Promising Therapeutic Efficacy of Kolaviron against Prenatal Valproate-Induced Autism Spectrum Disorder

Promising Therapeutic Efficacy of Kolaviron against Prenatal Valproate-Induced Autism Spectrum Disorder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Promising Therapeutic Efficacy of Kolaviron against Prenatal Valproate-Induced Autism Spectrum Disorder Vahid Khodashenas, Tourandokht Baluchnejadmojarad, Mitra Farbin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6783521/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Autism Spectrum Disorder (ASD) is a lifelong neurodevelopmental condition marked by impairments in social communication, language and behavior. Both genetic and environmental factors, including prenatal exposure to valproic acid (VPA), contribute to its pathogenesis. VPA exposure during critical periods of neurodevelopment induces oxidative stress, inflammation, and serotonergic dysregulation. Kolaviron (KV), a polyphenolic extract from Garcinia kola , exhibits potent antioxidant and anti-inflammatory properties, potentially offering Neuroprotection in ASD models . The aim of our study was to evaluate whether Kolaviron could improve the VPA-induced autism model in the areas of Mitochondrial dysregulation oxidative stress, inflammation, and behavior, and to compare its effects with oxytocin on the serotonergic system. Methods Pregnant Wistar rats received a single intraperitoneal dose of VPA (600 mg/kg) on gestational day 12.5 to induce autism-like features in offspring. Male pups were weaned on postnatal day (PND) 21 and randomly assigned to receive KV (50 or 100 mg/kg, oral), oxytocin (12 µg/kg, intranasal), or saline until PND 49. Behavioral tests were conducted on PNDs 42–49. Brain tissues were collected for ELISA analysis of hippocampal 5-HTT, 5-HTR7, TNF Alfa and IL-6 levels, Immunohistochemical staining for parvalbumin was performed to assess interneuron integrity. Results KV-treated VPA-exposed rats showed significant improvements in social interaction, reduced repetitive behavior, and attenuated. Biochemically, KV decreased IL-6 levels and modulated serotonergic markers (5-HTT, 5-HTR7). Histologically, KV preserved hippocampal architecture and parvalbumin-positive interneurons, suggesting Neuroprotection. Conclusions These effects were dose-dependent, indicating KV's potential as a complementary therapeutic agent in ASD. Further studies are warranted to clarify its mechanisms and clinical relevance. Autism Spectrum Disorder (ASD) Valproic Acid (VPA) Kolaviron (KV) Serotonin Hippocampus Oxytocin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Autism Spectrum Disorder (ASD) is a lifelong neurodevelopmental condition marked by persistent challenges in social communication, language acquisition, and the presence of restrictive or repetitive behaviors. Typically emerging in early childhood, ASD manifests through a heterogeneous array of symptoms such as delayed speech and motor milestones, difficulties interpreting social cues (e.g., tone of voice, body language), hyper- or hypo-reactivity to sensory stimuli, and sometimes self-injurious behaviors( 1 , 2 ). Although genetic factors contribute substantially to ASD risk, environmental influences including prenatal exposure to certain medications also play a critical role( 3 ). To date, no curative therapies exist, underpinning the urgent need for mechanistic studies and novel interventions( 4 ). Valproic acid (VPA) is a branched-chain fatty acid extensively used as an antiepileptic, mood stabilizer, and migraine prophylactic( 3 ). However, when administered during early gestation, VPA is linked to an elevated risk of autism spectrum disorder (ASD) in the offspring( 4 , 5 ). In rodent studies, a single intraperitoneal injection of 600 mg/kg VPA on gestational day 12.5 delays neural tube closure and triggers widespread molecular disruptions, including compromised mitochondrial function( 6 ). This critical exposure window overlaps with peak periods of neurogenesis and serotonergic neuron migration, making the developing brain particularly susceptible to excitatory-inhibitory imbalance and oxidative damage( 6 ). At the cellular level, VPA increases reactive oxygen species and proinflammatory mediators while diminishing inhibitory interneuron populations( 3 , 7 ). Behaviorally, VPA-exposed rodents faithfully reproduce core ASD phenotypes, social deficits in the three-chamber test, heightened repetitive actions, and atypical ultrasonic vocalizations( 1 , 8 ). In this work, pregnant dams received 600 mg/kg VPA on day 12.5 of gestation, and male offspring were subsequently assessed for behavioral, biochemical, and histopathological outcomes. Kolaviron (KV) is a natural mixture of Flavonoids obtained from Garcinia kola seeds, traditionally used in West African medicine ( 9 ). It is recognized for its antioxidant and anti-inflammatory activities, mainly due to its polyphenolic components ( 10 – 13 ). Beyond these general properties, Kolaviron (KV) has been extensively investigated for its specific neuroprotective potential in preclinical models. Numerous studies have demonstrated its ability to mitigate oxidative stress and inflammation in the central nervous system. For example, KV has been shown to reverse scopolamine-induced memory impairment and prevent brain microstructural derangements and cognitive deficits in rodents exposed to various neurotoxins, including methamphetamine and cuprizon ( 15 ), In a cuprizone-induced demyelination model, KV treatment significantly restored endogenous antioxidant enzyme levels (such as SOD and GPx), reduced lipid peroxidation (MDA), and preserved cerebellar neuronal morphology and astrocytic health, highlighting its robust neuroprotective and anti-inflammatory actions within the brain ( 16 ). Furthermore, in a rat model of maternal deprivation, Kolaviron successfully attenuated behavioral deficits, improved memory and cognitive performance, and restored oxidative balance in hippocampal and prefrontal cortex regions by enhancing SOD levels, suggesting its capacity to disrupt excitotoxic stimuli and prevent mitochondrial dysfunctions ( 17 ). These findings from diverse in vitro and in vivo models collectively provide strong experimental evidence for Kolaviron's therapeutic potential in neurological conditions characterized by oxidative stress, neuroinflammation, and neuronal damage. The relevance of Kolaviron's multifaceted actions extends to the complex pathophysiology of Autism Spectrum Disorder (ASD), where oxidative stress and neuroinflammation are recognized as key contributing factors. Given that Valproic Acid (VPA) is a well-established inducer of neurodevelopmental abnormalities in animal models, partly through the generation of oxidative stress and inflammatory responses, the compelling evidence of Kolaviron's ability to modulate these pathways in preclinical settings provides a strong rationale for its investigation in VPA-induced ASD models. Moreover, its reported influence on the serotonergic system ( 18 ), a critical neurotransmitter system often dysregulated in ASD, further supports its potential as a therapeutic agent. These properties allow KV to counteract oxidative damage, modulate immune responses, and support mitochondrial health. Considering that oxidative stress and inflammation contribute to the development of autism spectrum disorder (ASD), KV may help to protect against neural damage seen in this condition ( 14 ). It has also been shown to influence the serotonergic system, which plays a role in ASD pathophysiology. In this study, KV was administered orally at 50 and 100 mg/kg after weaning in a VPA-based rat model of autism to assess its effects on behavior, oxidative and inflammatory markers, and serotonin-related targets in the hippocampus. The aim of our study was to evaluate whether Kolaviron could improve the VPA-induced autism model in the areas of oxidative stress, inflammation, and behavior. We also assessed its effects on the serotonergic pathway and compared the results with oxytocin to determine how effective Kolaviron could be. 2 Materials and Methods 2.1 Animals Sexually mature male and female Wistar rats, including young adults, were obtained from the Experimental and Comparative Research Center of Iran University of Medical Sciences (Tehran, Iran). Breeding was carried out under controlled laboratory conditions, and the male offspring were used for the subsequent experimental procedures. The animals were housed in groups of no more than four per cage, maintained on a 12-hour light/dark cycle, at a constant temperature of 22°C and a relative humidity of 40–50%. Food and water were available to the animals at all times. All procedures were conducted in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Iran University of Medical Sciences. 2.2 Kolaviron A total of 100 grams of Garcinia kola seeds were processed by first removing Lipophilic components using 150 ml of petroleum ether over 24 hours in a Soxhlet apparatus. The defatted material was then air-dried at 25°C, followed by an initial extraction with 120 ml of acetone at 40°C. After concentration, a secondary purification step was carried out using ethyl acetate. This method resulted in the isolation of kolaviron, a compound predominantly enriched with Biflavonoid structures such as Garcinia Biflavonoid 1 and 2, as well as kolaflavanone( 19 , 20 ).Biochemical properties of kolaviron were assessed using standardized spectrophotometric assays. Antioxidant activity was measured via the DPPH radical scavenging method, calibrated with Trolox. Total flavonoid content was determined using aluminum chloride and potassium acetate, expressed as quercetin equivalents. Total phenolic content was measured by a modified Folin–Ciocalteu method, with Gallic acid as the standard. Flavonol content was evaluated after incubation with aluminum trichloride and sodium acetate, using rutin as the reference. All assays were performed in triplicate to ensure accuracy and reproducibility( 11 ).To date, no significant adverse effects have been documented in the scientific literature concerning the pharmacological actions of Garcinia kola. The limited toxicological assessments conducted suggest a high margin of safety, with the oral median lethal dose (LD₅₀) of the seed extracts reported to be approximately 5000 mg/kg body weight( 14 ). 2.3 Experimental Procedure Male Wistar rats were housed under standard conditions (21–23°C, 45–50% humidity, 12/12 h light/dark cycle) with free access to food and water. Female rats with synchronized estrous cycles were mated, and the day after mating was designated as gestational day (GD) 1. On GD12.5, pregnant dams received a single intraperitoneal injection of either sodium valproate (600 mg/kg)(Darou Pakhsh Co., Tehran, Iran) to induce autism-like features (VPA group) or normal saline (control group). Dams were housed individually until parturition, and postnatal development of pups was monitored. On postnatal day (PND) 21, pups were weaned, sexed, and only males were included in the study. From PND 21 to 49, male offspring received daily oral administration of either normal saline, kolaviron (50 or 100 mg/kg), or intranasal oxytocin (12 µg/kg)(Royan Co, Tehran, Iran). Animals were divided into eight groups (n = 6 each): ( 1 ) Control + Saline, ( 2 ) Control + Kolaviron50, ( 3 ) Control + Kolaviron100, ( 4 ) VPA + Saline, ( 5 ) VPA + Kolaviron50, ( 6 )VPA + Kolaviron100, and ( 7 ) VPA + Oxytocin. Behavioral assessments were performed during PNDs 42–49. On PND 49, five animals per group were deeply anesthetized (Ketamine/Xylazine) and perfused transcardially. Brains were fixed in 10% formalin for Immunohistochemical analysis of parvalbumin-positive interneurons in the hippocampus. Remaining brain samples were dissected, stored at − 80°C, and later analyzed by ELISA for hippocampal levels of serotonin transporter (5-HTT), serotonin receptor 7 (5-HTR7), and inflammatory cytokines such as IL-6. At the end of study, all animals were euthanized using CO₂ and disposed of following ethical protocols. 2.4 Behavioral Tests 2.4.1 Three Chamber Test The procedure for this test has been reported( 3 , 21 ). In this study, the three-chamber test was utilized to evaluate the social interaction of rats. Initially, the rats were placed in the center of the apparatus and given 10 minutes to explore their surroundings freely. Following this phase, an empty metal cage was positioned on one side of the apparatus, while a cage containing an unfamiliar rat was placed on the opposite side. The test rat was then allowed 10 minutes to assess its social interactions with the unfamiliar rat. The sociability index was calculated by subtracting the time spent in the non-social chamber from the time spent in the social chamber, and this result was divided by the total time spent in both chambers. Lastly, the time spent by the rats interacting with social and non-social stimuli such as cleaning, fighting, or other social behaviors was recorded. This index was utilized to analyze the social behaviors of the rats in response to various stimuli. 2.4.2 Self Grooming Test On the test day, each animal was individually placed in a Plexiglas cage measuring 30 × 30 × 45 cm. To familiarize them with the environment, the rats were acclimated to the empty cage for 10 minutes on the previous day. Following this, their total grooming time across all body areas was recorded over a 10-minute period( 22 ). 2.4.3 Marble Burying Test An apparatus measuring 40 cm × 40 cm × 40 cm was prepared, filled with nesting material and set up with 20 marbles arranged in 5 rows. Rats were then placed inside for 30 minutes, during which they could explore freely. The number of marbles they buried was recorded as an indicator of repetitive digging behavior( 3 , 21 ). 2.4.4 Social Interaction Test To measure social interactions, an open field apparatus (60 × 60 × 40 cm) was set up. Each rat was placed with an unfamiliar rat it had never encountered before, for a 10-minute session. The partner rats were matched in strain, sex, age, and weight to ensure consistency. To help reduce stress from the new environment, each rat was placed in the test cage individually for 5 minutes the day before testing. Social behaviors, including sniffing, licking, crawling or mounting, approaching, or following the other rat, were observed, with the total time spent on these activities counted as active social behavior( 22 ). 2.5 ELISA Assay The concentrations of TNF-α(Cat # sc-52746, Santa Cruz Biotechnology, Inc., USA), IL-6(Cat # sc-57315, Santa Cruz Biotechnology, Inc., USA), 5-HTT(Cat # MBS2533556, MyBioSource, Inc., USA), and 5-HT7R(Cat # MBS282566), MyBioSource, Inc., USA) in hippocampal tissues were determined using enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturers protocols. In brief, supernatants obtained from homogenized hippocampal samples were loaded onto pre-coated microplates and incubated at 4°C for 24 hours. After washing the plates three times with PBS to remove unbound components, HRP-conjugated secondary antibodies were added and incubated for 2 hours at room temperature. Then, Tetramethylbenzidine (TMB) substrate solution containing hydrogen peroxide was applied, and the reaction was allowed to proceed for 15 minutes in the dark. The enzymatic reaction was terminated with 0.18 M sulfuric acid, and optical density was measured at 450 nm using a microplate reader. 2.6 Hippocampal Biochemical Assessments 2.6.1 Assessment of hippocampal Malondialdehyde (MDA) The amount of Malon-di-aldehyde (Cat # KMDA96, Kiazist, Hamadan, Iran), a marker for assessing lipid peroxidation, was evaluated using 0.5% Thiobarbituric acid and 20% Trichloroacetic acid. The experiment was conducted in a thermoblock set at 95 degrees Celsius for 30 minutes. After this period, the samples were allowed to cool, and then centrifuged for 5 minutes at 25 degrees Celsius with a speed of 1500×g. Finally, the optical absorbance was measured at a wavelength of 535 nanometers, using tetraethoxypropane as the standard for this measurement( 23 ). 2.6.2 Assessment of hippocampal catalase activity (CAT) The amount of catalase activity (Cat # KCAT96, Kiazist, Hamadan, Iran), In this method, the enzyme activity is assessed by first mixing 20 µL of supernatant with 100 µL of buffer and 20 µL of diluted substrate. This mixture is allowed to react for 20 min, facilitating the enzymatic reaction. After this period, the reaction is halted by adding 30 µL of diluted KOH. Next, 10 µL of KIO4 are introduced to the solution, and after waiting for 5 min, the absorbance is measured at 540 nm using a spectrophotometer. The amount of breakdown of H2O2 with catalase (CAT) is the basis of this assessment, providing valuable information about the enzyme activity in the presence of H2O2, KOH, and KIO4( 11 ). 2.6.3 Assessment of hippocampal nitrite levels (NIT) For The nitrite content(Cat # KNIT96, Kiazist, Hamadan, Iran) in the tissue was measured using Griess reagent, which consisted of 2.5% phosphoric acid, 1% sulfanilamide, and 0.1% naphthyl ethylenediamine dihydrochloride. After a 10-minute incubation, the optical density was measured at 540 nm, with sodium nitrite used as the calibration standard in the assay( 24 , 25 ). 2.6.4 Assessment of Mitochondrial Membrane Potential (MMP) The assessment of mitochondrial membrane potential (MMP), which serves as a key diagnostic marker for mitochondrial integrity and health, was performed using Rhodamine 123 (Cat # R8004, SigmaAldrich,USA). This compound is a cationic fluorescent stain that preferentially accumulates in the mitochondria upon entering the cells. In this procedure, samples were first subjected to centrifugation at 10,000 rpm. Subsequently, 20 µL of Rhodamine 123 prepared at a concentration of 0.5 mg/5 mill DMSO from (SigmaAldrich, USA) was combined with 180 µL of phosphate-buffered saline (PBS) at a pH of 7.4 and After a 30-min incubation then added to the resulting pellet( 26 , 27 ). 2.7 Immunohistochemical Evaluation of Parvalbumin Interneurons in the Hippocampus Paraffin embedding and standard histological processing were performed on the hippocampal tissue blocks, after which serial sections of 5 µm thickness were obtained for further experimental analyses. Tissue sections were initially deparaffinized, rehydrated through descending concentrations of ethanol, and rinsed thoroughly in phosphate-buffered saline (PBS). To permeabilize the tissue, the slides were incubated with Triton X-100 in PBS for 15 minutes. Non-specific binding was blocked by exposing the sections to 10% normal goat serum (NGS) in PBS at room temperature for 1 hour. Subsequently, the sections were incubated overnight (24 h) in a humidified environment at room temperature with a rabbit polyclonal anti-parvalbumin primary antibody (Cat # P3088, SigmaAldrich, USA). After PBS washing steps, the slides were incubated for 2 hours with a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP)(Cat # sc-542741, MyBioSource, Inc., USA). For visualization, the Chromogenic reaction was developed by incubating the sections with 3,3′-diaminobenzidine (DAB)(Cat # D5637, SigmaAldrich, USA) and 0.01% hydrogen peroxide (H₂O₂) in PBS for 10 minutes in the dark. Following this step, the sections were rinsed, gently counterstained with Hematoxylin, dehydrated through graded alcohols, cleared in xylene, and coverslipped using Entellan. Microscopic evaluation was performed to identify and quantify parvalbumin-immunoreactive interneurons. 2.8 Statistical Methods Statistical analyses were performed using GraphPad Prism 9. Normality of data distribution was assessed via the Kolmogorov-Smirnov test, and outliers were identified and excluded using Grubbs' test. Group differences were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Results are presented as mean ± SEM, and statistical significance was defined as P < 0.05. 3 Results 3.1 Behavioral Findings 3.1.1 Self-grooming test Findings The results of the self-grooming test, which reflect stereotyped and repetitive behaviors in animals, are presented in Fig. 2 a. A one-way ANOVA analysis revealed significant differences among the experimental groups [F (6,44) = 21.04, P < 0.01]. Further evaluation using Tukey's Multiple Range Testindicated that intraperitoneal(IP) administration of valproic acid (VPA) significantly increased repetitive behaviors compared to the control group (P < 0.001).Moreover, treatment with Kolaviron (KV) in VPA model groups at both doses of 50 and 100 mg/kg led to a marked improvement compared to the VPA-damaged group. This improvement was significant for both doses, with a statistical level of (P < 0.001). This increase was 33.78%for the 50 mg/kg dose and 44.04% for the 100 mg/kg dose. Similarly, the group treated with Oxytocin showed a significant reduction in repetitive behaviors compared to the VPA group, aligning with the effects observed in KV-treated groups (P < 0.001) Oxytocin also induced a 45.66% reduction. These findings highlight the potential of Kolaviron and Oxytocin in mitigating VPA-induced stereotyped behaviors. 3.1.2 Three-Chamber test Findings Figure 2bpresents the results of the three-chamber test, which is used to assess social interaction and social avoidance behaviors. The statistical analysis using one-way ANOVA showed a significant difference between the groups [F (6, 41) = 4.456, P < 0.001]. Further analysis revealed that the VPA model group exhibited a significant reduction in social interaction compared to the control group (P < 0.001). In the KV-treated groups, the 100 mg/kg dose showed a significant improvement in social interaction compared to the VPA group (P < 0.05), This increase is equivalent to 341.91%. On the other hand, the Oxytocin-treated group showed a significant improvement compared to the VPA group(P < 0.05) The increase induced by oxytocin was also 342.64%. These findings suggest that Kolaviron treatment, especially at the 100 mg/kg dose, can enhance social interaction in VPA-induced social deficits. 3.1.3Open Field Test Findings Figure 2 c represents the Time in Social Zone, which assesses social behavior. One-way ANOVA statistical analysis indicates a significant difference among the studied groups [F (6,40) = 20.79,P < 0.0001]. Furthermore, our additional analyses reveal that the diseased group (VPA group) exhibited a significantly lower social behavior time compared to the control group (P < 0.001), with a reduction of 71.07%. In the VPA groups treated with Kolaviron, it was observed that the VPA + KV 50 mg/kg group showed a significant increase (P < 0.01) in social behavior time, with an improvement of 132.12%. Similarly, the VPA + KV 100 mg/kg group exhibited a comparable pattern, significantly increasing the index compared to the VPA group (P < 0.01), with an improvement of 118.01%. 3.1.4 Marble Burying Test Findings Figure 2 d shows the results of the Marble Burying Test, which is used to assess anxiety and repetitive behaviors in rats. The one-way ANOVA analysis revealed a significant difference among the groups [F (6,38) = 5.251, P < 0.001]. Further evaluation with Tukey's post-hoc test indicated that the VPA group exhibited significantly higher anxiety and repetitive behaviors compared to the control group (P < 0.01). In the KV-treated groups, the 50 mg/kg dose did not show a significant change in anxiety or repetitive behaviors. However, the 100 mg/kg dose of KV significantly reduced these behaviors compared to the VPA group (P < 0.05) This decrease is equivalent to 35.57%. Similarly, the Oxytocin-treated group also demonstrated a significant reduction in anxiety and repetitive behaviors compared to the VPA group (P < 0.05) Oxytocin also exhibited a 40% reduction. These findings suggest that Kolaviron at the 100 mg/kg dose and Oxytocin can effectively reduce anxiety and repetitive behaviors induced by VPA. 3.2 Biochemical Assessment In this study, our team assessed parameters related to oxidative stress, mitochondrial dysregulation, inflammation, and two factors associated with the serotonergic system. 3.2.1 Oxidative Stress Assessment 3.2.1.1 Malondialdehyde (MDA) Assessment In these assessments, for MDA a significant increase was observed in the VPA group compared to the control group in the hippocampus (P < 0.01). Additionally, in the treated disease group with 100 mg/kg Kolaviron, a significant reduction was observed compared to the disease group (P < 0.05). This reduction was 31.25% in the hippocampus (Fig. 3 a). 3.2.1.2 Catalase Assessment In the continuation of oxidative stress parameter assessments, a significant decrease in the Cat parameter was observed in the VPA group compared to the control group (P < 0.05). Furthermore, the disease group treated with 100 mg/kg Kolaviron showed a significant increase compared to the disease group (P < 0.05).This increase was 73.30% in the hippocampus (Fig. 3 b). 3.2.1.3 Nitrite Assessment Finally, for the oxidative stress parameter Nit, a significant increase was observed in the VPA group compared to the control group in the hippocampus (P < 0.01). However, no significant changes were observed in the treated groups compared to the disease group (Fig. 3 c). 3.2.2 Inflammation Assessments 3.2.2.1 Tumor Necrosis Factor (TNF) Assessment In the assessment of inflammatory parameters, two parameters, TNF and IL-6, were evaluated. For TNF, the VPA group showed a significant increase compared to the control group in the hippocampus (P < 0.001). In the hippocampus, the disease group treated with 100 mg/kg Kolaviron showed a significant reduction (P < 0.05), which was 33.32% (Fig. 4 A). 3.2.2.2 Interleukin-6 (IL-6) Assessment Similarly, for IL-6, the VPA group showed a significant increase compared to the control group in the hippocampus (P < 0.001). In this parameter, the disease group treated with 100 mg/kg Kolaviron showed a significant reduction in the hippocampus (P < 0.05). This reduction was 32.80% (Fig. 4 B). 3.2.3 Mitochondrial Membrane Potential (MMP) Assessment In this study, we also assessed MMP, which reflects mitochondrial dysregulation, a key factor contributing to cellular metabolic imbalance. A significant decrease was observed in the VPA group compared to the control group in the hippocampus (P < 0.01). However, among the treatment groups, only the disease group treated with 100 mg/kg Kolaviron showed a significant increase in the hippocampus compared to the disease group (P < 0.05). This increase was 48.82% (Fig. 5 A). 3.3 Histological Assessment 3.3.1 Parvalbumin Interneurons For parvalbumin interneurons, the VPA group showed a significant decrease compared to the control group (P < 0.01). Additionally, the disease group treated with 100 mg/kg Kolaviron showed a significant increase compared to the disease group (P < 0.05), with an increase of 98%. The VPA group treated with 50 mg/kg Kolaviron also exhibited an increase of 25% compared to the VPA group; however, this change did not reach statistical significance. Notably, this same group still showed a significant reduction of 43.18% compared to the control group (Fig. 5 b). 3.4 Serotonergic System Assessment In the assessment of serotonin-related parameters, we measured two parameters: 5HTT and 5HTR7. 3.4.1 Serotonin Transporter (5HTT) Assessment For 5HTT, the VPA group showed a significant increase compared to the control group in the hippocampus, with an elevation of 120.7%. The disease group treated with 100 mg/kg Kolaviron also showed a significant reduction compared to the disease group in the hippocampus (P < 0.05). This reduction was 40.38% in the hippocampus. Additionally, the VPA group treated with 50 mg/kg Kolaviron exhibited a 18.83% reduction, although this change did not reach statistical significance. A significant increase was also observed in the hippocampal region of the oxytocin group (P < 0.001), with a percentage increase of 48.66% (Fig. 6 A). 3.4.2 Serotonin Receptor 7 (5HTR7) Assessment For 5HTR7, however, no significant results were observed in any of the groups in the hippocampus. Kolaviron administration led to a reduction in 5HT7R expression both in healthy animals receiving kolaviron and in VPA-exposed animals treated with kolaviron. This reduction was more prominent at the 50 mg/kg dose. Specifically, in the healthy group treated with 50 mg/kg kolaviron, a 32.45% decrease in 5HT7R levels was observed compared to the control group. Similarly, in the VPA-exposed group receiving 50 mg/kg kolaviron, a 7.56% reduction was noted compared to the untreated VPA group. Notably, the VPA group itself exhibited a 36.13% increase in 5HT7R expression compared to the control group. The pattern of Kolaviron's effect was comparable to that of oxytocin, which also reduced 5HT7R expression by 15.98% in the VPA + oxytocin group compared to the VPA group. However, none of these changes reached statistical significance, indicating the need for further investigation (Fig. 6 b). 4 Discussion In this study, we aimed to investigate the potential effects of Kolaviron on a valproic acid (VPA)-induced model of autism. Our primary focus was on the hippocampus. Among the various systems implicated in the pathophysiology of autism, the serotonergic system particularly drew our attention. We also evaluated markers of oxidative stress and inflammation. By integrating behavioral assessments with histological and biochemical analyses, we sought to better understand the relationship between molecular and structural alterations and the observed behavioral changes. VPA is a short-chain fatty acid that has been widely used to model autism in rodents. When administered intraperitoneal during pregnancy, VPA can disrupt neural tube closure, leading to structural and functional abnormalities in the developing nervous system( 6 ). This model is known to induce oxidative stress, mitochondrial dysfunction, and neuroinflammation factors commonly associated with autism spectrum disorders. Given the high energy demands of neurons and the critical role of mitochondrial integrity in neuronal health, disruptions in these pathways can have profound effects( 1 ). VPA also influences sodium channel activity, neurotransmitter systems, and the excitatory-inhibitory balance in the brain, contributing to a neurodevelopmental profile that mimics core features of autism, including increased reactive oxygen species (ROS) levels and altered pro-inflammatory cytokine expression( 6 ). Kolaviron, a bioactive compound derived from Garcinia kola, is known for its potent antioxidant and anti-inflammatory properties( 10 ). Previous studies have demonstrated that Kolaviron effectively scavenges free radicals and acts as a robust antioxidant( 14 , 28 ). By mitigating oxidative and inflammatory damage, Kolaviron may protect neuronal regions that are critically involved in behavioral regulation, memory, and anxiety functions typically impaired in autism models. Brain areas such as the hippocampus are particularly vulnerable to oxidative and inflammatory insults( 29 ). Moreover, Kolaviron has been reported to exert neuroprotective effects against various forms of neurotoxicity and may improve social behavior and memory deficits associated with neurodevelopmental disorders such as autism( 28 , 30 ). Consistent with previous findings, our results also demonstrated a significant increase in Malondialdehyde (MDA) levels in the hippocampus of VPA-exposed rats, indicating elevated lipid peroxidation and oxidative damage. These findings support the hypothesis that oxidative stress plays a critical role in the neurodevelopmental abnormalities observed in the VPA model of autism. Treatment with kolaviron (KV) at a dose of 100 mg/kg significantly reduced MDA levels in the hippocampus, suggesting a protective effect against lipid peroxidation and free radical-induced damage. Notably, a 42.08% reduction in the hippocampus may be attributed to KV’s polyphenolic antioxidant structure, previously shown to scavenge free radicals and inhibit reactive oxygen species (ROS) production( 28 ). In the VPA group, a marked reduction in the activity of catalase (CAT) an essential enzyme responsible for breaking down hydrogen peroxide, a major ROS was observed. This reduction indicates a compromised or impaired antioxidant defense system in the brain( 2 ). KV treatment restored CAT levels, with an 83.23% increase in the hippocampus, highlighting Kolaviron's role in enhancing endogenous antioxidant defenses. Moreover, nitrite levels (NO₂⁻), used as an indirect measure of nitric oxide production and nitrosative stress, were significantly elevated in the VPA group, suggesting ongoing neuroinflammation and potential mitochondrial damage. However, KV treatment did not significantly alter nitrite levels, implying that its neuroprotective effects may be more pronounced in ROS-mediated rather than reactive nitrogen species (RNS)-mediated pathways. To further assess the impact of oxidative stress on cellular health, mitochondrial membrane potential (MMP) was evaluated. VPA exposure led to a substantial decrease in MMP, indicative of mitochondrial dysfunction, a hallmark of oxidative stress in neurodevelopmental disorders. Interestingly, KV treatment partially restored MMP in the hippocampus, suggesting improved mitochondrial integrity and possibly partial recovery of neuronal energy balance( 1 , 28 ). The inflammatory markers TNF-α and IL-6 were also assessed. In the VPA model group, both cytokines were significantly elevated in the hippocampus, indicating an activated immune response and ongoing neuroinflammation. Treatment with kolaviron, especially at 100 mg/kg, significantly reduced these cytokines in the hippocampus. This anti-inflammatory effect of kolaviron may contribute to the behavioral improvements observed in the treated animals. Additionally, our study revealed a significant reduction in parvalbumin-positive (PV+) neurons in the hippocampus of the VPA group. Such a reduction may disrupt inhibitory control and neural circuit balance, potentially leading to behavioral and cognitive deficits( 8 ). Notably, KV treatment (100 mg/kg) significantly increased the number of PV + neurons in the hippocampus a 98% increase highlighting Kolaviron's neuroprotective effect in counteracting VPA-induced neuronal loss. Since PV + neurons play a crucial role in regulating excitatory-inhibitory balance in the hippocampus, their restoration may indicate improved regional function and potentially amelioration of ASD-related behavioral and cognitive symptoms. Previous studies have reported elevated blood serotonin levels in the valproic acid (VPA)-induced autism-like model, suggesting a possible association between increased serotonin and the pathophysiology of autism( 31 ). Consequently, growing attention has been directed toward serotonin due to its critical role in the nervous system and the inconsistent changes observed in different neural circuits and regions( 29 ). Current investigations have targeted various aspects of the serotonergic system, including serotonin itself, its transporters, receptors, and precursors. In rats, serotonergic neurons begin to develop between embryonic days 10.5 and 13, after which they migrate to different brain regions and initiate serotonin synthesis( 6 ). Given the known role of serotonin in mood regulation and its strong implication in autism spectrum disorders, and considering the overlap between the timing of VPA injection in our model and key developmental windows for serotonergic neurons, we focused on exploring this system. In the case of the serotonin transporter (5HTT), a significant increase in expression was observed in the VPA group compared to the control group in the hippocampus, suggesting altered reuptake mechanisms in this region. Interestingly, the group treated with 100 mg/kg kolaviron showed a significant reduction in 5HTT levels specifically, a 40.37% decrease in the hippocampus. These findings suggest that kolaviron treatment may modulate serotonin transporter expression and potentially restore serotonergic balance. A similar pattern was observed in the oxytocin-treated group, which exhibited a significant increase in hippocampal 5HTT levels (48.66%), possibly indicating a compensatory or restorative serotonergic mechanism. Regarding the 5HTR7 receptor, no significant differences were observed across all groups in the evaluated brain regions. Although prior research suggests that 5HTR7 may play a role in behavioral disorders and neuroplasticity( 31 ), our data did not support a direct involvement of this receptor in the observed changes within the VPA model. This lack of significant findings could be attributed to timing of measurement or the complex functions of this receptor in the VPA model. Collectively, these results suggest that kolaviron may have a beneficial modulatory effect on the serotonergic system, particularly by reducing 5HTT levels, which may contribute to behavioral improvements observed in the VPA-induced model of autism. However, further studies are necessary to elucidate the precise mechanisms through which kolaviron affects the serotonergic pathway and its potential therapeutic implications in neurodevelopmental disorders like autism. In the Self-Grooming test, an indicator of repetitive behavior, the kolaviron-treated groups (50 and 100 mg/kg) exhibited a significant reduction in stereotypic behaviors compared to the VPA model group. This improvement may be attributed to Kolaviron's antioxidant properties and reduction in oxidative stress, potentially restoring neuronal function in brain areas responsible for repetitive behaviors, such as the hippocampus. In the Three-Chamber test, which specifically assesses social interaction, kolaviron-particularly at 100 mg/kg-led to significant improvement in social engagement. These effects mirrored those observed with oxytocin treatment and highlight Kolaviron's potential in ameliorating VPA-induced social deficits, possibly due to its anti-inflammatory properties and improved neural function in social-related brain regions.In the Marble Burying test, which is used to assess anxiety and repetitive behaviors, treatment with 100 mg/kg of kolaviron significantly reduced both anxiety and stereotypic behaviors. These outcomes suggest that kolaviron may be effective in alleviating anxiety symptoms, possibly through its impact on brain oxidative and inflammatory stress pathways. However, no comparable effects were observed at the 50 mg/kg dose, indicating that this lower dose may be insufficient to influence these specific behavioral outcomes. Finally, in the Open Field test, which evaluates both social behavior and anxiety, kolaviron significantly increased the time spent in the social zone, further supporting its potential in improving social engagement and reducing anxiety-related behaviors. This aligns with results from other behavioral assays, reinforcing Kolaviron's positive impact on anxiety reduction and social behavior enhancement. Overall, these findings suggest that kolaviron, especially at a 100 mg/kg dose, can effectively ameliorate repetitive behaviors, anxiety, and social deficits in the VPA-induced autism model. The lack of effects at lower doses underscores the importance of determining an optimal therapeutic dose for behavioral intervention. 5 Conclusion Kolaviron demonstrated neuroprotective effects in the valproic acid (VPA)-induced rodent model of autism, primarily through its antioxidant and anti-inflammatory actions. By reducing oxidative damage, supporting endogenous defenses, and preserving mitochondrial function, kolaviron helped restore hippocampal neuronal integrity. These biochemical improvements were associated with reduced repetitive behaviors, lower anxiety, and enhanced social interaction. The dose-dependent nature of these effects highlights the importance of optimizing therapeutic strategies. While promising, further research is needed to fully understand Kolaviron's mechanisms and therapeutic potential in autism spectrum disorders. Scope and Limitations Clearly, conducting a comprehensive research study is challenging, and the limitations inherent in any research such as practical, temporal, and financial constraints are evident. In this particular study, the vastness and complexity of the changes observed in Autism Spectrum Disorder (ASD) required that assessments be conducted across various models. Additionally, further examination of the serotonergic system through diverse brain regions could provide more precise and transparent insights into this field. The wide range of characteristics and features of autism can seem overwhelming, and it might be beneficial to categorize the disorder into more specific subgroups, allowing for more focused and in-depth studies. Abbreviation Autism Spectrum Disorder (ASD) Valproic acid (VPA) Kolaviron (KV) Intraperitoneal (IP) Postnatal day (PND) Oxytocin (OT) Enzyme-Linked Immunosorbent Assay (ELISA) 5‑Hydroxytryptamine Transporter (5-HTT) Serotonin Transporter (SERT) 5‑Hydroxytryptamine Receptor 7 (5‑HTR7) Tumor Necrosis Factor alpha(TNF‑α) Interleukin‑6 (IL‑6) Parvalbumin interneurons (PV interneurons) 2,2‑Diphenyl‑1‑picrylhydrazyl (DPPH) Tetramethylbenzidine (TMB) Malondialdehyde (MDA) Catalase activity (CAT) Nitrite levels (NIT) Mitochondrial Membrane Potential (MMP) Dimethyl sulfoxide (DMSO) phosphate-buffered saline (PBS) Normal goat serum (NGS) 3,3′-diaminobenzidine (DAB) Reactive oxygen species (ROS) Reactive nitrogen species (RNS) Declarations Data availability statement All original contributions of this study are included in the main article and its supplementary materials. Further information can be obtained by contacting the corresponding author. Ethics statement This animal experiment was reviewed and approved by the Ethics Committee of Iran University of Medical Sciences (IR.IUMS.FMD.REC.1401.210). All procedures were carried out in compliance with institutional guidelines and relevant national regulations. Author contributions VK: Conducted formal analysis, investigation, developed methodology, prepared the original draft, and participated in reviewing and editing the manuscript. TB: Performed formal analysis, secured funding, supervised the project, contributed to the original draft, and reviewed and edited the manuscript. MF: Contributed to methodology development and participated in manuscript review and editing. SM: Involved in methodology design and manuscript review and editing. AKK: Assisted with methodology and took part in reviewing and editing the manuscript. MR: Carried out formal analysis, contributed to methodology, supervised the study, drafted the original manuscript, and engaged in review and editing. Funding The author(s) declare that financial support was received for the research and/or publication of this article. This study is part of a PhD thesis project that was approved and funded by Iran University of Medical Sciences, Tehran, Iran (Grant no. 1401-3-104-23915). Conflict of interest The authors state that there were no commercial or financial ties that might be perceived as a potential conflict of interest during the conduct of this research. Clinical trial number Clinical trial number: not applicable References Mehra S, Ul Ahsan A, Seth E, Chopra M. Critical Evaluation of Valproic Acid-Induced Rodent Models of Autism: Current and Future Perspectives. J Mol Neurosci. 2022;72(6):1259-73. Gouda B, Sinha SN, Sangaraju R, Huynh T, Patangay S, Venkata Mullapudi S, et al. Extraction, Phytochemical profile, and neuroprotective activity of Phyllanthus emblica fruit extract against sodium valproate-induced postnatal autism in BALB/c mice. Heliyon. 2024;10(15):e34992. Afshari M, Gharibzadeh S, Pouretemad H, Roghani M. Promising therapeutic effects of high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) in addressing autism spectrum disorder induced by valproic acid. Front Neurosci. 2024;18:1385488. Ukezono M, Kasahara Y, Yoshida C, Murakami Y, Okada T, Takano Y. Impairments of social interaction in a valproic acid model in mice. Front Behav Neurosci. 2024;18:1430267. Al-Dossari AM, Al-Harbi LN, Al-Otaibi NM, Almubarak A, Almnaizel AT, Alshammari GM, et al. The Potency of Goat Milk in Reducing the Induced Neurotoxic Effects of Valproic Acid in Rat Pups as a Rodent Model of Autism Spectrum Disorder. Metabolites. 2023;13(4). Kuo HY, Liu FC. Pathophysiological Studies of Monoaminergic Neurotransmission Systems in Valproic Acid-Induced Model of Autism Spectrum Disorder. Biomedicines. 2022;10(3). Farrag EAE, Askar MH, Abdallah Z, Mahmoud SM, Abdulhai EA, Abdelrazik E, et al. Comparative effect of atorvastatin and risperidone on modulation of TLR4/NF-kappaB/NOX-2 in a rat model of valproic acid-induced autism. Behav Brain Funct. 2024;20(1):26. Nicolini C, Fahnestock M. The valproic acid-induced rodent model of autism. Exp Neurol. 2018;299(Pt A):217-27. Attah CO, Alhaji UI, Ameh DA, Forcados GE, Muhammad A, Bashir M, Ibrahim S. In Vivo Chemosuppressive Effects of Kolaviron on 7,12-Dimethylbenzanthracene-Induced Mammary Lesions are Associated with Changes in Levels of Estrogen Receptor-α, CYP 1A1, Proinflammatory Cytokines, and Alterations to Metabolic Pathways Implicated in Mammary Carcinogenesis. J Med Food. 2024;27(10):940-50. Olatoye FJ, Akindele AJ, Awodele O. The role of Kolaviron, a bioflavonoid from Garcinia kola, in the management of cardiovascular diseases: A systematic review. Heliyon. 2024;10(5):e27333. Nazari-Serenjeh M, Baluchnejadmojarad T, Hatami-Morassa M, Fahanik-Babaei J, Mehrabi S, Tashakori-Miyanroudi M, et al. Kolaviron neuroprotective effect against okadaic acid-provoked cognitive impairment. Heliyon. 2024;10(3):e25564. Abarikwu SO, Ezim OE, Ikeji CN, Farombi EO. Atrazine: cytotoxicity, oxidative stress, apoptosis, testicular effects and chemopreventive Interventions. Frontiers in Toxicology. 2023;5. Okafor AI, Ogban NN, Odinigwe AA. Kolaviron alleviates haematological abnormalities and hepato-renal damage in Naja nigricollis nigricollis venom-treated rats. Toxicol Rep. 2022;9:1869-76. Tauchen J, Frankova A, Manourova A, Valterova I, Lojka B, Leuner O. Garcinia kola: a critical review on chemistry and pharmacology of an important West African medicinal plant. Phytochem Rev. 2023:1-47. Oyovwi MO, Ben-Azu B, Edesiri TP, Victor E, Rotu RA, Ozegbe QE, et al. Kolaviron abates busulfan-induced episodic memory deficit and testicular dysfunction in rats: the implications for neuroendopathobiological changes during chemotherapy. Biomedicine & Pharmacotherapy. 2021;142:112022. Omotoso GO, Arietarhire LO, Ukwubile II, Gbadamosi IT. The protective effect of kolaviron on molecular, cellular, and behavioral characterization of cerebellum in the rat model of demyelinating diseases. Basic and Clinical Neuroscience. 2020;11(5):609. Omotoso GO, Mutholib NY, Abdulsalam FA, Bature AI. Kolaviron protects against cognitive deficits and cortico-hippocampal perturbations associated with maternal deprivation in rats. Anatomy & Cell Biology. 2019;53(1):95. Pizzella A, Penna E, Liu Y, Abate N, Lacivita E, Leopoldo M, et al. Alterations of synaptic plasticity in Angelman syndrome model mice are rescued by 5-HT7R stimulation. Progress in Neurobiology. 2024;242:102684. Emmanuel O, Uche ME, Dike ED, Etumnu LR, Ugbogu OC, Ugbogu EA. A review on garcinia kola heckel: traditional uses, phytochemistry, pharmacological activities, and toxicology. Biomarkers. 2022;27(2):101-17. OR A, ME C, NL B, OO O. Kolaviron, a biflavonoid complex of Garcinia kola seeds modulates apoptosis by suppressing oxidative stress and inflammation in diabetes-induced nephrotoxic rats. Phytomedicine. 2014;21(14):1785–93. Afshari M, Gharibzadeh S, Pouretemad H, Roghani M. Reversing valproic acid-induced autism-like behaviors through a combination of low-frequency repeated transcranial magnetic stimulation and superparamagnetic iron oxide nanoparticles. Sci Rep. 2024;14(1):8082. Zahedi E, Sadr SS, Sanaeierad A, Roghani M. Chronic acetyl-L-carnitine treatment alleviates behavioral deficits and neuroinflammation through enhancing microbiota derived-SCFA in valproate model of autism. Biomed Pharmacother. 2023;163:114848. Feriyani F, Maulanza H, Lubis RR, Balqis U, Darmawi D. Effects of Binahong (Anredera cordifolia (Tenore) Steenis) Extracts on the Levels of Malondialdehyde (MDA) in Cataract Goat Lenses. ScientificWorldJournal. 2021;2021:6617292. Ghalami J, Baluchnejad Mojarad T, Mansouri M, Khamse S, Roghani M. Paeonol Protection Against Intrastriatal 6-Hydroxydopamine Rat Model of Parkinson's Disease. Basic Clin Neurosci. 2021;12(1):43-56. Teixeira FC, Gutierres JM, Soares MSP, da Siveira de Mattos B, Spohr L, do Couto CAT, et al. Inosine protects against impairment of memory induced by experimental model of Alzheimer disease: a nucleoside with multitarget brain actions. Psychopharmacology (Berl). 2020;237(3):811-23. Ding J, Yu HL, Ma WW, Xi YD, Zhao X, Yuan LH, et al. Soy isoflavone attenuates brain mitochondrial oxidative stress induced by β-amyloid peptides 1-42 injection in lateral cerebral ventricle. J Neurosci Res. 2013;91(4):562-7. Pourmohammadi S, Roghani M, Kiasalari Z, Khalili M. Paeonol Ameliorates Cuprizone-Induced Hippocampal Demyelination and Cognitive Deficits through Inhibition of Oxidative and Inflammatory Events. J Mol Neurosci. 2022;72(4):748-58. Omotoso GO, Mutholib NY, Abdulsalam FA, Bature AI. Kolaviron protects against cognitive deficits and cortico-hippocampal perturbations associated with maternal deprivation in rats. Anat Cell Biol. 2020;53(1):95-106. Chen S, Huang L, Liu G, Kang J, Qian Q, Wang J, et al. Acupuncture Ameliorated Behavioral Abnormalities in the Autism Rat Model via Pathways for Hippocampal Serotonin. Neuropsychiatr Dis Treat. 2023;19:951-72. Omotoso GO, Arietarhire LO, Ukwubile, II, Gbadamosi IT. The Protective Effect of Kolaviron on Molecular, Cellular, and Behavioral Characterization of Cerebellum in the Rat Model of Demyelinating Diseases. Basic Clin Neurosci. 2020;11(5):609-18. Rahdar M, Davoudi S, Dehghan S, Javan M, Hosseinmardi N, Behzadi G, Janahmadi M. Reversal of electrophysiological and behavioral deficits mediated by 5-HT7 receptor upregulation following LP-211 treatment in an autistic-like rat model induced by prenatal valproic acid exposure. Neuropharmacology. 2024;257:110057. Additional Declarations No competing interests reported. Supplementary Files graphicalabspng.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6783521","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":477061151,"identity":"5623298e-7e27-4495-8fbd-cf9e0ab0a862","order_by":0,"name":"Vahid Khodashenas","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Vahid","middleName":"","lastName":"Khodashenas","suffix":""},{"id":477061152,"identity":"187e3de0-7e03-417c-9aa5-0a6d64ecf81f","order_by":1,"name":"Tourandokht Baluchnejadmojarad","email":"data:image/png;base64,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","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Tourandokht","middleName":"","lastName":"Baluchnejadmojarad","suffix":""},{"id":477061153,"identity":"1ecaca75-c7de-4243-8276-c490ab657e99","order_by":2,"name":"Mitra Farbin","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mitra","middleName":"","lastName":"Farbin","suffix":""},{"id":477061154,"identity":"902435e5-4c16-4a04-b99a-b5e5d6e03f03","order_by":3,"name":"Ali Khodabakhshi Korelaei","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"Khodabakhshi","lastName":"Korelaei","suffix":""},{"id":477061155,"identity":"a1c497b3-3696-45f8-a340-4814aff10c3b","order_by":4,"name":"Soraya Mehrabi","email":"","orcid":"","institution":"Iran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Soraya","middleName":"","lastName":"Mehrabi","suffix":""},{"id":477061156,"identity":"9cd4121f-9052-4336-b2e1-4d4d9ff9b0e5","order_by":5,"name":"Mehrdad Roghani","email":"","orcid":"","institution":"Shahed University","correspondingAuthor":false,"prefix":"","firstName":"Mehrdad","middleName":"","lastName":"Roghani","suffix":""}],"badges":[],"createdAt":"2025-05-30 09:53:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6783521/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6783521/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85565642,"identity":"4c37e756-b273-454d-96e7-fa3ed7594fdb","added_by":"auto","created_at":"2025-06-27 14:25:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":275005,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of the experimental timeline.Valproic acid (600 mg/kg, i.p.) was administered to pregnant rats on gestational day 12.5 to establish the autism model. Male offspring were treated with kolaviron via oral gavage from postnatal day 21 to 49. Behavioral assessments were conducted between postnatal days 42 and 49. Following these tests, animals were perfused, brain tissues were collected, and subsequent histological and biochemical analyses were performed.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6783521/v1/a317fc60eb8729f4ec3f66ce.png"},{"id":85565977,"identity":"e84cd652-7832-4277-a630-d275d8abd2af","added_by":"auto","created_at":"2025-06-27 14:33:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121353,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral assessments: (a) Self-Grooming Test, (b) Open Field Test, (c) Three-Chamber Social Test, and (d) Marble Burying Test. s denotes a significant difference compared to the disease model group (P \u0026lt; 0.05), ss (P \u0026lt; 0.01), and sss (P \u0026lt; 0.001). Similarly, g represents significance compared to the control group (P \u0026lt; 0.05), gg (P \u0026lt; 0.01), and ggg (P \u0026lt; 0.001). Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6783521/v1/1a169c05b46047cf8d100c5e.png"},{"id":85565978,"identity":"015576b5-4f61-4df0-98e3-945ba9811f9e","added_by":"auto","created_at":"2025-06-27 14:33:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":124204,"visible":true,"origin":"","legend":"\u003cp\u003eOxidative stress assessments: (a) MDA levels, (b) Catalase (CAT) activity, and (c) Nitrite (NIT) levels across different experimental groups. s denotes a significant difference compared to the disease model group (P \u0026lt; 0.05), ss (P \u0026lt; 0.01), and sss (P \u0026lt; 0.001). Similarly, g represents significance compared to the control group (P \u0026lt; 0.05), gg (P \u0026lt; 0.01), and ggg (P \u0026lt; 0.001). Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6783521/v1/eec3708f6885bd95e1064bb3.png"},{"id":85565646,"identity":"be3dad82-0780-40de-89ff-b4d1880fc96e","added_by":"auto","created_at":"2025-06-27 14:25:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81044,"visible":true,"origin":"","legend":"\u003cp\u003eInflammation assessments: (a) TNF-α levels and (b) IL-6 levels across different experimental groups. s denotes a significant difference compared to the disease model group (P \u0026lt; 0.05), ss (P \u0026lt; 0.01), and sss (P \u0026lt; 0.001). Similarly, g represents significance compared to the control group (P \u0026lt; 0.05), gg (P \u0026lt; 0.01), and ggg (P \u0026lt; 0.001). Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6783521/v1/64af4df51c810d1bbf60c499.png"},{"id":85565980,"identity":"00cf1eca-06df-4cb7-bb90-30ffbe0c583a","added_by":"auto","created_at":"2025-06-27 14:33:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":285891,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of Mitochondrial Membrane Potential: Parvalbumin Interneurons: (a) MMP levels and (b) Parvalbumin interneuron expression across different experimental groups. s denotes a significant difference compared to the disease model group (P \u0026lt; 0.05), ss (P \u0026lt; 0.01), and sss (P \u0026lt; 0.001). Similarly, g represents significance compared to the control group (P \u0026lt; 0.05), gg (P \u0026lt; 0.01), and ggg (P \u0026lt; 0.001). Data are presented as mean ± SEM. Black solid arrows show parvalbumin-immunopositive interneurons.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6783521/v1/8933706895cd4bba0a0bc3af.png"},{"id":85565647,"identity":"18e3553e-660d-4908-851d-18d25aa8cf5f","added_by":"auto","created_at":"2025-06-27 14:25:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72254,"visible":true,"origin":"","legend":"\u003cp\u003eSerotonergic System Assessment: (a) 5HTT expression levels and (b) 5HT7R expression levels across different experimental groups. s denotes a significant difference compared to the disease model group (P \u0026lt; 0.05), ss (P \u0026lt; 0.01), and sss (P \u0026lt; 0.001). Similarly, g represents significance compared to the control group (P \u0026lt; 0.05), gg (P \u0026lt; 0.01), and ggg (P \u0026lt; 0.001). Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6783521/v1/1a629973928af275de055db9.png"},{"id":100881330,"identity":"5d3cf9cd-9e5f-44ca-b0e5-f1b6ad7a640a","added_by":"auto","created_at":"2026-01-22 11:13:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2728629,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6783521/v1/c62b40b4-04cf-4dbc-a95c-f11d7c964423.pdf"},{"id":85565652,"identity":"bad0f2bc-810a-4162-bd51-e319bfb64d95","added_by":"auto","created_at":"2025-06-27 14:25:05","extension":"png","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1129379,"visible":true,"origin":"","legend":"","description":"","filename":"graphicalabspng.png","url":"https://assets-eu.researchsquare.com/files/rs-6783521/v1/d0fd0cb34107b65eac8499e8.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Promising Therapeutic Efficacy of Kolaviron against Prenatal Valproate-Induced Autism Spectrum Disorder","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAutism Spectrum Disorder (ASD) is a lifelong neurodevelopmental condition marked by persistent challenges in social communication, language acquisition, and the presence of restrictive or repetitive behaviors. Typically emerging in early childhood, ASD manifests through a heterogeneous array of symptoms such as delayed speech and motor milestones, difficulties interpreting social cues (e.g., tone of voice, body language), hyper- or hypo-reactivity to sensory stimuli, and sometimes self-injurious behaviors(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Although genetic factors contribute substantially to ASD risk, environmental influences including prenatal exposure to certain medications also play a critical role(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). To date, no curative therapies exist, underpinning the urgent need for mechanistic studies and novel interventions(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Valproic acid (VPA) is a branched-chain fatty acid extensively used as an antiepileptic, mood stabilizer, and migraine prophylactic(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, when administered during early gestation, VPA is linked to an elevated risk of autism spectrum disorder (ASD) in the offspring(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In rodent studies, a single intraperitoneal injection of 600 mg/kg VPA on gestational day 12.5 delays neural tube closure and triggers widespread molecular disruptions, including compromised mitochondrial function(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). This critical exposure window overlaps with peak periods of neurogenesis and serotonergic neuron migration, making the developing brain particularly susceptible to excitatory-inhibitory imbalance and oxidative damage(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). At the cellular level, VPA increases reactive oxygen species and proinflammatory mediators while diminishing inhibitory interneuron populations(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Behaviorally, VPA-exposed rodents faithfully reproduce core ASD phenotypes, social deficits in the three-chamber test, heightened repetitive actions, and atypical ultrasonic vocalizations(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). In this work, pregnant dams received 600 mg/kg VPA on day 12.5 of gestation, and male offspring were subsequently assessed for behavioral, biochemical, and histopathological outcomes.\u003c/p\u003e \u003cp\u003eKolaviron (KV) is a natural mixture of Flavonoids obtained from Garcinia kola seeds, traditionally used in West African medicine (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). It is recognized for its antioxidant and anti-inflammatory activities, mainly due to its polyphenolic components (\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). \u003cb\u003eBeyond these general properties, Kolaviron (KV) has been extensively investigated for its specific neuroprotective potential in preclinical models. Numerous studies have demonstrated its ability to mitigate oxidative stress and inflammation in the central nervous system. For example, KV has been shown to reverse scopolamine-induced memory impairment and prevent brain microstructural derangements and cognitive deficits in rodents exposed to various neurotoxins, including methamphetamine and cuprizon\u003c/b\u003e (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), \u003cb\u003eIn a cuprizone-induced demyelination model, KV treatment significantly restored endogenous antioxidant enzyme levels (such as SOD and GPx), reduced lipid peroxidation (MDA), and preserved cerebellar neuronal morphology and astrocytic health, highlighting its robust neuroprotective and anti-inflammatory actions within the brain\u003c/b\u003e (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). \u003cb\u003eFurthermore, in a rat model of maternal deprivation, Kolaviron successfully attenuated behavioral deficits, improved memory and cognitive performance, and restored oxidative balance in hippocampal and prefrontal cortex regions by enhancing SOD levels, suggesting its capacity to disrupt excitotoxic stimuli and prevent mitochondrial dysfunctions\u003c/b\u003e (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). \u003cb\u003eThese findings from diverse\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003emodels collectively provide strong experimental evidence for Kolaviron's therapeutic potential in neurological conditions characterized by oxidative stress, neuroinflammation, and neuronal damage. The relevance of Kolaviron's multifaceted actions extends to the complex pathophysiology of Autism Spectrum Disorder (ASD), where oxidative stress and neuroinflammation are recognized as key contributing factors. Given that Valproic Acid (VPA) is a well-established inducer of neurodevelopmental abnormalities in animal models, partly through the generation of oxidative stress and inflammatory responses, the compelling evidence of Kolaviron's ability to modulate these pathways in preclinical settings provides a strong rationale for its investigation in VPA-induced ASD models. Moreover, its reported influence on the serotonergic system\u003c/b\u003e (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), \u003cb\u003ea critical neurotransmitter system often dysregulated in ASD, further supports its potential as a therapeutic agent.\u003c/b\u003e These properties allow KV to counteract oxidative damage, modulate immune responses, and support mitochondrial health. Considering that oxidative stress and inflammation contribute to the development of autism spectrum disorder (ASD), KV may help to protect against neural damage seen in this condition (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). It has also been shown to influence the serotonergic system, which plays a role in ASD pathophysiology. In this study, KV was administered orally at 50 and 100 mg/kg after weaning in a VPA-based rat model of autism to assess its effects on behavior, oxidative and inflammatory markers, and serotonin-related targets in the hippocampus. The aim of our study was to evaluate whether Kolaviron could improve the VPA-induced autism model in the areas of oxidative stress, inflammation, and behavior. We also assessed its effects on the serotonergic pathway and compared the results with oxytocin to determine how effective Kolaviron could be.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals\u003c/h2\u003e \u003cp\u003eSexually mature male and female Wistar rats, including young adults, were obtained from the Experimental and Comparative Research Center of Iran University of Medical Sciences (Tehran, Iran). Breeding was carried out under controlled laboratory conditions, and the male offspring were used for the subsequent experimental procedures. The animals were housed in groups of no more than four per cage, maintained on a 12-hour light/dark cycle, at a constant temperature of 22\u0026deg;C and a relative humidity of 40\u0026ndash;50%. Food and water were available to the animals at all times. All procedures were conducted in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Iran University of Medical Sciences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Kolaviron\u003c/h2\u003e \u003cp\u003eA total of 100 grams of Garcinia kola seeds were processed by first removing Lipophilic components using 150 ml of petroleum ether over 24 hours in a Soxhlet apparatus. The defatted material was then air-dried at 25\u0026deg;C, followed by an initial extraction with 120 ml of acetone at 40\u0026deg;C. After concentration, a secondary purification step was carried out using ethyl acetate. This method resulted in the isolation of kolaviron, a compound predominantly enriched with Biflavonoid structures such as Garcinia Biflavonoid 1 and 2, as well as kolaflavanone(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).Biochemical properties of kolaviron were assessed using standardized spectrophotometric assays. Antioxidant activity was measured via the DPPH radical scavenging method, calibrated with Trolox. Total flavonoid content was determined using aluminum chloride and potassium acetate, expressed as quercetin equivalents. Total phenolic content was measured by a modified Folin\u0026ndash;Ciocalteu method, with Gallic acid as the standard. Flavonol content was evaluated after incubation with aluminum trichloride and sodium acetate, using rutin as the reference. All assays were performed in triplicate to ensure accuracy and reproducibility(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).To date, no significant adverse effects have been documented in the scientific literature concerning the pharmacological actions of Garcinia kola. The limited toxicological assessments conducted suggest a high margin of safety, with the oral median lethal dose (LD₅₀) of the seed extracts reported to be approximately 5000 mg/kg body weight(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental Procedure\u003c/h2\u003e \u003cp\u003eMale Wistar rats were housed under standard conditions (21\u0026ndash;23\u0026deg;C, 45\u0026ndash;50% humidity, 12/12 h light/dark cycle) with free access to food and water. Female rats with synchronized estrous cycles were mated, and the day after mating was designated as gestational day (GD) 1. On GD12.5, pregnant dams received a single intraperitoneal injection of either sodium valproate (600 mg/kg)(Darou Pakhsh Co., Tehran, Iran) to induce autism-like features (VPA group) or normal saline (control group). Dams were housed individually until parturition, and postnatal development of pups was monitored. On postnatal day (PND) 21, pups were weaned, sexed, and only males were included in the study. From PND 21 to 49, male offspring received daily oral administration of either normal saline, kolaviron (50 or 100 mg/kg), or intranasal oxytocin (12 \u0026micro;g/kg)(Royan Co, Tehran, Iran). Animals were divided into eight groups (n\u0026thinsp;=\u0026thinsp;6 each): (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Control\u0026thinsp;+\u0026thinsp;Saline, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Control\u0026thinsp;+\u0026thinsp;Kolaviron50, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Control\u0026thinsp;+\u0026thinsp;Kolaviron100, (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) VPA\u0026thinsp;+\u0026thinsp;Saline, (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) VPA\u0026thinsp;+\u0026thinsp;Kolaviron50, (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)VPA\u0026thinsp;+\u0026thinsp;Kolaviron100, and (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) VPA\u0026thinsp;+\u0026thinsp;Oxytocin. Behavioral assessments were performed during PNDs 42\u0026ndash;49. On PND 49, five animals per group were deeply anesthetized (Ketamine/Xylazine) and perfused transcardially. Brains were fixed in 10% formalin for Immunohistochemical analysis of parvalbumin-positive interneurons in the hippocampus. Remaining brain samples were dissected, stored at \u0026minus;\u0026thinsp;80\u0026deg;C, and later analyzed by ELISA for hippocampal levels of serotonin transporter (5-HTT), serotonin receptor 7 (5-HTR7), and inflammatory cytokines such as IL-6. At the end of study, all animals were euthanized using CO₂ and disposed of following ethical protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Behavioral Tests\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Three Chamber Test\u003c/h2\u003e \u003cp\u003eThe procedure for this test has been reported(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In this study, the three-chamber test was utilized to evaluate the social interaction of rats. Initially, the rats were placed in the center of the apparatus and given 10 minutes to explore their surroundings freely. Following this phase, an empty metal cage was positioned on one side of the apparatus, while a cage containing an unfamiliar rat was placed on the opposite side. The test rat was then allowed 10 minutes to assess its social interactions with the unfamiliar rat. The sociability index was calculated by subtracting the time spent in the non-social chamber from the time spent in the social chamber, and this result was divided by the total time spent in both chambers. Lastly, the time spent by the rats interacting with social and non-social stimuli such as cleaning, fighting, or other social behaviors was recorded. This index was utilized to analyze the social behaviors of the rats in response to various stimuli.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Self Grooming Test\u003c/h2\u003e \u003cp\u003eOn the test day, each animal was individually placed in a Plexiglas cage measuring 30 \u0026times; 30 \u0026times; 45 cm. To familiarize them with the environment, the rats were acclimated to the empty cage for 10 minutes on the previous day. Following this, their total grooming time across all body areas was recorded over a 10-minute period(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Marble Burying Test\u003c/h2\u003e \u003cp\u003eAn apparatus measuring 40 cm \u0026times; 40 cm \u0026times; 40 cm was prepared, filled with nesting material and set up with 20 marbles arranged in 5 rows. Rats were then placed inside for 30 minutes, during which they could explore freely. The number of marbles they buried was recorded as an indicator of repetitive digging behavior(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4 Social Interaction Test\u003c/h2\u003e \u003cp\u003eTo measure social interactions, an open field apparatus (60 \u0026times; 60 \u0026times; 40 cm) was set up. Each rat was placed with an unfamiliar rat it had never encountered before, for a 10-minute session. The partner rats were matched in strain, sex, age, and weight to ensure consistency. To help reduce stress from the new environment, each rat was placed in the test cage individually for 5 minutes the day before testing. Social behaviors, including sniffing, licking, crawling or mounting, approaching, or following the other rat, were observed, with the total time spent on these activities counted as active social behavior(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5 ELISA Assay\u003c/h2\u003e \u003cp\u003eThe concentrations of TNF-α(Cat # sc-52746, Santa Cruz Biotechnology, Inc., USA), IL-6(Cat # sc-57315, Santa Cruz Biotechnology, Inc., USA), 5-HTT(Cat # MBS2533556, MyBioSource, Inc., USA), and 5-HT7R(Cat # MBS282566), MyBioSource, Inc., USA) in hippocampal tissues were determined using enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturers protocols. In brief, supernatants obtained from homogenized hippocampal samples were loaded onto pre-coated microplates and incubated at 4\u0026deg;C for 24 hours. After washing the plates three times with PBS to remove unbound components, HRP-conjugated secondary antibodies were added and incubated for 2 hours at room temperature. Then, Tetramethylbenzidine (TMB) substrate solution containing hydrogen peroxide was applied, and the reaction was allowed to proceed for 15 minutes in the dark. The enzymatic reaction was terminated with 0.18 M sulfuric acid, and optical density was measured at 450 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Hippocampal Biochemical Assessments\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Assessment of hippocampal Malondialdehyde (MDA)\u003c/h2\u003e \u003cp\u003eThe amount of Malon-di-aldehyde (Cat # KMDA96, Kiazist, Hamadan, Iran), a marker for assessing lipid peroxidation, was evaluated using 0.5% Thiobarbituric acid and 20% Trichloroacetic acid. The experiment was conducted in a thermoblock set at 95 degrees Celsius for 30 minutes. After this period, the samples were allowed to cool, and then centrifuged for 5 minutes at 25 degrees Celsius with a speed of 1500\u0026times;g. Finally, the optical absorbance was measured at a wavelength of 535 nanometers, using tetraethoxypropane as the standard for this measurement(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Assessment of hippocampal catalase activity (CAT)\u003c/h2\u003e \u003cp\u003eThe amount of catalase activity (Cat # KCAT96, Kiazist, Hamadan, Iran), In this method, the enzyme activity is assessed by first mixing 20 \u0026micro;L of supernatant with 100 \u0026micro;L of buffer and 20 \u0026micro;L of diluted substrate. This mixture is allowed to react for 20 min, facilitating the enzymatic reaction. After this period, the reaction is halted by adding 30 \u0026micro;L of diluted KOH. Next, 10 \u0026micro;L of KIO4 are introduced to the solution, and after waiting for 5 min, the absorbance is measured at 540 nm using a spectrophotometer. The amount of breakdown of H2O2 with catalase (CAT) is the basis of this assessment, providing valuable information about the enzyme activity in the presence of H2O2, KOH, and KIO4(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3 Assessment of hippocampal nitrite levels (NIT)\u003c/h2\u003e \u003cp\u003eFor The nitrite content(Cat # KNIT96, Kiazist, Hamadan, Iran) in the tissue was measured using Griess reagent, which consisted of 2.5% phosphoric acid, 1% sulfanilamide, and 0.1% naphthyl ethylenediamine dihydrochloride. After a 10-minute incubation, the optical density was measured at 540 nm, with sodium nitrite used as the calibration standard in the assay(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.6.4 Assessment of Mitochondrial Membrane Potential (MMP)\u003c/h2\u003e \u003cp\u003eThe assessment of mitochondrial membrane potential (MMP), which serves as a key diagnostic marker for mitochondrial integrity and health, was performed using Rhodamine 123 (Cat # R8004, SigmaAldrich,USA). This compound is a cationic fluorescent stain that preferentially accumulates in the mitochondria upon entering the cells. In this procedure, samples were first subjected to centrifugation at 10,000 rpm. Subsequently, 20 \u0026micro;L of Rhodamine 123 prepared at a concentration of 0.5 mg/5 mill DMSO from (SigmaAldrich, USA) was combined with 180 \u0026micro;L of phosphate-buffered saline (PBS) at a pH of 7.4 and After a 30-min incubation then added to the resulting pellet(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunohistochemical Evaluation of Parvalbumin Interneurons in the Hippocampus\u003c/h2\u003e \u003cp\u003eParaffin embedding and standard histological processing were performed on the hippocampal tissue blocks, after which serial sections of 5 \u0026micro;m thickness were obtained for further experimental analyses. Tissue sections were initially deparaffinized, rehydrated through descending concentrations of ethanol, and rinsed thoroughly in phosphate-buffered saline (PBS). To permeabilize the tissue, the slides were incubated with Triton X-100 in PBS for 15 minutes. Non-specific binding was blocked by exposing the sections to 10% normal goat serum (NGS) in PBS at room temperature for 1 hour. Subsequently, the sections were incubated overnight (24 h) in a humidified environment at room temperature with a rabbit polyclonal anti-parvalbumin primary antibody (Cat # P3088, SigmaAldrich, USA). After PBS washing steps, the slides were incubated for 2 hours with a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP)(Cat # sc-542741, MyBioSource, Inc., USA). For visualization, the Chromogenic reaction was developed by incubating the sections with 3,3\u0026prime;-diaminobenzidine (DAB)(Cat # D5637, SigmaAldrich, USA) and 0.01% hydrogen peroxide (H₂O₂) in PBS for 10 minutes in the dark. Following this step, the sections were rinsed, gently counterstained with Hematoxylin, dehydrated through graded alcohols, cleared in xylene, and coverslipped using Entellan. Microscopic evaluation was performed to identify and quantify parvalbumin-immunoreactive interneurons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical Methods\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism 9. Normality of data distribution was assessed via the Kolmogorov-Smirnov test, and outliers were identified and excluded using Grubbs' test. Group differences were analyzed by one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and statistical significance was defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Behavioral Findings\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Self-grooming test Findings\u003c/h2\u003e \u003cp\u003eThe results of the self-grooming test, which reflect stereotyped and repetitive behaviors in animals, are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. A one-way ANOVA analysis revealed significant differences among the experimental groups [F (6,44)\u0026thinsp;=\u0026thinsp;21.04, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01]. Further evaluation using Tukey's Multiple Range Testindicated that intraperitoneal(IP) administration of valproic acid (VPA) significantly increased repetitive behaviors compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).Moreover, treatment with Kolaviron (KV) in VPA model groups at both doses of 50 and 100 mg/kg led to a marked improvement compared to the VPA-damaged group. This improvement was significant for both doses, with a statistical level of (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This increase was 33.78%for the 50 mg/kg dose and 44.04% for the 100 mg/kg dose. Similarly, the group treated with Oxytocin showed a significant reduction in repetitive behaviors compared to the VPA group, aligning with the effects observed in KV-treated groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) Oxytocin also induced a 45.66% reduction. These findings highlight the potential of Kolaviron and Oxytocin in mitigating VPA-induced stereotyped behaviors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Three-Chamber test Findings\u003c/h2\u003e \u003cp\u003eFigure 2bpresents the results of the three-chamber test, which is used to assess social interaction and social avoidance behaviors. The statistical analysis using one-way ANOVA showed a significant difference between the groups [F (6, 41)\u0026thinsp;=\u0026thinsp;4.456, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. Further analysis revealed that the VPA model group exhibited a significant reduction in social interaction compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the KV-treated groups, the 100 mg/kg dose showed a significant improvement in social interaction compared to the VPA group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), This increase is equivalent to 341.91%. On the other hand, the Oxytocin-treated group showed a significant improvement compared to the VPA group(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) The increase induced by oxytocin was also 342.64%. These findings suggest that Kolaviron treatment, especially at the 100 mg/kg dose, can enhance social interaction in VPA-induced social deficits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3Open Field Test Findings\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec represents the Time in Social Zone, which assesses social behavior. One-way ANOVA statistical analysis indicates a significant difference among the studied groups [F (6,40)\u0026thinsp;=\u0026thinsp;20.79,P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001]. Furthermore, our additional analyses reveal that the diseased group (VPA group) exhibited a significantly lower social behavior time compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with a reduction of 71.07%. In the VPA groups treated with Kolaviron, it was observed that the VPA\u0026thinsp;+\u0026thinsp;KV 50 mg/kg group showed a significant increase (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in social behavior time, with an improvement of 132.12%. Similarly, the VPA\u0026thinsp;+\u0026thinsp;KV 100 mg/kg group exhibited a comparable pattern, significantly increasing the index compared to the VPA group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with an improvement of 118.01%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 Marble Burying Test Findings\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed shows the results of the Marble Burying Test, which is used to assess anxiety and repetitive behaviors in rats. The one-way ANOVA analysis revealed a significant difference among the groups [F (6,38)\u0026thinsp;=\u0026thinsp;5.251, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. Further evaluation with Tukey's post-hoc test indicated that the VPA group exhibited significantly higher anxiety and repetitive behaviors compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the KV-treated groups, the 50 mg/kg dose did not show a significant change in anxiety or repetitive behaviors. However, the 100 mg/kg dose of KV significantly reduced these behaviors compared to the VPA group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) This decrease is equivalent to 35.57%. Similarly, the Oxytocin-treated group also demonstrated a significant reduction in anxiety and repetitive behaviors compared to the VPA group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Oxytocin also exhibited a 40% reduction. These findings suggest that Kolaviron at the 100 mg/kg dose and Oxytocin can effectively reduce anxiety and repetitive behaviors induced by VPA.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Biochemical Assessment\u003c/h2\u003e \u003cp\u003eIn this study, our team assessed parameters related to oxidative stress, mitochondrial dysregulation, inflammation, and two factors associated with the serotonergic system.\u003c/p\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Oxidative Stress Assessment\u003c/h2\u003e \u003cdiv id=\"Sec27\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.1 Malondialdehyde (MDA) Assessment\u003c/h2\u003e \u003cp\u003eIn these assessments, for MDA a significant increase was observed in the VPA group compared to the control group in the hippocampus (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, in the treated disease group with 100 mg/kg Kolaviron, a significant reduction was observed compared to the disease group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This reduction was 31.25% in the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.2 Catalase Assessment\u003c/h2\u003e \u003cp\u003eIn the continuation of oxidative stress parameter assessments, a significant decrease in the Cat parameter was observed in the VPA group compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, the disease group treated with 100 mg/kg Kolaviron showed a significant increase compared to the disease group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).This increase was 73.30% in the hippocampus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section4\"\u003e \u003ch2\u003e3.2.1.3 Nitrite Assessment\u003c/h2\u003e \u003cp\u003eFinally, for the oxidative stress parameter Nit, a significant increase was observed in the VPA group compared to the control group in the hippocampus (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, no significant changes were observed in the treated groups compared to the disease group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Inflammation Assessments\u003c/h2\u003e \u003cdiv id=\"Sec31\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.1 Tumor Necrosis Factor (TNF) Assessment\u003c/h2\u003e \u003cp\u003eIn the assessment of inflammatory parameters, two parameters, TNF and IL-6, were evaluated. For TNF, the VPA group showed a significant increase compared to the control group in the hippocampus (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In the hippocampus, the disease group treated with 100 mg/kg Kolaviron showed a significant reduction (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which was 33.32% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section4\"\u003e \u003ch2\u003e3.2.2.2 Interleukin-6 (IL-6) Assessment\u003c/h2\u003e \u003cp\u003eSimilarly, for IL-6, the VPA group showed a significant increase compared to the control group in the hippocampus (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In this parameter, the disease group treated with 100 mg/kg Kolaviron showed a significant reduction in the hippocampus (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This reduction was 32.80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3 Mitochondrial Membrane Potential (MMP) Assessment\u003c/h2\u003e \u003cp\u003eIn this study, we also assessed MMP, which reflects mitochondrial dysregulation, a key factor contributing to cellular metabolic imbalance. A significant decrease was observed in the VPA group compared to the control group in the hippocampus (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, among the treatment groups, only the disease group treated with 100 mg/kg Kolaviron showed a significant increase in the hippocampus compared to the disease group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This increase was 48.82% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Histological Assessment\u003c/h2\u003e \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Parvalbumin Interneurons\u003c/h2\u003e \u003cp\u003eFor parvalbumin interneurons, the VPA group showed a significant decrease compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, the disease group treated with 100 mg/kg Kolaviron showed a significant increase compared to the disease group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with an increase of 98%. The VPA group treated with 50 mg/kg Kolaviron also exhibited an increase of 25% compared to the VPA group; however, this change did not reach statistical significance. Notably, this same group still showed a significant reduction of 43.18% compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Serotonergic System Assessment\u003c/h2\u003e \u003cp\u003eIn the assessment of serotonin-related parameters, we measured two parameters: 5HTT and 5HTR7.\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Serotonin Transporter (5HTT) Assessment\u003c/h2\u003e \u003cp\u003eFor 5HTT, the VPA group showed a significant increase compared to the control group in the hippocampus, with an elevation of 120.7%. The disease group treated with 100 mg/kg Kolaviron also showed a significant reduction compared to the disease group in the hippocampus (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This reduction was 40.38% in the hippocampus. Additionally, the VPA group treated with 50 mg/kg Kolaviron exhibited a 18.83% reduction, although this change did not reach statistical significance. A significant increase was also observed in the hippocampal region of the oxytocin group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with a percentage increase of 48.66% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Serotonin Receptor 7 (5HTR7) Assessment\u003c/h2\u003e \u003cp\u003eFor 5HTR7, however, no significant results were observed in any of the groups in the hippocampus. Kolaviron administration led to a reduction in 5HT7R expression both in healthy animals receiving kolaviron and in VPA-exposed animals treated with kolaviron. This reduction was more prominent at the 50 mg/kg dose. Specifically, in the healthy group treated with 50 mg/kg kolaviron, a 32.45% decrease in 5HT7R levels was observed compared to the control group. Similarly, in the VPA-exposed group receiving 50 mg/kg kolaviron, a 7.56% reduction was noted compared to the untreated VPA group. Notably, the VPA group itself exhibited a 36.13% increase in 5HT7R expression compared to the control group. The pattern of Kolaviron's effect was comparable to that of oxytocin, which also reduced 5HT7R expression by 15.98% in the VPA\u0026thinsp;+\u0026thinsp;oxytocin group compared to the VPA group. However, none of these changes reached statistical significance, indicating the need for further investigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eIn this study, we aimed to investigate the potential effects of Kolaviron on a valproic acid (VPA)-induced model of autism. Our primary focus was on the hippocampus. Among the various systems implicated in the pathophysiology of autism, the serotonergic system particularly drew our attention. We also evaluated markers of oxidative stress and inflammation. By integrating behavioral assessments with histological and biochemical analyses, we sought to better understand the relationship between molecular and structural alterations and the observed behavioral changes. VPA is a short-chain fatty acid that has been widely used to model autism in rodents. When administered intraperitoneal during pregnancy, VPA can disrupt neural tube closure, leading to structural and functional abnormalities in the developing nervous system(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). This model is known to induce oxidative stress, mitochondrial dysfunction, and neuroinflammation factors commonly associated with autism spectrum disorders. Given the high energy demands of neurons and the critical role of mitochondrial integrity in neuronal health, disruptions in these pathways can have profound effects(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). VPA also influences sodium channel activity, neurotransmitter systems, and the excitatory-inhibitory balance in the brain, contributing to a neurodevelopmental profile that mimics core features of autism, including increased reactive oxygen species (ROS) levels and altered pro-inflammatory cytokine expression(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Kolaviron, a bioactive compound derived from Garcinia kola, is known for its potent antioxidant and anti-inflammatory properties(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Previous studies have demonstrated that Kolaviron effectively scavenges free radicals and acts as a robust antioxidant(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). By mitigating oxidative and inflammatory damage, Kolaviron may protect neuronal regions that are critically involved in behavioral regulation, memory, and anxiety functions typically impaired in autism models. Brain areas such as the hippocampus are particularly vulnerable to oxidative and inflammatory insults(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Moreover, Kolaviron has been reported to exert neuroprotective effects against various forms of neurotoxicity and may improve social behavior and memory deficits associated with neurodevelopmental disorders such as autism(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Consistent with previous findings, our results also demonstrated a significant increase in Malondialdehyde (MDA) levels in the hippocampus of VPA-exposed rats, indicating elevated lipid peroxidation and oxidative damage. These findings support the hypothesis that oxidative stress plays a critical role in the neurodevelopmental abnormalities observed in the VPA model of autism. Treatment with kolaviron (KV) at a dose of 100 mg/kg significantly reduced MDA levels in the hippocampus, suggesting a protective effect against lipid peroxidation and free radical-induced damage. Notably, a 42.08% reduction in the hippocampus may be attributed to KV\u0026rsquo;s polyphenolic antioxidant structure, previously shown to scavenge free radicals and inhibit reactive oxygen species (ROS) production(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In the VPA group, a marked reduction in the activity of catalase (CAT) an essential enzyme responsible for breaking down hydrogen peroxide, a major ROS was observed. This reduction indicates a compromised or impaired antioxidant defense system in the brain(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). KV treatment restored CAT levels, with an 83.23% increase in the hippocampus, highlighting Kolaviron's role in enhancing endogenous antioxidant defenses. Moreover, nitrite levels (NO₂⁻), used as an indirect measure of nitric oxide production and nitrosative stress, were significantly elevated in the VPA group, suggesting ongoing neuroinflammation and potential mitochondrial damage. However, KV treatment did not significantly alter nitrite levels, implying that its neuroprotective effects may be more pronounced in ROS-mediated rather than reactive nitrogen species (RNS)-mediated pathways. To further assess the impact of oxidative stress on cellular health, mitochondrial membrane potential (MMP) was evaluated. VPA exposure led to a substantial decrease in MMP, indicative of mitochondrial dysfunction, a hallmark of oxidative stress in neurodevelopmental disorders. Interestingly, KV treatment partially restored MMP in the hippocampus, suggesting improved mitochondrial integrity and possibly partial recovery of neuronal energy balance(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The inflammatory markers TNF-α and IL-6 were also assessed. In the VPA model group, both cytokines were significantly elevated in the hippocampus, indicating an activated immune response and ongoing neuroinflammation. Treatment with kolaviron, especially at 100 mg/kg, significantly reduced these cytokines in the hippocampus. This anti-inflammatory effect of kolaviron may contribute to the behavioral improvements observed in the treated animals. Additionally, our study revealed a significant reduction in parvalbumin-positive (PV+) neurons in the hippocampus of the VPA group. Such a reduction may disrupt inhibitory control and neural circuit balance, potentially leading to behavioral and cognitive deficits(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Notably, KV treatment (100 mg/kg) significantly increased the number of PV\u0026thinsp;+\u0026thinsp;neurons in the hippocampus a 98% increase highlighting Kolaviron's neuroprotective effect in counteracting VPA-induced neuronal loss. Since PV\u0026thinsp;+\u0026thinsp;neurons play a crucial role in regulating excitatory-inhibitory balance in the hippocampus, their restoration may indicate improved regional function and potentially amelioration of ASD-related behavioral and cognitive symptoms. Previous studies have reported elevated blood serotonin levels in the valproic acid (VPA)-induced autism-like model, suggesting a possible association between increased serotonin and the pathophysiology of autism(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Consequently, growing attention has been directed toward serotonin due to its critical role in the nervous system and the inconsistent changes observed in different neural circuits and regions(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Current investigations have targeted various aspects of the serotonergic system, including serotonin itself, its transporters, receptors, and precursors. In rats, serotonergic neurons begin to develop between embryonic days 10.5 and 13, after which they migrate to different brain regions and initiate serotonin synthesis(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Given the known role of serotonin in mood regulation and its strong implication in autism spectrum disorders, and considering the overlap between the timing of VPA injection in our model and key developmental windows for serotonergic neurons, we focused on exploring this system. In the case of the serotonin transporter (5HTT), a significant increase in expression was observed in the VPA group compared to the control group in the hippocampus, suggesting altered reuptake mechanisms in this region. Interestingly, the group treated with 100 mg/kg kolaviron showed a significant reduction in 5HTT levels specifically, a 40.37% decrease in the hippocampus. These findings suggest that kolaviron treatment may modulate serotonin transporter expression and potentially restore serotonergic balance. A similar pattern was observed in the oxytocin-treated group, which exhibited a significant increase in hippocampal 5HTT levels (48.66%), possibly indicating a compensatory or restorative serotonergic mechanism. Regarding the 5HTR7 receptor, no significant differences were observed across all groups in the evaluated brain regions. Although prior research suggests that 5HTR7 may play a role in behavioral disorders and neuroplasticity(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), our data did not support a direct involvement of this receptor in the observed changes within the VPA model. This lack of significant findings could be attributed to timing of measurement or the complex functions of this receptor in the VPA model. Collectively, these results suggest that kolaviron may have a beneficial modulatory effect on the serotonergic system, particularly by reducing 5HTT levels, which may contribute to behavioral improvements observed in the VPA-induced model of autism. However, further studies are necessary to elucidate the precise mechanisms through which kolaviron affects the serotonergic pathway and its potential therapeutic implications in neurodevelopmental disorders like autism. In the Self-Grooming test, an indicator of repetitive behavior, the kolaviron-treated groups (50 and 100 mg/kg) exhibited a significant reduction in stereotypic behaviors compared to the VPA model group. This improvement may be attributed to Kolaviron's antioxidant properties and reduction in oxidative stress, potentially restoring neuronal function in brain areas responsible for repetitive behaviors, such as the hippocampus. In the Three-Chamber test, which specifically assesses social interaction, kolaviron-particularly at 100 mg/kg-led to significant improvement in social engagement. These effects mirrored those observed with oxytocin treatment and highlight Kolaviron's potential in ameliorating VPA-induced social deficits, possibly due to its anti-inflammatory properties and improved neural function in social-related brain regions.In the Marble Burying test, which is used to assess anxiety and repetitive behaviors, treatment with 100 mg/kg of kolaviron significantly reduced both anxiety and stereotypic behaviors. These outcomes suggest that kolaviron may be effective in alleviating anxiety symptoms, possibly through its impact on brain oxidative and inflammatory stress pathways. However, no comparable effects were observed at the 50 mg/kg dose, indicating that this lower dose may be insufficient to influence these specific behavioral outcomes. Finally, in the Open Field test, which evaluates both social behavior and anxiety, kolaviron significantly increased the time spent in the social zone, further supporting its potential in improving social engagement and reducing anxiety-related behaviors. This aligns with results from other behavioral assays, reinforcing Kolaviron's positive impact on anxiety reduction and social behavior enhancement. Overall, these findings suggest that kolaviron, especially at a 100 mg/kg dose, can effectively ameliorate repetitive behaviors, anxiety, and social deficits in the VPA-induced autism model. The lack of effects at lower doses underscores the importance of determining an optimal therapeutic dose for behavioral intervention.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eKolaviron demonstrated neuroprotective effects in the valproic acid (VPA)-induced rodent model of autism, primarily through its antioxidant and anti-inflammatory actions. By reducing oxidative damage, supporting endogenous defenses, and preserving mitochondrial function, kolaviron helped restore hippocampal neuronal integrity. These biochemical improvements were associated with reduced repetitive behaviors, lower anxiety, and enhanced social interaction. The dose-dependent nature of these effects highlights the importance of optimizing therapeutic strategies. While promising, further research is needed to fully understand Kolaviron's mechanisms and therapeutic potential in autism spectrum disorders.\u003c/p\u003e \u003cp\u003e \u003cb\u003eScope and Limitations\u003c/b\u003e \u003c/p\u003e \u003cp\u003eClearly, conducting a comprehensive research study is challenging, and the limitations inherent in any research such as practical, temporal, and financial constraints are evident. In this particular study, the vastness and complexity of the changes observed in Autism Spectrum Disorder (ASD) required that assessments be conducted across various models. Additionally, further examination of the serotonergic system through diverse brain regions could provide more precise and transparent insights into this field. The wide range of characteristics and features of autism can seem overwhelming, and it might be beneficial to categorize the disorder into more specific subgroups, allowing for more focused and in-depth studies.\u003c/p\u003e"},{"header":"Abbreviation","content":"\u003cp\u003eAutism Spectrum Disorder (ASD)\u003c/p\u003e\n\u003cp\u003eValproic acid (VPA)\u003c/p\u003e\n\u003cp\u003eKolaviron (KV)\u003c/p\u003e\n\u003cp\u003eIntraperitoneal (IP)\u003c/p\u003e\n\u003cp\u003ePostnatal day (PND)\u003c/p\u003e\n\u003cp\u003eOxytocin (OT)\u003c/p\u003e\n\u003cp\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/p\u003e\n\u003cp\u003e5‑Hydroxytryptamine Transporter (5-HTT)\u003c/p\u003e\n\u003cp\u003eSerotonin Transporter (SERT)\u003c/p\u003e\n\u003cp\u003e5‑Hydroxytryptamine Receptor\u0026nbsp;7 (5‑HTR7)\u003c/p\u003e\n\u003cp\u003eTumor Necrosis Factor alpha(TNF‑α)\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Interleukin‑6 (IL‑6)\u003c/p\u003e\n\u003cp\u003eParvalbumin interneurons (PV interneurons)\u003c/p\u003e\n\u003cp\u003e2,2‑Diphenyl‑1‑picrylhydrazyl (DPPH)\u003c/p\u003e\n\u003cp\u003eTetramethylbenzidine (TMB)\u003c/p\u003e\n\u003cp\u003eMalondialdehyde (MDA)\u003c/p\u003e\n\u003cp\u003eCatalase activity (CAT)\u003c/p\u003e\n\u003cp\u003eNitrite levels (NIT)\u003c/p\u003e\n\u003cp\u003eMitochondrial Membrane Potential (MMP)\u003c/p\u003e\n\u003cp\u003eDimethyl sulfoxide (DMSO)\u003c/p\u003e\n\u003cp\u003ephosphate-buffered saline (PBS)\u003c/p\u003e\n\u003cp\u003eNormal goat serum (NGS)\u003c/p\u003e\n\u003cp\u003e3,3′-diaminobenzidine (DAB)\u003c/p\u003e\n\u003cp\u003eReactive oxygen species (ROS)\u003c/p\u003e\n\u003cp\u003eReactive nitrogen species (RNS)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eAll original contributions of this study are included in the main article and its supplementary materials. Further information can be obtained by contacting the corresponding author.\u003c/p\u003e\n\u003cp\u003eEthics statement\u003c/p\u003e\n\u003cp\u003eThis animal experiment was reviewed and approved by the Ethics Committee of Iran University of Medical Sciences (IR.IUMS.FMD.REC.1401.210). All procedures were carried out in compliance with institutional guidelines and relevant national regulations.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eVK: Conducted formal analysis, investigation, developed methodology, prepared the original draft, and participated in reviewing and editing the manuscript. TB: Performed formal analysis, secured funding, supervised the project, contributed to the original draft, and reviewed and edited the manuscript. MF: Contributed to methodology development and participated in manuscript review and editing. SM: Involved in methodology design and manuscript review and editing. AKK: Assisted with methodology and took part in reviewing and editing the manuscript. MR: Carried out formal analysis, contributed to methodology, supervised the study, drafted the original manuscript, and engaged in review and editing.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe author(s) declare that financial support was received for the research and/or publication of this article. This study is part of a PhD thesis project that was approved and funded by Iran University of Medical Sciences, Tehran, Iran (Grant no. 1401-3-104-23915).\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors state that there were no commercial or financial ties that might be perceived as a potential conflict of interest during the conduct of this research.\u003c/p\u003e\n\u003cp\u003eClinical trial number\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMehra S, Ul Ahsan A, Seth E, Chopra M. Critical Evaluation of Valproic Acid-Induced Rodent Models of Autism: Current and Future Perspectives. J Mol Neurosci. 2022;72(6):1259-73.\u003c/li\u003e\n \u003cli\u003eGouda B, Sinha SN, Sangaraju R, Huynh T, Patangay S, Venkata Mullapudi S, et al. Extraction, Phytochemical profile, and neuroprotective activity of Phyllanthus emblica fruit extract against sodium valproate-induced postnatal autism in BALB/c mice. Heliyon. 2024;10(15):e34992.\u003c/li\u003e\n \u003cli\u003eAfshari M, Gharibzadeh S, Pouretemad H, Roghani M. Promising therapeutic effects of high-frequency repetitive transcranial magnetic stimulation (HF-rTMS) in addressing autism spectrum disorder induced by valproic acid. Front Neurosci. 2024;18:1385488.\u003c/li\u003e\n \u003cli\u003eUkezono M, Kasahara Y, Yoshida C, Murakami Y, Okada T, Takano Y. Impairments of social interaction in a valproic acid model in mice. Front Behav Neurosci. 2024;18:1430267.\u003c/li\u003e\n \u003cli\u003eAl-Dossari AM, Al-Harbi LN, Al-Otaibi NM, Almubarak A, Almnaizel AT, Alshammari GM, et al. The Potency of Goat Milk in Reducing the Induced Neurotoxic Effects of Valproic Acid in Rat Pups as a Rodent Model of Autism Spectrum Disorder. Metabolites. 2023;13(4).\u003c/li\u003e\n \u003cli\u003eKuo HY, Liu FC. Pathophysiological Studies of Monoaminergic Neurotransmission Systems in Valproic Acid-Induced Model of Autism Spectrum Disorder. Biomedicines. 2022;10(3).\u003c/li\u003e\n \u003cli\u003eFarrag EAE, Askar MH, Abdallah Z, Mahmoud SM, Abdulhai EA, Abdelrazik E, et al. Comparative effect of atorvastatin and risperidone on modulation of TLR4/NF-kappaB/NOX-2 in a rat model of valproic acid-induced autism. Behav Brain Funct. 2024;20(1):26.\u003c/li\u003e\n \u003cli\u003eNicolini C, Fahnestock M. The valproic acid-induced rodent model of autism. Exp Neurol. 2018;299(Pt A):217-27.\u003c/li\u003e\n \u003cli\u003eAttah CO, Alhaji UI, Ameh DA, Forcados GE, Muhammad A, Bashir M, Ibrahim S. In Vivo Chemosuppressive Effects of Kolaviron on 7,12-Dimethylbenzanthracene-Induced Mammary Lesions are Associated with Changes in Levels of Estrogen Receptor-\u0026alpha;, CYP 1A1, Proinflammatory Cytokines, and Alterations to Metabolic Pathways Implicated in Mammary Carcinogenesis. J Med Food. 2024;27(10):940-50.\u003c/li\u003e\n \u003cli\u003eOlatoye FJ, Akindele AJ, Awodele O. The role of Kolaviron, a bioflavonoid from Garcinia kola, in the management of cardiovascular diseases: A systematic review. Heliyon. 2024;10(5):e27333.\u003c/li\u003e\n \u003cli\u003eNazari-Serenjeh M, Baluchnejadmojarad T, Hatami-Morassa M, Fahanik-Babaei J, Mehrabi S, Tashakori-Miyanroudi M, et al. Kolaviron neuroprotective effect against okadaic acid-provoked cognitive impairment. Heliyon. 2024;10(3):e25564.\u003c/li\u003e\n \u003cli\u003eAbarikwu SO, Ezim OE, Ikeji CN, Farombi EO. Atrazine: cytotoxicity, oxidative stress, apoptosis, testicular effects and chemopreventive Interventions. Frontiers in Toxicology. 2023;5.\u003c/li\u003e\n \u003cli\u003eOkafor AI, Ogban NN, Odinigwe AA. Kolaviron alleviates haematological abnormalities and hepato-renal damage in Naja nigricollis nigricollis venom-treated rats. Toxicol Rep. 2022;9:1869-76.\u003c/li\u003e\n \u003cli\u003eTauchen J, Frankova A, Manourova A, Valterova I, Lojka B, Leuner O. Garcinia kola: a critical review on chemistry and pharmacology of an important West African medicinal plant. Phytochem Rev. 2023:1-47.\u003c/li\u003e\n \u003cli\u003eOyovwi MO, Ben-Azu B, Edesiri TP, Victor E, Rotu RA, Ozegbe QE, et al. Kolaviron abates busulfan-induced episodic memory deficit and testicular dysfunction in rats: the implications for neuroendopathobiological changes during chemotherapy. Biomedicine \u0026amp; Pharmacotherapy. 2021;142:112022.\u003c/li\u003e\n \u003cli\u003eOmotoso GO, Arietarhire LO, Ukwubile II, Gbadamosi IT. The protective effect of kolaviron on molecular, cellular, and behavioral characterization of cerebellum in the rat model of demyelinating diseases. Basic and Clinical Neuroscience. 2020;11(5):609.\u003c/li\u003e\n \u003cli\u003eOmotoso GO, Mutholib NY, Abdulsalam FA, Bature AI. Kolaviron protects against cognitive deficits and cortico-hippocampal perturbations associated with maternal deprivation in rats. Anatomy \u0026amp; Cell Biology. 2019;53(1):95.\u003c/li\u003e\n \u003cli\u003ePizzella A, Penna E, Liu Y, Abate N, Lacivita E, Leopoldo M, et al. Alterations of synaptic plasticity in Angelman syndrome model mice are rescued by 5-HT7R stimulation. Progress in Neurobiology. 2024;242:102684.\u003c/li\u003e\n \u003cli\u003eEmmanuel O, Uche ME, Dike ED, Etumnu LR, Ugbogu OC, Ugbogu EA. A review on garcinia kola heckel: traditional uses, phytochemistry, pharmacological activities, and toxicology. Biomarkers. 2022;27(2):101-17.\u003c/li\u003e\n \u003cli\u003eOR A, ME C, NL B, OO O. Kolaviron, a biflavonoid complex of Garcinia kola seeds modulates apoptosis by suppressing oxidative stress and inflammation in diabetes-induced nephrotoxic rats. Phytomedicine. 2014;21(14):1785\u0026ndash;93.\u003c/li\u003e\n \u003cli\u003eAfshari M, Gharibzadeh S, Pouretemad H, Roghani M. Reversing valproic acid-induced autism-like behaviors through a combination of low-frequency repeated transcranial magnetic stimulation and superparamagnetic iron oxide nanoparticles. Sci Rep. 2024;14(1):8082.\u003c/li\u003e\n \u003cli\u003eZahedi E, Sadr SS, Sanaeierad A, Roghani M. Chronic acetyl-L-carnitine treatment alleviates behavioral deficits and neuroinflammation through enhancing microbiota derived-SCFA in valproate model of autism. Biomed Pharmacother. 2023;163:114848.\u003c/li\u003e\n \u003cli\u003eFeriyani F, Maulanza H, Lubis RR, Balqis U, Darmawi D. Effects of Binahong (Anredera cordifolia (Tenore) Steenis) Extracts on the Levels of Malondialdehyde (MDA) in Cataract Goat Lenses. ScientificWorldJournal. 2021;2021:6617292.\u003c/li\u003e\n \u003cli\u003eGhalami J, Baluchnejad Mojarad T, Mansouri M, Khamse S, Roghani M. Paeonol Protection Against Intrastriatal 6-Hydroxydopamine Rat Model of Parkinson\u0026apos;s Disease. Basic Clin Neurosci. 2021;12(1):43-56.\u003c/li\u003e\n \u003cli\u003eTeixeira FC, Gutierres JM, Soares MSP, da Siveira de Mattos B, Spohr L, do Couto CAT, et al. Inosine protects against impairment of memory induced by experimental model of Alzheimer disease: a nucleoside with multitarget brain actions. Psychopharmacology (Berl). 2020;237(3):811-23.\u003c/li\u003e\n \u003cli\u003eDing J, Yu HL, Ma WW, Xi YD, Zhao X, Yuan LH, et al. Soy isoflavone attenuates brain mitochondrial oxidative stress induced by \u0026beta;-amyloid peptides 1-42 injection in lateral cerebral ventricle. J Neurosci Res. 2013;91(4):562-7.\u003c/li\u003e\n \u003cli\u003ePourmohammadi S, Roghani M, Kiasalari Z, Khalili M. Paeonol Ameliorates Cuprizone-Induced Hippocampal Demyelination and Cognitive Deficits through Inhibition of Oxidative and Inflammatory Events. J Mol Neurosci. 2022;72(4):748-58.\u003c/li\u003e\n \u003cli\u003eOmotoso GO, Mutholib NY, Abdulsalam FA, Bature AI. Kolaviron protects against cognitive deficits and cortico-hippocampal perturbations associated with maternal deprivation in rats. Anat Cell Biol. 2020;53(1):95-106.\u003c/li\u003e\n \u003cli\u003eChen S, Huang L, Liu G, Kang J, Qian Q, Wang J, et al. Acupuncture Ameliorated Behavioral Abnormalities in the Autism Rat Model via Pathways for Hippocampal Serotonin. Neuropsychiatr Dis Treat. 2023;19:951-72.\u003c/li\u003e\n \u003cli\u003eOmotoso GO, Arietarhire LO, Ukwubile, II, Gbadamosi IT. The Protective Effect of Kolaviron on Molecular, Cellular, and Behavioral Characterization of Cerebellum in the Rat Model of Demyelinating Diseases. Basic Clin Neurosci. 2020;11(5):609-18.\u003c/li\u003e\n \u003cli\u003eRahdar M, Davoudi S, Dehghan S, Javan M, Hosseinmardi N, Behzadi G, Janahmadi M. Reversal of electrophysiological and behavioral deficits mediated by 5-HT7 receptor upregulation following LP-211 treatment in an autistic-like rat model induced by prenatal valproic acid exposure. Neuropharmacology. 2024;257:110057.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Autism Spectrum Disorder (ASD), Valproic Acid (VPA), Kolaviron (KV), Serotonin, Hippocampus, Oxytocin","lastPublishedDoi":"10.21203/rs.3.rs-6783521/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6783521/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\n\u003cp\u003eAutism Spectrum Disorder (ASD) is a lifelong neurodevelopmental condition marked by impairments in social communication, language and behavior. Both genetic and environmental factors, including prenatal exposure to valproic acid (VPA), contribute to its pathogenesis. VPA exposure during critical periods of neurodevelopment induces oxidative stress, inflammation, and serotonergic dysregulation. Kolaviron (KV), a polyphenolic extract from \u003cem\u003eGarcinia kola\u003c/em\u003e, exhibits potent antioxidant and anti-inflammatory properties, potentially offering Neuroprotection in ASD models\u003cem\u003e. The aim of our study was to evaluate whether Kolaviron could improve the VPA-induced autism model in the areas of \u003c/em\u003eMitochondrial dysregulation \u003cem\u003eoxidative stress, inflammation, and behavior, and to compare its effects with oxytocin on the serotonergic system.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMethods\u003c/p\u003e\n\u003cp\u003ePregnant Wistar rats received a single intraperitoneal dose of VPA (600 mg/kg) on gestational day 12.5 to induce autism-like features in offspring. Male pups were weaned on postnatal day (PND) 21 and randomly assigned to receive KV (50 or 100 mg/kg, oral), oxytocin (12 µg/kg, intranasal), or saline until PND 49. Behavioral tests were conducted on PNDs 42–49. Brain tissues were collected for ELISA analysis of hippocampal 5-HTT, 5-HTR7, TNF Alfa and IL-6 levels, Immunohistochemical staining for parvalbumin was performed to assess interneuron integrity.\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003eKV-treated VPA-exposed rats showed significant improvements in social interaction, reduced repetitive behavior, and attenuated. Biochemically, KV decreased IL-6 levels and modulated serotonergic markers (5-HTT, 5-HTR7). Histologically, KV preserved hippocampal architecture and parvalbumin-positive interneurons, suggesting Neuroprotection.\u003c/p\u003e\n\u003cp\u003eConclusions\u003c/p\u003e\n\u003cp\u003eThese effects were dose-dependent, indicating KV's potential as a complementary therapeutic agent in ASD. Further studies are warranted to clarify its mechanisms and clinical relevance.\u003c/p\u003e","manuscriptTitle":"Promising Therapeutic Efficacy of Kolaviron against Prenatal Valproate-Induced Autism Spectrum Disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-27 14:25:00","doi":"10.21203/rs.3.rs-6783521/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7f0bda80-722c-4a21-b6b0-85098b8ddaef","owner":[],"postedDate":"June 27th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-22T11:11:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-27 14:25:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6783521","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6783521","identity":"rs-6783521","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}