Abstract: BACKGROUND: Previous studies found that Semen Ziziphi Spinosae extract prolongs slow-wave sleep and promotes growth hormone secretion in mice by increasing the expression of brain tissue serotonin 1A receptor (5-HT1AR), which binds to serotonin, thus leading to bone growth. Serotonin 1A receptor acts as a protein, and its expression abundance is regulated by miRNAs. The authors hypothesized that Semen Ziziphi Spinosae extract may regulate the expression of 5-HT1AR through miRNAs and thus exert drug effects. 
OBJECTIVE: To observe the effect of Semen Ziziphi Spinosae extract on bone growth by regulating the miR-7b-3p/5-HT1AR pathway in mouse brain tissue. 
METHODS: (1) Kunming mice were divided into a normal control group, a drug administration group (gavage administration of Semen Ziziphi Spinosae extract 0.320 mg/g), a positive control group (gavage administration of jujuboside 0.013 mg/g), a Semen Ziziphi Spinosae extract+5-HT1AR selective inhibitor group 
(8 μg of P-MPPF, a 5-HTIAR selective inhibitor, was injected into the lateral ventricle every day for the last 3 days during the gavage administration of Semen Ziziphi Spinosae extract). After 25 days, the effects of Semen Ziziphi Spinosae extract on bone growth, serum growth hormone level and brain 5-HT1AR expression were observed. (2) Then the chip method was used to observe differentially expressed miRNAs in the brain tissues of growing mice and ordinary mice. PCR validation and dual luciferase reporter gene assay confirmed the regulatory relationship between the screened Mir-7B-3p and 5-HT1AR. (3) Mouse cerebral cortical cells were cultured and identified in vitro, and the effect of Semen Ziziphi Spinosae extract on 5-HT1AR expression in cerebral cortical cells was observed using western blot. (4) Kunming mice were divided into a normal control group, a medication group, a miR-7b-3p inhibitor group, a medication +5-HT1AR inhibitor group, and a positive control group. The expression of 5-HT1AR in brain tissues of mice and the binding activity of 5-HT and 5-HT1AR were observed. (5) Sprague-Dawley rats were divided into a normal control group, a medication group, a miR-7b-3p inhibitor group, a medication+miR-7b-3p mimics group and a positive control group. Changes in slow wave sleep in mice were observed. 
RESULTS AND CONCLUSION: (1) Semen Ziziphi Spinosae extract could promote the growth of body length and tibia, growth hormone secretion, and brain tissue 5-HT1AR level in mice. (2) The number of differentially expressed miRNAs screened by GeneChip was 16, of which 13 were up-regulated and 3 were down-regulated. Bioinformatics results predicted that down-regulation of miR-7b-3p could regulate 5-HT1AR expression, and dual-luciferase reporter gene experiments confirmed a direct regulatory relationship between the two. (3) Semen Ziziphi Spinosae extract and silencing of miR-7b-3p expression in cerebral cortical cells could cause high expression of 5-HT1AR. After silencing of miR-7b-3p, 5-HT1AR expression, binding activity of serotonin and 5-HT1AR and growth hormone secretion in mouse brain tissue were all elevated. After overexpression of miR-7b-3p, 5-HT1AR expression, binding activity of serotonin and 5-HT1AR and growth hormone secretion were all reduced. Accordingly, the slow-wave sleep period in mice was also prolonged or shortened. To conclude, Semen Ziziphi Spinosae extract can reduce the level of miR-7b-3p and increase the expression of 5-HT1AR in brain tissue to prolong slow wave sleep and promote the secretion of growth hormone, thereby playing a postive effect on bone growth. These findings provide a scientific basis for the use of Semen Ziziphi Spinosae extract as a potential measure to promote bone growth. 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Semen Ziziphi Spinosae extract, miR-7b-3p, brain tissue, serotonin, serotonin 1A receptor, growth hormone