Abstract: BACKGROUND: Hypothyroidism is often accompanied by inadequate secretion of pituitary synthetic secretory hormones. Some studies have shown that icariin has an exact effect on pituitary hormone secretion deficiency caused by hypothyroidism, but the mechanism is still unclear.
OBJECTIVE: To investigate the effect and mechanism of icariin on pituitary hormone synthesis and secretion after hypothyroidism in rats. 
METHODS: (1) Forty newborn female Sprague-Dawley rats were randomized into control, model, 75, 150, 300 mg/kg icariin groups with 8 rats in each group. The rats in the latter four groups were intragastrically treated with propylthiouracil (1.5 mg/kg/day) to construct a hypothyroid rat model, and treated with different doses (0, 75, 150, and 300 mg/kg) of icariin. The expression of miR-17-5p, LIM homeobox 4 (LHX4), prop paired end homology 1 protein (PROP1), and HESX1 was detected, as well as hormone content synthetized and secreted by the pituitary gland. (2) Co-cultured rat thyrocytes and anterior pituitary cells were treated with lithium salt, and different concentrations of icariin (10, 30, 50, 70, 100 μmol/L) were administered. Anterior pituitary cell viability was assessed by cell counting kit-8. Follicle-stimulating hormone, growth hormone, and thyroid-stimulating hormone levels were detected by ELISA kits. miR-17-5p expression was detected by real-time fluorescence quantitative PCR. PROP1 and HESX1 protein levels were measured by western blot assay. The binding relationship between miR-17-5p and LHX4 was examined by RNA binding protein immunoprecipitation and dual luciferin reporter assays. 
RESULTS AND CONCLUSION: In vivo results showed that the treatment with 150 mg/kg icariin decreased the contents of thyroid-stimulating hormone and significantly increased the contents of growth hormone and follicle-stimulating hormone compared with the model group. In addition, the hormone secretion levels of serum free triiodothyronine, serum free thyroxine, serum triiodothyronine, and serum thyroxine were increased in the 150 mg/kg icariin group compared with the model group. Intervention with 50, 70 and 100 μmol/L icariin could enhance the cell viability of rat anterior pituitary cells (P < 0.01), among which 70 μmol/L icariin had the best effect (P < 0.01). Icariin treatment inhibited the increased expression of miR-17-5p and upregulated the expression of PROP1 and HESX1 in lithium salt-treated rat anterior pituitary cells (P < 0.05). The contents of growth hormone and follicle-stimulating hormone in the cell supernatant of the lithium salt treated group were obviously decreased, and the content of thyrotropin was significantly increased compared with the control (P < 0.01). When the cells were intervened with different doses of icariin, the contents of growth hormone (P < 0.01) and follicle stimulating hormone (P < 0.01) increased, the level of thyroid-stimulating hormone (P < 0.01) decreased significantly, and the best intervention effect was achieved at 70 μmol/L, with a significant difference (P < 0.01). Compared with the control group, transfection with miR-17-5p mimic inhibited cell viability of anterior pituitary cells (P < 0.05), downregulated the protein expression of PROP1 and HESX1 in the cells, increased thyrotropin content (P < 0.05), and decreased growth hormone (P < 0.01) and follicle-stimulating hormone (P < 0.01) contents in the cells (P < 0.01), whereas transfection with miR-17-5p inhibitor had the opposite effect as transfection with miR-17-5p mimic. LHX4 is a target gene of miR-17-5p. Overexpression of LHX4 increased cell viability, promoted PROP1 and HESX1 protein expression (P < 0.01), reduced thyrotropin content (P < 0.05), and increased growth hormone (P < 0.01) and follicle-stimulating hormone (P < 0.05) contents. To conclude, icariin may restore the level of secretory hormones synthesized by dysregulated pituitary gland through the miR-17-5p/LHX4 axis.

Key words: icariin, hypothyroidism, miR-17-5p/LHX4 axis, growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone