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. 2026 Jul 27;21:342–351. doi: 10.1016/j.ibneur.2026.07.017

Therapeutic potential of edaravone and flurbiprofen in valproic acid-induced autism: Targeting oxidative stress and neuroinflammation

Anahita Hejazi a, Razieh Hajisoltani b, Mitra Farbib a, Hossein Sadr c,d,⁎, Fatemeh Mehrabi e, Tourandokht Baluchnejadmojarad f, Soraya Mehrabi g,⁎⁎
PMCID: PMC13451920  PMID: 42571262

Abstract

Background and objectives

Autism Spectrum Disorder (ASD) presents significant therapeutic challenges, with growing evidence implicating neuroinflammation and oxidative stress in its pathophysiology. This study aimed to investigate the protective effects of edaravone and flurbiprofen, administered alone and in combination, in a valproic acid (VPA)-induced autism model in male rats.

Materials and methods

An autism model was established through prenatal VPA exposure. Social behavior, anxiety, and memory were assessed using a battery of standardized behavioral tests. Pro-inflammatory and oxidative stress markers were evaluated via ELISA, and hippocampal neuronal density was assessed by Nissl staining.

Results

All treatment groups (edaravone, flurbiprofen, and their combination) showed significant improvements in autism-related behaviors, including enhanced social interaction, reduced memory deficits, and decreased anxiety. The treatments lowered pro-inflammatory cytokines (IL-6, TNF-α) and MDA levels, while restoring the activity of antioxidant enzymes GPX and SOD.

Conclusions

Edaravone and flurbiprofen effectively improved autism-like behaviors by targeting shared pathways of oxidative stress and neuroinflammation. However, the combination therapy offered no additive benefits over monotherapy. This finding suggests that monotherapy with either drug is sufficient to achieve maximal therapeutic effects, a point with significant implications for future treatment strategies in ASD.

Significance statement

This study advances neurodevelopmental neuroscience by demonstrating that independently targeting oxidative stress or neuroinflammation achieves maximal behavioral and hippocampal rescue in an autism model, revealing a convergent therapeutic ceiling that challenges combinatorial polypharmacy and refines our understanding of how overlapping pathological pathways govern neural circuit dysfunction and functional recovery.

Keywords: Edaravone, Flurbiprofen, Oxidative stress, Neuroinflammation, Autism

Graphical Abstract

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Introduction

Autism Spectrum Disorder (ASD), derived from the Greek root autos (meaning "self"), is a neurodevelopmental disorder first described by psychiatrist Leo Kanner in children with impaired social communication (Kanner, 1965). ASD is characterized by lifelong challenges in social interaction, communication, and repetitive behaviors, typically manifesting before age 3. Global prevalence approaches 1%, with males diagnosed 2–3 times more frequently than females (Takano, 2015).

Core symptoms include pervasive deficits in social communication and interaction, such as avoidance of eye contact, delayed language development, and difficulty interpreting nonverbal cues (e.g., facial expressions, gestures). Restricted/repetitive behaviors, including insistence on rigid routines, sensory hypersensitivity, and stereotyped movements (e.g., hand flapping), are also hallmark features (Lauritsen, 2013, Sadr et al., 2024).

While ASD’s exact cause remains unclear, genetic and environmental factors are implicated (Sealey et al., 2016). Genetic/epigenetic mechanisms, such as DNA methylation, histone modifications, and microRNA dysregulation, contribute to neurodevelopmental disruptions (Kubota and Mochizuki, 2016). Environmental triggers, including advanced parental age, zinc deficiency, and prenatal viral infections, may exacerbate risk (Christensen et al., 2013). Neuroinflammation, characterized by elevated TNFα, IL-6, and disrupted endocannabinoid signaling, correlates with hypersensitivity and repetitive behaviors (Nicolini and Fahnestock, 2018). Animal studies using valproic acid-induced autism models show increased TNFα, IL-6, and IL1-β, alongside reduced non-inflammatory cytokines (Taleb et al., 2021, Liao et al., 2020, Haramshahi et al., 2025).

Prostaglandin E2 (PGE2), synthesized via cyclooxygenase (COX), modulates neuronal proliferation and differentiation. Clinical studies of ASD have identified disruptions in the COX2/PGE2 pathway, often linked to genetic or environmental factors (Tahmasebi et al., 2021). For example, elevated phospholipase A2 (PLA2) activity, reduced arachidonic acid levels, and increased PGE2 concentrations have been observed in blood samples from autistic individuals (Kazlauskas et al., 2016). The COX2/PGE2 signaling pathway is vulnerable to inflammation, oxidative stress, pollution, and heavy metals, all of which are implicated in ASD pathogenesis (Tahmasebi et al., 2021, Moghadam et al., 2026).

Molecular evidence highlights the COX2/PGE2 pathway’s role in brain development and ASD. Elevated PGE2 levels alter intracellular calcium dynamics, enhancing neuronal proliferation and differentiation in neural stem cells (Yui et al., 2022). Dysregulated expression of autism-related genes (e.g., Ptgs2, Mmp9, Ctnnb1, Wnt3) correlates with increased prostaglandin levels (El-Ansary et al., 2022). These findings underscore prostaglandin’s significance as a critical factor in ASD.

Flurbiprofen, a potent aryl propionic acid (Khullar, 2020), is a nonsteroidal anti-inflammatory drug (NSAID) that blocks prostaglandin production via non-selective COX inhibition (Mahesh et al., 2021). It demonstrates neuroprotective effects in neurodegenerative diseases, including improved spatial learning and memory in Alzheimer’s disease models (Tian et al., 2020, Flores-Cordero et al., 2022). It is noteworthy that neuroinflammation and oxidative stress are deeply interconnected cellular damage processes that often exacerbate one another, making them compelling therapeutic targets for complex disorders like ASD.

Oxidative stress is a key pathophysiological condition in ASD, contributing to neurodevelopmental disorders due to the brain’s heightened susceptibility to oxidative damage (Chen et al., 2021). Autistic children exhibit reduced superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, alongside elevated malondialdehyde (MDA) (Nayan et al., 2020).

Edaravone, a free-radical scavenger, demonstrates neuroprotective effects in acute cerebral infarction, amyotrophic lateral sclerosis (ALS), and ischemic stroke (Herbet et al., 2019). In ALS patients, Edaravone acts as an antioxidant, slows physical decline, inhibits oxidative stress-induced motor neuron death, and reduces hippocampal cell loss in status epilepticus by suppressing iNOS (Kamida et al., 2009).

Accordingly, we hypothesized that attenuating neuroinflammation with Flurbiprofen and oxidative stress with Edaravone would ameliorate autism-like behaviors in the VPA rat model. Furthermore, we predicted that a combined treatment might exert synergistic effects by simultaneously targeting these two interconnected pathways.

The objective of this study is to evaluate the impact of Edaravone (Eda), Flurbiprofen (Flur), or their combination on improving behavioral outcomes in rats with autism by modulating inflammation and oxidative stress.

Material and methods

Drugs and chemicals

Sodium valproate powder (Sigma-Aldrich Co., Saint Louis, MO, USA; CAS # 1069–66–5) was dissolved in 0.9% saline to achieve a concentration of 600 mg/kg. Edaravone was obtained from Simcere Pharmaceuticals (Nanjing, Jiangsu, China). Analytical-grade flurbiprofen was purchased from Sigma-Aldrich (Milwaukee, USA).

Animals and ethical statements

Protocols and experiments were conducted in accordance with the Guide for Care and Use of Laboratory Animals and approved by the ethics committee of Iran University of Medical Sciences (Ethics Code: IR.IUMS.FMD.REC.1400.636). Animals were housed at 22 ± 2 °C, 60% humidity, and a 12-h light–dark cycle, with free access to water and standard chow pellets.

Experimental design

Female and male Wistar rats (200–250 g, 2:1 ratio) were purchased from the animal laboratory of Iran University of Medical Sciences and housed overnight. Pregnant females (vaginal plug confirmed on gestational day 0, GD 0) were divided into:

  • 1)

    Control group: Single intraperitoneal (i.p.) injection of 0.9% saline on GD 12.5.

  • 2)

    VPA group: Single i.p. injection of VPA (600 mg/kg) on GD 12.5 to induce an autistic rat model in the offspring.

Male offspring (n = 48) were allocated into:

  • 1)

    Control: 8 pups from saline-treated dams.

  • 2)
    VPA subgroups: 40 pups from VPA-treated dams divided into:
    • 1.
      VPA: No treatment.
    • 2.
      VPA + Eda: Daily Edaravone (3 mg/kg, i.p.) for 7 days pre-behavioral tests.
    • 3.
      VPA + Flur: Daily Flurbiprofen (10 mg/kg, i.p.) for 7 days pre-behavioral tests, a dosage shown to be effective in previous neuroprotective studies.
    • 4.
      VPA + Eda + Flur: Combined Edaravone (3 mg/kg) and Flurbiprofen (10 mg/kg) for 7 days pre-behavioral tests.

The summary of experimental design and timeline, from VPA administration through behavioral testing and final analysis, is summarized in Table 1.

Table 1.

Summary of Experimental Design and Timeline.

Phase Timeline / Timing Key Actions & Descriptions
Phase 1: Prenatal Gestational Day 0 (GD 0) & GD 12.5 Mating and pregnancy confirmation occur on GD 0. A single dose of Valproic Acid (VPA) is administered to pregnant dams on GD 12.5 to induce the autism model.
Phase 2: Postnatal & Treatment Postnatal Days 38–44 (PND 38–44) Male offspring are divided into 5 groups: Control, VPA, VPA + Edaravone (Eda), VPA + Flurbiprofen (Flur), and VPA + Eda + Flur. A 7-day daily treatment regimen is initiated.
Phase 3: Behavioral Assessment Beginning on PND 45 A battery of behavioral tests is conducted to assess social behavior, anxiety, and memory. Tests include: Social Interaction, Marble Burying, Elevated Plus Maze (EPM), Novel Object Recognition (NOR), and Passive Avoidance.
Phase 4: Final Analysis Following all behavioral tests Animals are sacrificed, and hippocampal tissue is collected. Biochemical analysis is performed to measure inflammatory cytokines and oxidative stress markers via ELISA. Histological analysis is conducted to assess neuronal density via Nissl Staining.
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Drug dosage justification

The dosages for Valproic Acid (VPA), Flurbiprofen, and Edaravone were selected based on pharmacokinetic profiles and therapeutic windows established in previous preclinical literature. Specifically, VPA was administered at 200 mg/kg to achieve therapeutic plasma levels equivalent to clinical ranges, accounting for the rapid drug clearance rate in rodents (Löscher, 1999). Flurbiprofen was utilized at 10 mg/kg as this dosage is well-documented to effectively suppress inflammatory markers and COX activation while avoiding gastrointestinal toxicity (Jamali et al., 1988). Lastly, Edaravone was administered at 10 mg/kg, which falls within the established therapeutic window for free radical scavenging, lipid peroxidation reduction, and blood-brain barrier protection (Hall, 1997).

Tissue collection for biochemical and histological examinations

At the end of the experimental period, the brain tissues were collected using distinct protocols depending on the subsequent analysis. For biochemical assays, the animals were deeply anesthetized with a mixture of ketamine and xylazine and subsequently euthanized by decapitation. The brains were rapidly extracted on ice, rinsed in cold phosphate-buffered saline (PBS) to remove residual blood, and the specific regions of interest were immediately dissected. The samples were snap-frozen in liquid nitrogen and stored at −80°C to prevent protein degradation until further biochemical processing.

For histological examinations, the animals were deeply anesthetized and subjected to transcardial perfusion. The systemic circulation was first cleared with ice-cold PBS (pH 7.4), followed by perfusion with a fixative solution of 4% paraformaldehyde (PFA) in PBS. The whole brains were carefully removed from the skull, post-fixed in the same 4% PFA solution for 24 h at 4°C, and subsequently cryoprotected by immersion in a 30% sucrose solution until they sank to the bottom of the container, ready for cryosectioning and staining.

Social interaction test

The social behavior of rats was evaluated using a three-chambered social test. The testing apparatus is a clear plexiglass box (45 cm × 30 cm × 21.5 cm) with three equal chambers and two walls between them. Two small round doorways (3.5 cm diameter) connect the compartments (1, 2, and 3). To permit visual, olfactory, auditory, and tactile contact while preventing sexual interactions, the apparatus includes two wire cups (8 cm diameter, 9.5 cm tall) with broadly spaced wire bars. This setup measures simple interest in approaching and maintaining physical proximity to another rat. We performed the experiment on postnatal day (PND) 45. To habituate the animal, it was placed in the central chamber (with doorways blocked) for 10 min. A new rat unfamiliar to the test rat was placed in one wire enclosure and termed the "Stranger." The test rat was allowed 10 min to habituate to the Stranger’s presence. A second unfamiliar rat ("Novel") was introduced to the opposite wire enclosure (previously empty). The test rat, now considered the first rat familiar and was given 10 min to interact with the Novel rat. Time spent near each cage (Stranger or Novel) was recorded. Social indices were calculated as: social index = time in Stranger zone 1 / time in empty area, and social preference index = time in Novel zone 2 / time in Stranger zone 1.

Marble burying test

Rats were habituated separately in a cage filled with 3 cm of clean bedding for 10 min. After habituation, the animals were removed, and 20 black marbles (1.5 cm) were arranged equidistantly in a 4 × 5 pattern on the bedding surface. The rats were returned to the cage for 10 min, and the number of marbles buried (>50%) was counted by two observers. Marbles were cleaned with 70% ethanol after each test.

Elevated plus maze test

The apparatus for the elevated-plus maze test consists of two open arms (50 × 10 cm) and two enclosed arms (50 × 10 × 50 cm), with a central platform (10 × 10 cm) elevated 50 cm above the floor. Each male pup was placed individually in the center, facing an open arm. After a 5-min exploration period, time spent in open/enclosed arms and anxiety index were measured.

AnxietyIndex=TopenarmTopenarm+Tclosedarm

Novel object recognition test

Next, learning and memory abilities were evaluated using novel object recognition. The experiment consisted of three stages: Habituation stage: The rat was placed in an empty test chamber for 5 min of free exploration. Training stage: Two identical objects were positioned in the northwest and southeast corners. The rat explored for 10 min, with exploration time recorded for each object. Afterward, the rat was moved to a clean cage, and the chamber was cleaned with 70% alcohol. Test stage: One hour post-training, one familiar object was placed in the northwest corner, and a novel object in the southeast corner. The rat explored for 10 min, and the exploration time for each object was measured. The Discrimination index was calculated using the following equations:

Discriminationindex=TnovelTnovel+Tfamiliar

Passive avoidance test

The passive avoidance test assessed learning/memory using a two-compartment apparatus (dark/light). The dark floor delivered a 50-Hz, 1 mA shock for 1.5 s. The test spanned three stages over two days. During the adaptation stage, the rat was placed in the bright compartment. After 10 s, the door opened, allowing free exploration of both environments for 2 min. Thirty minutes later, in the training stage, the rat received a 50-Hz square wave shock (1 mA for 1.5 s) upon entering the dark compartment. After 15 s, it was moved to a holding area. This process was repeated every 2 min until the rat avoided the dark compartment for ≤ 120 s or was excluded after three failed trials. Trials to acquisition and Step-through latency (STLa) were recorded. Twenty-four hours post-training, the recall stage began: the rat was placed in the bright compartment, and after 10 s, the door opened for 300 s. Step-through latency during the recall stage (STLr), indicating the time taken for the animal to enter the dark area for the first time, and time in the dark compartment (TDC) were measured. Non-entry was scored as 300 s.

Oxidative stress and inflammatory cytokine analysis

At the end of the experimental period, animals were sacrificed, and hippocampus tissues were collected, homogenized, and analyzed for oxidative stress factors (MDA, GPX, SOD) and inflammatory cytokines (IL-6, TNF-α) using commercially available kits per manufacturer guidelines.

Nissl staining

At the end of the experimental period, animals were perfused intracardially with PBS (0.1 M) followed by 4% (w/v) paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Brain tissues were collected, fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned into 4 μm coronal slices. The sections were then deparaffinized, rehydrated, and stained with cresyl violet to evaluate neuronal density in the hippocampus.

Statistical analysis

All data are expressed as mean ± SD. Differences between three or more groups were analyzed using one-way or two-way ANOVA, followed by Tukey’s post hoc test. Statistical significance was set at P < 0.05.

Results

Social interaction test

The three-chamber social interaction test was employed to assess alterations in social behavior among autistic rats. Results revealed that valproic acid (VPA)-exposed rats exhibited impaired sociability compared to controls, spending significantly less time with stranger rats and more time in the center zone (P < 0.0001). Treatment with edaravone, flurbiprofen, or their combined administration failed to restore the reduced sociability index (P > 0.05; Fig. 1A).

Fig. 1.

Fig. 1

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Sociability index (A) and social preference index (B) in the social interaction test. *** P < 0.001 and **** P < 0.0001 in comparision with control group. + + P > 0.01 and + + + P > 0.001 compared with the VPA group. VPA: valproic acid; Eda: Edaravone; Flur: Flurbiprofen.

Additionally, the social preference index was markedly decreased in autistic rats compared to controls (P < 0.001). This reduction in social preference index was rescued by all treatments, including individual drugs and their combination, reaching control group levels when compared to the VPA group (P < 0.01, P < 0.001, and P < 0.001, respectively; Fig. 1B).

Marble burying test

The marble burying test was conducted to evaluate stereotypical behaviors in rats. Results demonstrated that valproic acid (VPA)-exposed rats, modeling autistic-like behaviors, buried significantly fewer marbles than the control group (P < 0.0001). However, treatment with edaravone, flurbiprofen, and their combined administration markedly increased the number of buried marbles compared to the VPA group (P < 0.0001, P < 0.001, and P < 0.0001, respectively; Fig. 2).

Fig. 2.

Fig. 2

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The number of buried marbles in the marble burying test. **** P < 0.0001 compared to the control group. + + + P > 0.001 and + + + + P > 0.0001 compared to the VPA group. VPA: valproic acid; Eda: Edaravone; Flur: Flurbiprofen.

Elevated plus maze test

Anxiety-like behavior, commonly observed in individuals with autism spectrum disorder (ASD), was assessed using the elevated plus maze (EPM) test. Results showed that VPA-exposed rats spent significantly less time in the open arms compared to the control group (P < 0.0001). Treatment with edaravone, flurbiprofen, or their combination significantly increased the time spent in the open arms compared to the VPA group (P < 0.0001; Fig. 3A).

Fig. 3.

Fig. 3

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Time spent in the open arm (A) and anxiety index (B) in the elevated plus maze test. ** P < 0.01, *** P < 0.001 and **** P < 0.0001 in comparision with control group. + + + + P > 0.0001 in comparison with the VPA group. VPA: valproic acid; Eda: Edaravone; Flur: Flurbiprofen.

The anxiety index was significantly higher in VPA-exposed rats compared to the control group (VPA: P < 0.0001; Eda: P < 0.01; Flur: P < 0.05; Eda+Flur: P < 0.001). However, this index was significantly reduced in all treated groups compared to the VPA group (P < 0.0001; Fig. 3B). Despite this reduction, no significant differences were observed between the different treatment groups (P > 0.05).

Novel object recognition test

The novel object recognition test was conducted to assess recognition memory. Rats in the VPA group exhibited a significantly lower discrimination index (DI) compared to the control group (P < 0.001). However, treatment with edaravone, flurbiprofen, or their combined administration markedly increased the DI relative to the VPA group (P < 0.001; Fig. 4).

Fig. 4.

Fig. 4

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Time spent near novel vs. familiar objects in the novel object recognition test. *** P < 0.001 compared with the control group. + + + P > 0.001 in comparison with the VPA group. VPA: valproic acid; Eda: Edaravone; Flur: Flurbiprofen.

The passive avoidance test

The passive avoidance test was employed to evaluate learning and memory retention deficits. During the training/acquisition phase, no significant differences were observed among any of the experimental groups in either step-through latency during acquisition (STLa; Fig. 5A) or the number of trials required to reach the acquisition criterion (NTa; Fig. 5B) (P>0.05). This indicates that prenatal VPA exposure and subsequent drug treatments did not impair baseline learning acquisition or motor/sensory capabilities.

Fig. 5.

Fig. 5

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Passive avoidance test for evaluation of memory retention deficit. Evaluation of learning and memory retention in the passive avoidance test. (A) Step-through latency during the acquisition phase (STLa), (B) Number of trials to acquisition (NTa), (C) Step-through latency during the recall phase (STLr), and (D) Time spent in the dark compartment during the recall phase (TDC). Data are expressed as mean ± SD. *** P<0.001 compared to the control group; * P<0.05 compared to the control group. + P<0.05, + + P<0.01, and + + + P<0.001 compared to the VPA group. VPA: valproic acid; Eda: Edaravone; Flur: Flurbiprofen.

In the retention (recall) test conducted 24 h post-training, a significant memory impairment was evident. Autistic rats in the VPA group exhibited a marked reduction in step-through latency during recall (STLr) compared to the control group (P<0.001; Fig. 5C). However, daily treatment with edaravone (P<0.01), flurbiprofen (P<0.001), or their combination (P<0.001) significantly restored the STLr compared to the VPA group (Fig. 5C).

Furthermore, the time spent in the dark compartment (TDC) during the recall phase was significantly elevated in the VPA group compared to the control group (P<0.001; Fig. 5D). Consistent with the STLr results, daily treatment with edaravone (P<0.001), flurbiprofen (P<0.05), or the combination therapy (P<0.001) led to a significant reduction in TDC compared to the VPA group (Fig. 5D). No significant differences in memory recovery were observed among the various treatment groups (P>0.05).

Evaluation of oxidative stress markers

The ELISA results demonstrated an elevation in malondialdehyde (MDA) levels and decreased activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in autistic rats (VPA group) compared to the control group (P < 0.001, P < 0.001, and P < 0.0001, respectively). Treatment with edaravone, flurbiprofen, or their combination significantly reduced MDA levels compared to the VPA group (P < 0.01, P < 0.05, and P < 0.01, respectively), restoring them to control group levels (P > 0.05). GPx activity and SOD activity were significantly elevated in all treated groups compared to the VPA group (GPx: P < 0.01 for Eda/Flur, and P < 0.001 for Eda+Flur; SOD: P < 0.0001). No significant differences were observed among the treatment groups (P > 0.05; Fig. 6).

Fig. 6.

Fig. 6

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Evaluation of oxidative stress factors in experimental groups. (A) The level of MDA, (B) GPx activity and (C) SOD activity. *** P < 0.001 and **** P < 0.0001 in comparision with control group. + P > 0.05, + + P < 0.01, + + + P < 0.001 and + + + + P < 0.0001 in comparision with VPA group. VPA: valproic acid; Eda: Edaravone; Flur: Flurbiprofen.

Evaluation of pro-inflammatory cytokines

TNF-α and IL-6 levels were markedly elevated in the VPA group compared to the control group (P < 0.001). Treatment with edaravone, flurbiprofen, or their combination significantly reduced TNF-α levels compared with VPA rats (P < 0.001, P < 0.05, and P < 0.01, respectively). Similarly, IL-6 levels decreased significantly in all treated groups compared to the VPA group (P < 0.05, P < 0.01, and P < 0.01, respectively). No significant differences were observed between treatment groups (P > 0.05; Fig. 7).

Fig. 7.

Fig. 7

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Evaluation of pro-inflammatory factors IL-6 (A) and TNF-α (B). Data are presented as mean ± SEM. ***P<0.001 compared to the Control group, +P<0.05 compared to the VPA group.

Neuronal density in the hippocampus region

To assess neuronal density, the percentage of dark cells (indicative of neuronal death) was quantified. Nissl-stained hippocampal sections revealed a marked increase in dark cells in the VPA groups compared to the control group (P < 0.0001). Rats treated with edaravone, flurbiprofen, or their combination showed reduced dark cell counts compared to the VPA group (P < 0.01, P < 0.05, and P < 0.01, respectively; Fig. 8).

Fig. 8.

Fig. 8

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Investigation of hippocampal neuronal density using Nissl staining. **** P < 0.0001 in comparison with the control group. + P > 0.05 and + + P < 0.01 compared with the VPA group. VPA: valproic acid; Eda: Edaravone; Flur: Flurbiprofen.

Discussion

Edaravone, a free radical scavenger with recognized antioxidant, neuroprotective, and anti-inflammatory properties, has demonstrated therapeutic potential across multiple disease models (Yoshida et al., 2006). In this study, we confirmed these effects and further observed that Edaravone improved behavioral outcomes in autistic rats. Consistent with our findings, Dang et al. (2022) reported that Edaravone enhanced social interaction in depressed rats. Additionally, anxiety behaviors assessed via EPM and OFT tests revealed significantly reduced anxiety in Edaravone-treated depressed rats compared to controls (Dang et al., 2022). Earlier work by Jiao et al. (2015) highlighted its role in improving cognitive memory in Alzheimer’s mouse models (Jiao et al., 2015), while Ding et al. (2021) noted enhanced spatial learning and memory in trauma-injured rats using the Morris water maze test (Ding et al., 2021). These studies collectively attribute behavioral improvements to Edaravone’s antioxidant capacity and mitigation of oxidative stress, aligning with our oxidant factor measurements.

Further supporting our results, prior research underscores Edaravone’s ability to reduce inflammatory markers (Yoshida et al., 2006, Yuan et al., 2014). Its neuroprotective efficacy, evident in our study through reduced hippocampal damage and cell death, mirrors findings by Wang et al., who noted decreased neuronal death in traumatic brain injury models via oxidative stress inhibition and anti-inflammatory action. This convergence of evidence reinforces Edaravone’s multifaceted role in addressing core pathological mechanisms in neurodevelopmental disorders (Wang et al., 2011).

Yoshida et al. (2006) elucidated that Edaravone’s chemical structure enables it to scavenge hydroxyl radicals (OH) and inhibit lipid peroxidation. The drug also suppresses peroxidation systems caused by water-soluble and fat-soluble peroxyl radicals, exhibiting effects comparable to combined vitamins C and E. Through inhibition of the lipoxygenase pathway and non-enzymatic lipid peroxidation, Edaravone exerts antioxidant and anti-inflammatory effects, thereby protecting neurons (Yoshida et al., 2006).

Previous studies have highlighted the critical role of oxidative stress in autism. The human brain, as the body’s largest oxygen consumer, contains abundant fatty acids and active metals like copper and iron, rendering it highly vulnerable to oxidative damage (Thompson et al., 2001). Elevated oxidative biomarkers, such as malondialdehyde, correlate directly with autism severity, and research indicates that antioxidant therapies can mitigate behavioral symptoms (Chen et al., 2013, Wu et al., 2018). In line with these findings, our study demonstrates that Edaravone reduces free radicals by enhancing superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity while lowering malondialdehyde levels, thus preventing lipid peroxidation and preserving cellular integrity. This protective effect was evident in hippocampal neurons, underscoring Edaravone’s neuroprotective role in autism. By shielding the brain from oxidative stress, Edaravone improves behavioral outcomes, including social interaction, anxiety, memory, and spatial learning. Given the hippocampus’s central role in memory, learning, spatial cognition, and emotional regulation, its protection against oxidative damage via Edaravone administration likely underpins these behavioral improvements (Codagnone et al., 2015).

Flurbiprofen, a non-steroidal anti-inflammatory drug (NSAID), also showed promise in this study, with its effects investigated in relation to autism. We observed that Flurbiprofen had favorable effects on autistic behaviors in rats. Consistent with the findings of Farhat et al (Farhat et al., 2021)., our results demonstrated a reduction in anxiety levels in treated rats. Furthermore, similar to studies on Alzheimer’s disease, this study showed improvements in memory and learning (Wilcock et al., 2008, Wassmann et al., 2004). These beneficial effects are further supported by our findings that Flurbiprofen significantly decreased the levels of inflammatory factors IL-6 and TNF-α, as well as the oxidative biomarker malondialdehyde (MDA), by reducing oxidative stress. Concurrently, it increased the activity of antioxidant enzymes, superoxide dismutase (SOD), and glutathione peroxidase (GPx). By attenuating neuroinflammation and oxidative stress, Flurbiprofen demonstrated neuroprotective properties, protecting hippocampal neurons from destruction, necrosis, and cell death.

Several studies have corroborated the neuroprotective effects of Flurbiprofen. For instance, Sun et al. (2011) and Wu et al. (2018) reported that Flurbiprofen provided significant neuroprotection in cerebral ischemia models by reducing neuronal damage and oxidative stress during reperfusion injury (Wu et al., 2018, Sun et al., 2011). Similarly, Wong et al. highlighted that Flurbiprofen improved hippocampal function and memory in Alzheimer’s disease by limiting neuroinflammation and preserving neuronal integrity (Wong and Ho, 2018).

A noteworthy finding in this study was the result of the marble burying test, in which the VPA-exposed group buried significantly fewer marbles than the control group. This finding contrasts with some reports where marble burying, often used as a proxy for repetitive and anxiety-like behavior, is increased in models of autism. One possible explanation for this phenomenon is that the VPA model may induce motor deficits or a state of apathy, leading to reduced engagement with the task rather than a decrease in the repetitive behavior itself. Regardless of the reason for this baseline behavior, it is significant that treatment with both Edaravone and Flurbiprofen successfully reversed this deficit, bringing the number of buried marbles close to control levels. This suggests that these treatments may help improve general motivation and exploratory behaviors, which are often impaired in neurodevelopmental disorders.

Neuroinflammation is considered a major contributor to many behavioral symptoms of autism, affecting various systems and organs in the body (Tahmasebi and Barati, 2023). Recent studies have highlighted the critical role of IL-6 in the nervous system. IL-6 promotes the differentiation of astrocytes, dorsal ganglion neurons, hippocampal neurons, and Schwann cells. Sun et al. reported that IL-6 is essential for central nervous system (CNS) function during the embryonic period but may also contribute to neurodegenerative and neurodevelopmental disorders. Depending on its concentration, brain region, and cell type, IL-6 can either induce neuronal death or promote neuronal growth (Bastard et al., 2006). Wei et al. developed a model in which mice were injected with high levels of IL-6 into the lateral ventricles. This study revealed that the mice exhibited autism-like behaviors, including reduced cognitive ability, memory and learning impairments, and anxiety-related symptoms (Wei et al., 2012). Similarly, TNF-α, another inflammatory factor essential for brain function, is elevated in autism. Huang et al. (2022) stated that increased TNF-α levels can enhance blood-brain barrier permeability and disrupt brain function. Furthermore, TNF-α levels have been shown to correlate with the severity of autism symptoms (Huang et al., 2022).

Consistent with our findings, numerous studies have confirmed elevated levels of inflammatory biomarkers in children and autism model rats, suggesting that controlling neuroinflammation can mitigate autism symptoms. Neuroinflammation is known to disrupt synaptic stability and alter dendritic branching in neurons, ultimately leading to changes in connectivity within specific brain regions. These neuronal alterations caused by neuroinflammation are considered a major contributor to many autistic behaviors (Hegazy et al., 2015).

In this study, Flurbiprofen demonstrated significant improvements in behavioral symptoms in autistic rats. The observed behavioral enhancements can be attributed to Flurbiprofen’s ability to reduce neuroinflammation, primarily by decreasing levels of inflammatory factors such as TNF-α and IL-6. Additionally, since IL-6 promotes superoxide production and exacerbates oxidative stress, Flurbiprofen indirectly reduces oxidative stress by lowering IL-6 levels. By attenuating both neuroinflammation and oxidative stress, Flurbiprofen helps preserve cellular integrity, resulting in reduced neuronal death and increased survival of hippocampal cells. This preservation of hippocampal activity is critical, as the hippocampus plays a central role in memory, learning, spatial cognition, and emotional regulation. Consequently, Flurbiprofen-treated autistic rats exhibited improved memory and learning abilities, enhanced social interactions, and reduced anxiety behaviors.

However, while each drug individually improved autistic behaviors, the combined administration of edaravone and flurbiprofen improved social interactions, anxiety, memory, and learning in autistic rats to a similar extent. Contrary to expectations of synergistic effects, the combination did not significantly enhance symptom improvement compared to monotherapy. This lack of additive benefit may be attributed to both drugs exhibiting anti-inflammatory and antioxidant properties, potentially reaching a ceiling effect in ameliorating inflammatory markers, oxidative stress, and neuroprotection to levels comparable with the control group. The absence of synergy suggests that the combined treatment did not activate additional mechanisms or pathways beyond those targeted by each drug individually for further behavioral improvement in this autism model.

Limitations of the study

First, it is important to acknowledge the limitations of this study. The findings are based on a single neurodevelopmental model—the valproic acid (VPA)-induced rat model of autism. While this model is well-established for studying certain ASD-like behaviors, autism in humans is a highly heterogeneous disorder with diverse genetic and environmental causes, so these results may not be universally generalizable. Second, this study was conducted exclusively on male offspring. Given the known sex differences in the prevalence and presentation of ASD, these findings cannot be assumed to apply to females, and further research is needed to explore potential sex-specific therapeutic effects. Additionally, the treatment was administered for a relatively short duration of seven days and at a single dose for each drug. A dose-response study over a longer period would be necessary to determine the optimal dosage and long-term efficacy and safety of these interventions. Finally, while the behavioral tests are standard, they remain proxies for complex human behaviors and may not capture the full spectrum of ASD-related challenges.

Moreover, it must be mentioned that although the current study provides robust insights into the neuroprotective effects of the administered treatments, our focus was restricted to the hippocampus. ASD is well-established as a distributed whole-brain disorder affecting multiple interconnected regions, including the cerebral cortex and cerebellum, which govern executive functions and motor coordination, respectively. The hippocampus was selected as the primary target in this investigation due to its definitive role in modulating social memory networks and its high vulnerability to oxidative stress and neuroinflammation in the VPA prenatal model. Nevertheless, evaluating the structural and molecular alterations in the cerebellum and cerebral cortex remains an essential avenue for future research to fully elucidate the comprehensive therapeutic spectrum of these agents.

Conclusion

Edaravone, with its antioxidant properties, prevented oxidative stress-induced damage and necrosis in hippocampal neurons by reducing oxidant activity, enhancing antioxidant enzyme activity (SOD, GPx), and lowering inflammatory factors (IL-6, TNF-α). These effects improved behavioral symptoms in autistic rats, including stereotyped behaviors, social interactions, memory, and learning. Flurbiprofen, by limiting neuroinflammation and oxidative stress, protected hippocampal neurons from necrosis and similarly improved autism-related behaviors. However, co-administration of the two drugs did not yield greater improvements in autism symptoms compared to monotherapy. This suggests that both drugs independently achieved maximal therapeutic effects by targeting overlapping pathways—oxidative stress and inflammation—without synergistic interactions.

However, to build upon these promising findings, future research should be directed toward several key areas. Specifically, conducting dose-response studies to identify the optimal therapeutic window, investigating the more precise downstream molecular pathways affected by these drugs, and validating these results in other preclinical models of autism, such as genetic models, will be the next crucial steps in translating these findings toward potential clinical applications. Leveraging Artificial Intelligence (AI) to analyze the complex behavioral and molecular data from these future studies could significantly accelerate the translation of these findings toward potential clinical applications.

CRediT authorship contribution statement

Razieh Hajisoltani: Writing – review & editing, Project administration, Methodology. Anahita Hejazi: Writing – original draft, Formal analysis, Data curation, Conceptualization. Soraya Mehrabi: Writing – review & editing, Writing – original draft, Supervision, Project administration, Investigation, Conceptualization. Fatemeh Mehrabi: Investigation, Conceptualization. Tourandokht Baluchnejadmojarad: Methodology, Investigation, Conceptualization. Mitra Farbib: Writing – review & editing, Visualization, Formal analysis. sadr hossein: Writing – original draft, Methodology, Conceptualization.

Author contribution

A.H., S.M., and T.B. conceived the study and designed the experimental framework. A.H. was responsible for the prenatal VPA model induction, execution of the behavioral test battery, and hippocampal tissue collection, with technical advisory from H.S. regarding statistical analysis and data interpretation. R.H. and M.F. supervised the biochemical assays (ELISA) and histological evaluations, while F.M. oversaw the validation of oxidative stress and neuroinflammatory markers. A.H. drafted the original manuscript. All authors critically reviewed, edited, and approved the final version of the manuscript for submission.

Ethical approval

The project was found to be in accordance with the ethical principles and national norms and standards for conducting Medical Research in Iran (Approval ID: IR.IUMS.FMD.REC.1400.636).

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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.

Contributor Information

Hossein Sadr, Email: hosein.sadr@gmail.com.

Soraya Mehrabi, Email: soraya.mehrabi@gmail.com.

Data availability

All data generated and analyzed during this study, including behavioral assessments, biochemical assays, and histological evaluations, are included in the published article and its corresponding figures. The raw datasets supporting the findings of this study are available from the corresponding author upon reasonable request. All experimental materials, including pharmacological compounds, ELISA kits, and histological reagents, were sourced from commercial suppliers as detailed in the Materials and Methods section.

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Associated Data

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

Data Availability Statement

All data generated and analyzed during this study, including behavioral assessments, biochemical assays, and histological evaluations, are included in the published article and its corresponding figures. The raw datasets supporting the findings of this study are available from the corresponding author upon reasonable request. All experimental materials, including pharmacological compounds, ELISA kits, and histological reagents, were sourced from commercial suppliers as detailed in the Materials and Methods section.

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