Abstract

Long-term antibiotics treatment can induce anxiety-like behavior in mice. • Colonic Trp metabolism is the key mechanism in mice of anxiety-like behavior. • Mice exhibiting anxiety-like behavior show inflammation in both the gut and the brain. The widespread and often excessive use of antibiotics has raised concerns about its long-term impact on host health. Growing evidence suggests that antibiotics-induced gut microbiota dysbiosis may contribute to neuropsychiatric conditions, including anxiety. However, the mechanistic pathways linking chronic antibiotic exposure, microbial disruption, and anxiety-like behaviors remain largely unclear. To investigate the effects of antibiotic exposure on gut-brain axis function, mice were administered antibiotic-containing drinking water for either short-term or long-term durations. Behavioral assessments, 16S rRNA gene sequencing, biochemical analyses, histological staining, and Western blotting were used to evaluate anxiety-like behaviors, microbiota composition, tryptophan (Trp) metabolism, intestinal barrier integrity, and inflammation in both the colon and hippocampus. Long-term, but not short-term, antibiotic treatment induced pronounced anxiety-like behaviors in mice. Microbiota analysis revealed that long-term antibiotic exposure significantly reduced microbial diversity and altered the abundance of key bacterial genera. These changes were associated with disrupted colonic Trp metabolism, reflected by decreased Trp and 5-HT levels in the colon and serum, along with suppressed expression of the Trp metabolic enzyme TPH1 and 5-HT4R. Additionally, long-term antibiotic treatment impaired intestinal barrier integrity, downregulated tight junction proteins and MUC2, and activated colonic TLR4/NF-κB/NLRP3 inflammatory signaling pathways. Neuroinflammation was also observed in the hippocampus. Our results reveal a time-dependent effect of antibiotic-induced anxiety-like behaviors and suggest that gut microbiota dysbiosis, disrup