**Background:** Epilepsy is a chronic CNS disorder characterized by recurrent seizures due to an imbalance between excitation and inhibition. Over 30% of patients are drug-resistant, and current antiseizure medications lack disease-modifying properties. The gut microbiota has emerged as a potential modulator of epilepsy, with studies showing differences in microbiota composition between epileptic patients and healthy controls, and between drug-resistant and drug-sensitive patients. Probiotics, including multi-strain formulations, have shown promise in reducing seizure frequency and severity in both animal models and human studies. Lactobacillus helveticus R0052 is a well-characterized probiotic strain with anti-inflammatory and psychotropic effects, but its effect on seizure susceptibility and antiseizure drug pharmacokinetics had not been studied. This study aimed to evaluate the effect of 28-day supplementation with L. helveticus R0052 on seizure thresholds, antiseizure activity of sodium valproate, anxiety-like behavior, motor function, and cecal short-chain fatty acid (SCFA) levels in mice.
**Methods:** Male albino Swiss mice (n=210) were randomly assigned to receive either 2 × 10^9 cfu of L. helveticus R0052 in 200 μl PBS or PBS alone by oral gavage daily for 28 days. Seizure thresholds were assessed on day 28 using three acute seizure tests: maximal electroshock (MES) test (tonic hindlimb extension), 6 Hz psychomotor seizure test (CC50 values), and intravenous pentylenetetrazol (ivPTZ) test (thresholds for myoclonic twitch, generalized clonus, and tonic forelimb extension). The antiseizure potency of sodium valproate was evaluated in the subcutaneous PTZ (scPTZ) test by determining ED50 values. Serum and brain valproate concentrations were measured by HPLC 30 min after oral valproate (380 mg/kg). Behavioral tests (elevated plus maze, light/dark box, locomotor activity, accelerating rotarod, grip strength) were performed on day 27. Cecal SCFAs (acetate, propionate, butyrate, isobutyrate) and lactate were quantified by HPLC before and after treatment. Statistical analyses included Student's t-test, Mann-Whitney test, and Wilcoxon matched-pairs signed rank test, with significance set at p<0.05.
**Key Results:** L. helveticus R0052 significantly increased the threshold for 6 Hz-induced psychomotor seizures, raising the CC50 value by ~60% (t=8.225, df=16, p<0.0001). In the ivPTZ test, the probiotic slightly but significantly increased the threshold for the first myoclonic twitch by ~13% (U=57.5, p=0.021) and for generalized clonus by ~11% (U=62, p=0.036), but did not affect the threshold for tonic forelimb extension (U=98, p=0.772). No effect was observed on the MES threshold (t=0.395, df=17, p=0.698). The antiseizure potency of valproate in the scPTZ test was unchanged (ED50: t=0.479, df=38, p=0.635). However, L. helveticus R0052 significantly increased serum valproate concentration by ~30% (U=18, p=0.015), while brain valproate concentration was not significantly altered (U=33, p=0.218). No significant effects were found on anxiety-like behavior (elevated plus maze: % open arm entries U=63, p=0.630; % time in open arms U=70, p=0.932; light/dark box: entries U=54, p=0.475; time in light U=70, p=0.932; latency to dark U=56, p=0.369), locomotor activity (U=68, p=0.843), neuromuscular strength (U=44, p=0.110), or motor coordination (rotarod latency: first trial U=57, p=0.410; second U=65, p=0.713; third U=54, p=0.319). Cecal SCFA analysis showed that L. helveticus R0052 significantly increased total SCFAs (U=16, p=0.009), acetate (U=19, p=0.019), and butyrate (U=20.5, p=0.025) compared to controls, while propionate (U=36, p=0.306) and lactate (U=49, p=0.971) were unchanged. Pairwise before-after comparisons within the probiotic group showed no significant changes, but in PBS-treated mice, lactate increased (p=0.020) and propionate decreased (p=0.047).
**Clinical Implications:** This study provides evidence that a single-strain probiotic, L. helveticus R0052, can reduce seizure susceptibility in mice, particularly for psychomotor seizures (a model of human partial seizures) and, to a lesser extent, myoclonic and clonic seizures. The effect may be mediated by increased production of SCFAs (acetate and butyrate), which are known to modulate neuroinflammation, oxidative stress, and neurotransmission. The lack of effect on maximal electroshock seizures suggests specificity for certain seizure types. Importantly, the probiotic did not interfere with the antiseizure efficacy of valproate but increased its serum concentration, indicating potential pharmacokinetic interactions that could affect drug bioavailability. This finding warrants further investigation into the mechanisms (e.g., modulation of intestinal β-glucuronidase activity) and clinical relevance, especially for patients on valproate therapy. The absence of anxiolytic or motor effects suggests that L. helveticus R0052 is behaviorally neutral in healthy mice, which is favorable for a potential adjunctive therapy. Overall, these results support the gut-brain axis as a therapeutic target in epilepsy and highlight the need for human studies to confirm the efficacy and safety of probiotic supplementation in epilepsy management.