Jiannao pills mitigate chronic restraint stress-induced anxiety in mice through the regulation of intestinal microflora.

Objective: To investigate the effects of Jiannao pills on mice with chronic restraint stress-induced anxiety and its mechanisms.

Methods: Anxiety-like behaviors were induced in mice by exposing them to chronic restraint stress (8 h/day for 21 days). Subsequently, Jiannao pills were given to these mice for the treatment of the induced anxiety. Following treatment, the intestinal microflora of the mice were analyzed using the 16S rRNA sequencing method.

Results: Under positive electrospray ionization mode, a total of 68 chemical compositions were found in Jiannao pills, and under negative electrospray ionization mode, the number was 18. With these chemical compositions in effect, it was observed that Jiannao pills alleviated chronic restraint induced anxiety-like behaviors in mice by extending their dwelling time, standing time, and grooming time in the central area, as well as the percentage of entries and time spent in the open arms. This effect was similar to that of alprazolam. In addition, Jiannao pills significantly improved neural functions in mice with chronic restraint-induced anxiety, diminished the levels of 5-hydroxytryptamine and glutamate, and increased the levels of γ-aminobutyric acid. Furthermore, Jiannao pills decreased the expressions of the corticotropin-releasing factor and cholecystokinin protein, while elevating neuropeptide Y protein levels. The results of 16S rRNA sequencing analysis revealed both Jiannao pills and alprazolam altered the composition of intestinal microflora in mice, with Jiannao pills exhibiting a more pronounced effect. Specifically, there was a significant increase in the abundance of S24-7 in mice following treatment. Besides, significant differences were observed in a total of 632 operational taxonomic units in mice after Jiannao pill treatment. The functions of the intestinal microflora of mice were primarily associated with their betalain biosynthesis and classification levels.

Conclusion: Jiannao pills effectively ameliorated chronic restraint anxiety-like behaviors in mice and enhanced their neural functions potentially through the regulation of their intestinal microflora.