2022, Vol. 26 ›› Issue (35): 5658-5664

miRNA screening for targeted regulation of bone growth by semen ziziphi spinosae extract

Luo Shiren, Xie Yan, Zhang Li, Yin Na   

Abstract: BACKGROUND: Growth hormone is secreted during slow-wave sleep in large proportion, and the initiation and prolongation of slow-wave sleep is closely linked with serotonin. Previous studies have shown that semen ziziphi spinosae extract can increase the expression of serotonin 1A receptor. The combination of serotonin 1A receptor and serotonin can prolong slow-wave sleep, thereby promoting the secretion of growth hormone and bone growth. In addition, semen ziziphi spinosae extract can down-regulate the expression of serotonin 2A receptor in brain tissue, which can promote the combination of serotonin 1A receptor and serotonin, thereby promoting the prolongation of slow-wave sleep, the secretion of growth hormone, and bone growth.
OBJECTIVE: To prove that semen ziziphi spinosae extract can promote bone growth in mice by down-regulating the expression of serotonin 2A receptor and to screen miRNAs targeting serotonin 2A receptor and promoting bone growth.
METHODS: (1) Kunming mice were divided into blank control group, high- and low-dose groups of semen ziziphi spinosae extract (hereinafter referred to as high- and low-dose groups), positive control group, and serotonin 2A receptor selective inhibitor group (hereinafter referred to as inhibitor group), with 10 mice in each group. Mice in the blank control group were gavaged with deionized water every day, the high- and low-dose groups were gavaged with 0.16 and 0.32 mg/g semen ziziphi spinosae extract suspension respectively, the positive control group was gavaged with semen ziziphi spinosae saponin A standard solution, and the inhibitor group was gavaged with semen ziziphi spinosae extract aqueous solution. Gavage was administered to each group at the same period, and 10 μg of M100907 was injected into the lateral ventricle of each mouse once a day for the last 3 days. Changes in the body length of mice were observed, and changes in the expression of serum growth hormone and brain tissue serotonin 2A receptor were observed by ELISA. (2) Sprague-Dawley rats were divided into blank control group, medication group (given semen ziziphi spinosae extract), positive control group and serotonin 2A receptor selective inhibitor group (hereinafter referred to as the inhibitor group). The effects of drugs on slow-wave sleep were observed at the 3rd day after intragastric administration. (3) The chip method was used to detect the differentially expressed miRNAs in the brain tissues of mice with bone growth and ordinary mice. The miRNAs targeting serotonin 2A receptor were selected and verified by real-time fluorescent quantitative PCR.
RESULTS AND CONCLUSION: (1) After 25 days of treatment, the body length of mice in the high-dose group was significantly longer than that in the blank control group (P < 0.01). The serum growth hormone level of mice was significantly higher in the high-dose group than the blank control and inhibitor groups (P < 0.01, P < 0.01). Compared with the inhibitor group, semen ziziphi spinosae extract significantly lowered the expression of serotonin 2A receptor in brain tissue (P < 0.01). (2) The duration of slow-wave sleep in the medication and positive control groups was longer than that in the blank control group (P < 0.01, P < 0.01) and in the inhibitor group (P < 0.05, P < 0.05). There was no difference in the effects of drugs on paradoxical sleep periods. (3) A total of 16 differentially expressed miRNAs met the screening conditions, 13 of which were up-regulated and 3 were down-regulated. (4) It was verified by real-time fluorescent quantitative PCR that the up-regulated miR-34a-5p could regulate serotonin 2A receptor. (5) All these findings indicate that semen ziziphi spinosae extract can inhibit the expression of serotonin 2A receptor and prolong slow-wave sleep, thereby increasing the secretion of growth hormone and promoting bone growth in mice. The possible mechanism is that semen ziziphi spinosae extract up-regulates the expression of miR-34a-5p which results in the lowered expression of serotonin 2A receptor.
Key words: semen ziziphi spinosae extract, serotonin 2A receptor, mouse, bone growth, microRNA, rat, slow-wave sleep