Circadian disruption promotes epileptic seizures via the Ruminococcus gnavus-arginine-NOX4-ferroptosis axis

Circadian rhythm disruption (CRD) exacerbates epileptic seizures, yet the underlying molecular mechanisms remain unclear. Using a pentylenetetrazol (PTZ) kindling rat model combined with continuous light exposure, we found that CRD significantly shortened seizure latency and increased seizure frequency. 16S rRNA sequencing revealed that CRD substantially increased the abundance of Ruminococcus gnavus (R. gnavus) in the gut microbiota. Antibiotic-mediated microbiota depletion, fecal microbiota transplantation (FMT), and R. gnavus monocolonization experiments confirmed that R. gnavus enrichment is a critical factor driving seizure aggravation. Metabolomic analysis demonstrated that elevated R. gnavus suppressed the expression of argininosuccinate synthase 1 (ASS1), a key enzyme for arginine biosynthesis in the kidney, leading to impaired L-citrulline-to-L-arginine conversion and consequently decreased L-arginine levels in serum and hippocampal tissues. Arginine deficiency subsequently activated NADPH oxidase 4 (NOX4) upregulation in the hippocampus, triggering enhanced oxidative stress (elevated malondialdehyde (MDA) and reduced superoxide dismutase (SOD) activity), which ultimately induced ferroptosis (characterized by mitochondrial cristae reduction, acyl-CoA synthetase long-chain family member 4 (ACSL4) upregulation, and glutathione peroxidase 4 (GPX4) downregulation). Pharmacological intervention with the NOX4-specific inhibitor GLX351322 ameliorated oxidative stress, suppressed ferroptosis, and alleviated seizure severity. Importantly, L-arginine supplementation significantly prolonged seizure latency and reduced seizure frequency by reversing ferroptosis activation through downregulating NOX4-ACSL4 expression and upregulating GPX4 expression. This study unveils a complete signaling axis whereby CRD promotes epileptic seizures through the R. gnavus-ASS1-arginine-NOX4-ferroptosis cascade, providing novel therapeutic targets for clinical intervention.