no. 24-0734), indicating their highly crystalline nature. X-ray photoelectron spectroscopy (XPS) was applied to analyze the chemical states of the as-prepared Mn₃O₄ NZs. Fig. 1D shows the elemental mapping result using energy-dispersive X-ray spectroscopy, indicating the uniform distribution of Mn and O elements in Mn₃O₄ NZs. In the XPS Mn 2p spectra of Mn₃O₄ NZs, two prominent peaks were observed at approximately 653.4 and 641.6 eV, assigned to Mn 2p_1/2 and Mn 2p_3/2 (Fig. 1E). After deconvoluted analysis of Mn 2p_3/2, four characteristic peaks were obtained. The peaks at 654.3 and 643.3 eV were typically ascribed to Mn³⁺, and peaks at 652.8 and 641.5 eV were attributed to Mn²⁺. In XPS O 1s spectra of Mn₃O₄, there were three peaks at 533.1, 531.4 and 529.4 eV, which were ascribed to –OH, surface adsorbed oxygen species and lattice oxygen species, respectively (Fig. 1F).

3.2 Evaluation of nanozyme activity

The catalase-like (CAT-like) activity of Mn₃O₄ NZs was validated by monitoring the fluorescence intensity of 2-hydroxyterephthalic acid (TAOH). The results demonstrated a significant reduction in fluorescence intensity in the presence of Mn₃O₄ NZs compared to the control group, indicating their superior H₂O₂ scavenging capability (Fig. 2A). The SOD-like enzyme activity of Mn₃O₄ NZs was subsequently evaluated using a commercial SOD assay kit. In this system, the xanthine/xanthine oxidase reaction generates superoxide anion radicals (O₂˙⁻), which oxidize hydroxylamine to form nitrite, resulting in characteristic absorbance at 550 nm. As shown in Fig. 2B, while the control group exhibited a distinct absorption peak at 550 nm, the Mn₃O₄ NZs showed significantly attenuated absorbance. These findings demonstrate that Mn₃O₄ NZs possess remarkable superoxide-scavenging capability with high specificity, confirming their intrinsic SOD-mimicking activity. Subsequently, the intracellular ROS scavenging activity of Mn₃O₄ NZs was investigated using the SH-SY5Y cell model. First, the toxicity of Mn₃O₄ NZs was explored using the MTT assay. As shown in Fig. 2C, cells treated with Mn₃O₄ NZs (0–80 μg ml⁻¹) exhibited a dose-dependent decrease in cell viability. Mn₃O₄ NZ concentrations of 5, 10, and 15 μg ml⁻¹ significantly increased cell viability compared to the model group. Therefore, 5 μg ml⁻¹ of Mn₃O₄ NZs was used in subsequent experiments (Fig. 2D). As shown in Fig. 2E–G, the ROS laser confocal experiments revealed a significant fluorescence enhancement in the model group compared to the control group. When Mn₃O₄ NZs were added, there was a clear reduction in fluorescence. Quantitative analysis of the fluorescence intensity confirmed these findings (Fig. 2H). The results indicate that Mn₃O₄ NZs have the capacity to scavenge intracellular ROS.

3.3 Mn₃O₄ NZs alter abnormal behaviors in BTBR mice

The Morris water maze test was used to assess memory and spatial learning impairments in BTBR mice.²⁹ The results showed that BTBR mice treated with Mn₃O₄ NZs exhibited significantly reduced escape latency compared to untreated BTBR mice, especially on days 3 and 4. These results suggest that treatment with Mn₃O₄ NZs enhanced the learning ability of BTBR mice. In the Mn₃O₄ NZ-treated BTBR mice group, the number of passing times through the target quadrant increased. These results indicate that Mn₃O₄ NZ treatment ameliorated memory and spatial learning impairments in BTBR mice (Fig. 3A–D). Social interaction behaviors were monitored in a three-chamber test.³⁰ BTBR mice showed no significant difference between the empty cage and the cage with unfamiliar stranger 1 in the social ability phase. However, BTBR mice treated with Mn₃O₄ NZs spent more time interacting with stranger 1 during the social ability phase. BTBR mice treated with Mn₃O₄ NZs exhibited a strong preference for the stranger 1 and stranger 2 chambers during the social phase and social preference phase. These results suggest that Mn₃O₄ NZs improve social dysfunction in BTBR mice (Fig. 3E–H). The open field test was used to assess repetitive anxiety behaviors. Anxious mice typically have longer movement durations and distances. Normal mice usually show a reduction in movement time and distance as they adapt to a new environment.³¹ We measured the total movement time and distance. The results indicate that supplementation with Mn₃O₄ NZs reduced the time spent and distance moved by BTBR mice in the open field, which may suggest a decrease in repetitive anxiety behaviors (Fig. 3I and J). Self-grooming and marble burying were used as measures of anxiety in rodents and repetitive behavior, respectively.^32,33 The results showed that BTBR mice exhibited longer grooming times and buried significantly more marbles. However, BTBR mice on Mn₃O₄ NZs exhibited lower self-grooming times and frequency. The Mn₃O₄ NZ treated BTBR mice buried significantly fewer marbles compared to BTBR mice, which suggests that Mn₃O₄ NZs reduce the occurrence and frequency of repetitive behaviors (Fig. 3K–M).

3.4 Mn₃O₄ nanozymes played a protective role in the brain of BTBR mice

In the study, cerebral blood flow (CBF) was evaluated through spatial vascular contours. Compared to the BTBR mice group, the Mn₃O₄ NZs group showed increased CBF in heat maps. This suggests that Mn₃O₄ NZs can restore impaired cerebral vessels and positively affect cerebral blood flow in BTBR mice (Fig. 4A). The hippocampus plays a critical role in learning and memory processes within the brain.³⁴ To explore the impact of Mn₃O₄ NZs on hippocampal neurons in BTBR mice, Nissl staining was utilized to examine neuronal morphology across different experimental groups. The distribution of neurons in the CA1 area of the hippocampus of BTBR mice is sparse, with irregular or ruptured nuclei. The number of Nissl bodies is significantly reduced, with pale cytoplasmic staining, cellular atrophy, and extensive vacuolization. The cytoplasm is lightly stained, with a large number of damaged pyramidal neurons present. The cells in the CA1 area of the hippocampus in BTBR mice treated with Mn₃O₄ NZs are arranged neatly, and an increase in the number of normal cells can be observed (Fig. 4B). Hippocampal neurons in different mouse groups were observed through immunofluorescence labeling of NeuN and γH2AX. Immunofluorescence of NeuN showed that the number of NeuN-positive cells in the Mn₃O₄ NZ group was higher than that in the BTBR group, indicating that Mn₃O₄ NZs significantly increased the proportion of normal neurons. The results of γH2AX immunofluorescence indicated that Mn₃O₄ NZs prevented neuronal DNA damage, suggesting that Mn₃O₄ NZs had a protective effect against damage in the BTBR group (Fig. 4C). These findings reveal that Mn₃O₄ NZs mitigated neuronal damage caused by BTBR. Therefore, the study suggests that Mn₃O₄ NZs have a positive effect on the neuronal damage induced by BTBR and protect hippocampal neurons in BTBR mice.

3.5 Effects of Mn₃O₄ NZs on oxidative stress in BTBR mice

We assessed the levels of MDA and ROS content in hippocampal tissue and measured the levels of antioxidant enzymes involved in oxidative stress balance, including catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD). The results showed that BTBR mice treated with Mn₃O₄ NZs had lower levels of MDA and ROS compared to BTBR mice. CAT, GSH, GSH-Px, SOD and Mn-SOD were significantly upregulated after treatment with Mn₃O₄ nanozymes (Fig. 5A–G). Taken together, these results indicate that Mn₃O₄ NZs effectively attenuated oxidative stress in the brains of BTBR mice. Compared to the control group, the ROS level in the BTBR group was upregulated, but it was significantly downregulated after treatment with Mn₃O₄ NZs.

3.6 Mn₃O₄ NZs inhibited inflammation in BTBR mice

Mn₃O₄ NZs suppress inflammation in BTBR mice by scavenging ROS, as previous literature has shown significant impacts of inflammation on ASD.^35,36 Therefore, we assessed the levels of inflammatory markers. The role of the cGAS-STING signaling pathway in inflammation was explored by analyzing the expression of cGAS and STING in the hippocampus. The results showed that treatment with Mn₃O₄ NZs significantly reduced the immunofluorescence intensity of cGAS and STING compared to the BTBR group (Fig. 6A and B). ELISA results demonstrated that Mn₃O₄ NZ treatment significantly downregulated the levels of IL-1β, IL-6, and TNF-α in the serum and hippocampus of BTBR mice (Fig. 6C–H).

4. Conclusion

In summary, we successfully synthesized Mn₃O₄ NZs that exhibit exceptional ROS scavenging activity. In vitro experiments demonstrated that Mn₃O₄ NZs effectively scavenged intracellular ROS. The outstanding antioxidant properties of Mn₃O₄ nanozymes observed in vitro prompted us to evaluate their ROS scavenging ability in vivo. Notably, in vivo experiments confirmed that Mn₃O₄ NZs can suppress oxidative stress responses in BTBR mice, effectively alleviating inflammation, indicating their potent ROS scavenging activity in vivo. This study not only demonstrated that Mn₃O₄ NZs may play a protective role in the hippocampus of BTBR mice by inhibiting oxidative stress and inflammation and improving repetitive stereotyped behavior, social behavior, and cognitive impairments, but also provided a promising therapeutic strategy for treating ASD using redox active nanozymes (Scheme 1).

Author contributions

Minghui Li: data curation, conceptualization, visualization and writing – original draft. Song Wang: methodology, investigation, formal analysis and writing – original draft. Lingling Chang: formal analysis, methodology and software. Ruiting Chen: methodology and software. Yuhang Liu: Methodology and software. Zhengjie Ye: methodology. Yuhang Zhao: methodology. YiFan Ma: software. Jing Yang: investigation and methodology. Xinyu Gan: investigation and software. Yongzhi Zhuang: validation, supervision and writing – review & editing. Peng Wang: project administration, resources, supervision and writing – review & editing. All authors read and approved the final version of the manuscript.

Ethical approval statement

All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Experimental Animals (NIH Publication no. 8023, revised 1978) and approved by the Experimental Animal Ethics Committee of Harbin Medical University, China (Animal Experimental Ethical Inspection Protocol NO. HMUDQ20241023002). The BTBR mice used in this study were housed in a specific pathogen free environment (ventilated room, 24 ± 2 °C, 55–65% humidity) and had free access to standard water and food at the Animal Experiment Center of the Daqing Campus of Harbin Medical University.

Conflicts of 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.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

Acknowledgements

This study was supported by the Natural Science Foundation of Heilongjiang Province, China: LH2024H030, the Fundamental Research Funds for the Provincial Universities (JFJCTD202202, JFJC202201), the Special Projects for Guiding Local Science and Technology Development by the Central Government (ZY22D052), and the Joint Guiding Funds for the Innovation and Entrepreneurship Ecosystem around Universities, Institutes, and Research Centers in Heilongjiang Province (DQ23KJYD004).

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Footnote

† These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2025