The Mechanism Of SRAGE In The Regulation Of VEGF Production In Granulosa Cells Of PCOS

Polycystic ovary syndrome(PCOS) is a common endocrine and metabolic disorder syndrome in women of reproductive age, it affects about 6% of women of reproductive age, it can lead to ovulation disorders, infertility, insulin resistance(IR) and hyperandrogenism,et al. However, the pathogenesis of PCOS still remains unclear. Studies suggest that the pathogenesis of PCOS may have involvement of disorders in regulatory mechanisms of angiogenesis, Doppler ultrasound color flow imaging found that the ovarian stromal blood flow is increased in women with PCOS. Vascular endothelial growth factor(VEGF) is the most important member of a family of angiogenic factors. In female reproductive system, it had found that VEGF were expressed in ovarian luteinizing granulosa cells, ovarian stroma and endometrial cells. VEGF plays a major role in the regulation of ovarian cyclic angiogenesis and vascular permeability by stimulating endothelial cell proliferation, and migration. Since the PCOS patients has a higher expression of VEGF in follicular fluid and ovarian tissue, it suggests that VEGF is involved in the physiological and pathogenesis processes of PCOS by influencing the angiogenesis.Soluble advanced glycation end products receptor(sRAGE) is the endogenous secretory form of advanced glycation end products receptor(RAGE). sRAGE act as a decoy receptor that competitive binding to RAGE ligands, inhibits RAGE-induced cell signal pathways. sRAGE playing a crucial role in modulating the ligand-RAGE axis, thus has a protective effect on both physiological and pathological states of many disease. The close relationship between sRAGE and inflammatory cytokines, insulin resistance, and ovarian reserve indicated that sRAGE may be involved in the occurrence of PCOS. Our previous study found that the sRAGE concentration in serum and follicular fluid of PCOS patients were lower than nomal women, and the follicular fluid sRAGE level was negatively correlated with VEGF. Therefore, we hypothesized that sRAGE may affect the development of PCOS by reducing VEGF expression, but the specific mechanism needs further study.Amphiregulin(AREG), β cytokine(BTC) and epiregulin(EREG) are members of the epidermal growth factor(EGF) family, they were called EGF-related growth factors due to the similar structures and functions with EGF. EGF family and its receptor family play an important role in the female reproductive system, they participate in ovarian function through autocrine and(or) paracrine action. Studies confirmed that these factors can promote VEGF expression in a variety of cells. However, whether sRAGE could inhibits the expression of EGF-related growth factors in PCOS ovarian granulosa cells is still unknown.This experiment will preliminary studies whether sRAGE could reduced VEGF synthesis through EGF-related growth factor signal pathway in PCOS ovarian granulosa cells. Objective1. To determine the effects of sRAGE on VEGF expression in PCOS ovarian granulosa cells.2. To investigate whether sRAGE could regulate the VEGF expression through EGF-related growth factor signal pathway in PCOS ovarian granulosa cells. Material and MethodsWe selected 10 PCOS patients who have in vitro fertilization(IVF) at the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University between March 2015 and July 2015. All patients were treated with conventional luteal phase long protocol. On the day of oocyte retrieval, collected the follicular fluid of patients, and extracted granulosa cells then cultured in vitro for 48 h. After that, each patient’s ovarian granulosa cells were divided into four groups, each cultured in the medium added with different concentrations of sRAGE(0μg/ml,0.6μg/ml,0.9μg/ml,1.2μg/ml) for 24 h. Detected the EGF-related factors: AREG, BTC and EREG protein concentration in the culture medium by enzyme-linked immunosorbent assay(ELISA). The cultured granulosa cells were divided into two parts, in the first part total RNA was extracted using Trizol, and then used RT-qPCR to measure the mRNA expression of VEGF and EGF-related factors(AREG, BTC and EREG); the other part was used to determine the VEGF protein expression levels by Western blotting. Results1. For 0μg / ml, 0.6μg / ml, 0.9μg / ml, 1.2μg / ml group, the relative expression of VEGF mRNA were 1.349 ± 0.347, 0.817 ± 0.163, 0.472 ± 0.101, 0.321 ± 0.106, respectively. The expression of VEGF mRNA were decreased with increasing concentration of sRAGE, the difference between the four groups was statistically significant(P <0.05).2. For 0μg / ml, 0.6μg / ml, 0.9μg / ml, 1.2μg / ml group, the relative expression of VEGF protein were 0.49 ± 0.037, 0.335 ± 0.018, 0.120 ± 0.012, 0.102 ± 0.005, respectively. The expression of VEGF protein were decreased with increasing concentration of sRAGE, the difference between the four groups was statistically significant(P <0.05).3. After PCOS ovarian granulosa cells were cultured in the medium added with different concentrations of sRAGE, the expression of AREG, BTC and EREG mRNA were decreased, and the expression of mRNA was dependent on the concentrations of sRAGE, the difference between the four groups was statistically significant(P <0.05).4. After ovarian granulosa cells were cultured in the medium added with different concentrations of sRAGE, the expression of AREG, BTC and EREG protein were decreased, the AREG, BTC and EREG protein expression were dependent on the concentrations of sRAGE, the difference between the four groups was statistically significant(P <0.05). Conclusions1. sRAGE may reduce the expression of VEGF mRNA and protein in granulosa cells of PCOS patients.2. In PCOS ovarian granulosa cells, sRAGE may be involved in the regulation of VEGF by blocking EGF-related growth factor signal pathway.