The influence of Yishen Ningxin formula on the behavioral and neurotransmitter changes in a zebrafish model of generalized anxiety

This study evaluated the anxiolytic effects of Yishen Ningxin formula (YSNX) in caffeine-induced zebrafish models and explored underlying mechanisms. Using 7-day post-fertilization AB zebrafish, anxiety-like models were established with 100μmol/L caffeine exposure. Groups included normal control, caffeine-only, and YSNX-treated cohorts (200/550/1600μg/mL). Behavioral assessments included thigmotaxis, vertical preference, and locomotor activity using automated tracking, with GABA/5-HT levels measured by ELISA. Compared with normal controls, caffeine-exposed zebrafish showed increased total movement time (P<0.05), movement distance (P<0.05), and large movement distance (P<0.05), accompanied by reduced central zone time (-28%, P<0.05) and distance (-31%, P<0.05), with significantly decreased bottom-dwelling time (P<0.05). YSNX administration (1600μg/mL and 550μg/mL) reversed these effects, significantly decreasing movement distance/time (vs caffeine group, P<0.05) while increasing central zone exploration (P<0.05). The high-dose group restored bottom-dwelling time to control levels (P<0.05). Neurochemical analysis revealed caffeine-induced GABA reduction (vs normal group: P<0.05) and 5-HT elevation (P<0.05) in caffeine, YSNX-L, and YSNX-M groups. YSNX-H treatment increased GABA (P<0.05 vs caffeine group) and decreased 5-HT (P<0.05), while YSNX-M specifically reduced 5-HT (P<0.05). These results demonstrate YSNX's ability to ameliorate anxiety-like behaviors in zebrafish, potentially through GABA/5-HT pathway modulation. This study provides experimental evidence supporting YSNX's therapeutic potential for anxiety disorders.