Discussion

The current findings demonstrate that both VPA and LPS prenatal exposures induce significant behavioral alterations characteristic of ASD, including reduced social interaction and increased repetitive behaviors (grooming) as well as anxiety-like behavior. Interventions with Artichoke (as a prebiotic), alone or in combination with probiotics and/or omega-3, consistently ameliorated these behavioral phenotypes.

Starting with social interaction duration and frequency (Figure 2A, B, C), the VPA-exposed group (VPA-0) showed a marked impairment in social interaction and shows a noticeable drop in the mean time spent in the social chamber, confirming the toxic effect of in utero VPA exposure, Similarly, Figure 3 (A, C, E) reveal a significant increase in locomotion activity, immobility and repetitive grooming behaviors, respectively, further demonstrating behavioral alterations relevant to ASD.

These results are in line with previous reports [7], 8], 35], 44], 45], which have consistently shown that prenatal exposure to VPA produces behavioral deficits resembling those observed in individuals with ASD and animal models, including pronounced social impairments and stereotyped behaviors.

Several mechanisms have been proposed to explain the antiseizure effects of VPA, including the enhancement of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in the brain, and the modulation of sodium and calcium ion channel influx and efflux. In addition, VPA acts as a histone deacetylase (HDAC) inhibitor, thereby inducing epigenetic modifications that alter cell proliferation and differentiation in developing tissues, including the fetal nervous system [46], 47]. Importantly, VPA can cross the placental barrier and enter fetal circulation, with fetal concentrations typically ranging from 70 to 100 % of maternal levels. Moreover, VPA is excreted into breast milk at concentrations comparable to plasma levels [8], 48], raising concerns about its teratogenic potential and developmental risks.

Additionally, offspring from mothers exposed to maternal immune activation (MIA) induced by LPS (LPS-0) showed significantly reduced social interaction duration and frequency (Figure 2 D, E), indicating that maternal infection during early gestation could be a risk factor for ASD. While chamber occupancy reflects spatial preference for social proximity, direct investigation measures provide a more specific index of active social engagement with the conspecific [49]. The percentage of time spent in the social chamber relative to the total test duration did not reveal statistically significant differences between groups, although a non-significant trend toward reduced social preference was observed in the VPA-exposed animals (Figure 2C). This apparent discrepancy between normalized chamber occupancy and direct social interaction measures may be explained by differences in general activity levels and exploratory behavior. Indeed, VPA and LPS exposed animals exhibited altered locomotor activity [50], [51], [52].

Likewise, Figure 3 (B, D, F) show hypo locomotion activity, increased immobility and repetitive grooming behaviors, further demonstrating ASD-relevant phenotypes. These findings are consistent with previous studies [13], 14], 20], 38] which report that prenatal LPS-induced MIA initiates neuroinflammatory processes in the developing fetal brain. Elevated levels of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-17) during gestation are strongly associated with behavioral impairments (social deficits, repetitive behaviors, and cognitive inflexibility in offspring) and hypomyelination, thereby providing a mechanistic link between maternal infection, neuroinflammation, and ASD-like outcomes.

Embryonic exposure to VPA or LPS offspring exhibited an increased immobility time compared to the control as mentioned in Figure 3 C, D, which might indicate an increased anxiety-like behavior, which is a behavioral feature of ASD models [53], 54]. However, validated tests of anxiety need to be performed in future studies to better assess anxiety-like behavior in this model of autism.

The therapeutic efficacy of Cynara scolymus (Artichoke) has been recognized since antiquity and is primarily attributed to its rich content of bioactive compounds, particularly polyphenols (e.g., caffeoylquinic acid derivatives such as chlorogenic acid, cynarin, neochlorogenic acid, and cryptochlorogenic acid) and flavonoids (e.g., luteolin, apigenin, kaempferol) [27], 55], 56]. Additionally, Artichoke is considered a prebiotic due to its high inulin content. Inulin, a soluble dietary fiber, undergoes fermentation by gut microbiota, promoting the growth of beneficial bacteria and thereby supporting gut health [26], 27].

Behavioral analyses revealed that artichoke alone (VPA-AR) did not significantly improve VPA-induced deficits in social interaction duration and frequency (Figure 2 A, B), nor in repetitive grooming behavior frequency (Figure 3E). However, it did ameliorate immobility and locomotion activity (Figure 3A, C). In contrast, the combination of artichoke as a prebiotic with probiotics (VPA-AR.PRO) significantly ameliorated social interaction deficits and repetitive behaviors induced by VPA, as shown in Figures 2 (A, C) and 3 (A, C, E). These results suggest that Artichoke’s modulatory effects may be enhanced through synergistic interactions with probiotics.

Mechanistically, VPA exposure during pregnancy is known to induce profound alterations in the gut, including inflammation and microbiota dysbiosis, thereby disrupting the gut–brain axis and influencing ASD-related behaviors [57], [58], [59]. The observed rescue effects of Artichoke–probiotic combinations support the growing evidence for targeting the microbiota–gut–brain axis as a therapeutic strategy for ASD.

In 2024, Prince and colleagues provided strong evidence that a prebiotic diet consisting of 3 % galacto-oligosaccharide/fructo-oligosaccharide (GOS/FOS; 9:1) normalized both immune and behavioral deficits in a VPA-induced mouse model of ASD. In contrast, the current investigation found that Artichoke alone had no corrective benefits as a prebiotic in improving VPA-induced social interaction deficiencies. This disparity could be related to changes in dosage (400 mg/kg in the current investigation), duration of treatment (23 days beginning on postnatal day 7), or timing of intervention as compared to Prince et al. who began food supplementation at birth and continued for 49 days [60].

On the other hand, previous research has demonstrated that combined probiotic and prebiotic interventions can alleviate a broad range of autistic-like symptoms in prenatal VPA-induced rodent models. These therapeutic effects appear to be mediated not only by behavioral improvements but also by modulation of inflammatory responses (e.g., IL-6, IL-10), neurotransmitter systems (serotonin, GABA), and restoration of gut microbiota balance [33], 61], 62]. Collectively, these findings support the use of synbiotic interventions as a promising therapeutic strategy for subsets of individuals with autism, acting through immune regulation, neurotransmitter modulation, and microbiota–gut–brain axis restoration.

As highlighted in previous studies, ASD-like behaviors are strongly associated with gut dysbiosis, which can increase intestinal permeability and allow the translocation of LPS into the bloodstream. Circulating LPS can activate immune responses within the brain, thereby contributing to neuroinflammation and the behavioral manifestations of ASD [7], 15], 38], 63]. In the present study, offspring from MIA induced by LPS that received Artichoke as a prebiotic alone (LPS-AR) or in combination with probiotics and omega-3 fatty acids (LPS-AR.PRO.ω3) exhibited remarkable improvements in social interaction behavior (Figure 2D, E, F) as well as reductions in immobility and repetitive behaviors (Figure 3D, F) also significantly increase in activity (Figure 3B). These findings suggest that prebiotics, probiotics, and omega-3 fatty acids mitigate the detrimental effects of prenatal LPS exposure and inflammation by modulating the gut microbiota and immune responses.

Mechanistically, probiotics and omega-3s reduce gut permeability, thereby lowering systemic LPS translocation and subsequent neuroinflammation, while prebiotics promote the growth of beneficial bacteria that further suppress LPS production. Through these microbiota-mediated processes, such interventions influence brain function and alleviate ASD-related behaviors [19], 22], 24], 25], 64], 65]. The present findings are in line with this evidence, further supporting the gut–brain axis as a critical therapeutic target.

The corrective role of omega-3 supplementation in MIA offspring was specifically evaluated in this study to clarify its contribution as part of a combined intervention with prebiotics and probiotics. Leyrolle et al. reported that omega-3s, together with prebiotics and probiotics, can positively modulate the gut–brain axis and mitigate MIA as risk of ASD behavioral features in rat offspring [66]. Thus, synergistic effects have been reported when omega-3s are combined with prebiotics and probiotics, with greater efficacy than single interventions alone. Such combinations have been shown to reduce inflammation, improve gut barrier integrity, and enhance neurodevelopmental outcomes in animal models [65], 67], 68]. These findings correlate with the present results, in which the combined intervention significantly improved social behaviors in the LPS-induced model of ASD.

Because MIA during pregnancy is strongly associated with an increased risk of ASD, it is of particular interest to evaluate the potential protective role of Artichoke supplementation during gestation in preventing the development of ASD-like behaviors in offspring [12], 20]. In the present study, dams administered artichoke as a prebiotic from the first day of gestation (protective dose) and offspring received postnatal therapeutic doses. As shown in Figures 2 and 3, this protective intervention alleviated ASD-related behavioral abnormalities. These findings suggest that protection against VPA-induced toxicity during pregnancy is more readily achieved compared with LPS-induced MIA. This highlights the fact that MIA represents a stronger risk factor for ASD, as maternal protection during gestation is less effective against immune-mediated toxicity. Mechanically, while both VPA and LPS are powerful preclinical models of ASD, the direct teratogenic effect of VPA is more amenable to prevention than the complex, multifactorial immune-inflammatory processes triggered by LPS [8], [11], [12], [13].

In summary, both VPA and LPS are widely used to model ASD, through either direct chemical toxicity or indirect immune-mediated mechanisms, respectively. Although both models result in overlapping behavioral and neurobiological phenotypes, their mechanistic differences shape distinct therapeutic challenges. Artichoke supplementation as prebiotic alone shows promise in mitigating grooming and immobility behavioral deficits induced by both VPA and LPS. However, the addition of probiotics enhances the therapeutic efficacy of Artichoke in the VPA model, particularly in restoring social interaction and reducing repetitive behaviors. In contrast, in the LPS model, the combination of Artichoke, probiotics, and omega-3 fatty acids effectively reduced immobility and normalized grooming behaviors but did not enhance social interaction beyond the effect of Artichoke alone. This suggests a complex interaction between dietary interventions and the immune-mediated pathways underlying MIA, indicating that tailored strategies may be required depending on the etiology of ASD.