ObjectiveTo explore mice sleep improvement by Shehanshi water extract and its effect on calcium signaling pathways in brain tissues based on the “spleen-brain” correlation. MethodsThe components of Shehanshi water extract were analyzed via X-ray photoelectron spectroscopy, and the effects of low-dose (50 mg/kg) and high-dose (100 mg/kg) Shehanshi water extract in improving mice sleep were evaluated through such behavior tests as pentobarbital sodium subthreshold sleep test, suprathreshold sleep test, and autonomic activity test. Furthermore, the transcriptome sequencing and non-target metabonomics analysis were carried out on the spleen tissue and brain tissue of mice. Then, the concentration of metal ions in serum and brain tissue of mice was detected. Next, molecular docking was performed to analyze the binding of key pathway targets enriched in brain tissue to metallic elements, followed by relevant immunohistochemical experiments. ResultsShehanshi water extract contained elements like Na, Fe, Cd, Ca, and Al, and it could reduce the sleep latency, increase the sleep time, and elevate the sleep rate of mice. In the open field experiment, the movement distance of mice was shortened, and the central residence time and rest time were lengthened. Immuno-infiltration analysis and immunohistochemical verification of spleen tissue showed that compared with the control group, the immune abundance of neutrophils in the administration groups increased (P<0.05). Transcriptome data analysis revealed that Atp1b2 gene was the intersection of spleen and brain and positively correlated with neutrophil expression but negatively correlated with the expression of metabolite oleic acid in brain tissue. The metal ion detection results showed that compared with the control group, the concentration of Fe ions in the brain tissue and serum of the mice in the administration groups increased significantly (P<0.05). According to molecular docking experiments, Fe3+ exhibited strong binding activity with Plcg1 and Atp2a3. Immunohistochemical results showed that the expression of Atp2a3 protein decreased and the expression of Plcg1 protein increased in the high-dose group, and the difference was statistically significant (P<0.05). The Atp1b2 protein level in spleen tissue was positively correlated with that in brain tissue of mice (R=0.829, P=0.038). Western blotting results showed that the level of Atp1b2 protein in brain and spleen increased significantly in the high-dose group (P<0.05). ConclusionShehanshi water extract can improve mice sleep, and its mechanism may be associated with related genes on spleen-brain axis and calcium signaling pathways in brain tissue.