AGEs induce apoptosis and inhibits proliferation in KGN cells.
KGN cells cultured in vitro were treated with different concentrations of AGEs (0, 25, 50, 100, 200µg/ml) for 24 hours, or with 100µg/ml AGEs for different times (0, 24, 48, 72, 96h). The results showed that compared with the control group, the viability of KGN cells was significantly reduced after AGEs treatment in a concentration-dependent and time-dependent manner (Fig. 1A). Subsequently, we examined the effect of AGEs on the apoptosis and proliferation levels in KGN cells. As demonstrated, with the extension of time and concentration, the results revealed that AGEs significantly increased the ratio of Bax/Bcl2 and inhibited the expression of PCNA (Fig. 1B and C).
AGEs inhibit KGN cell proliferation and induces apoptosis by up-regulating the expression of AMH.
Serum AMH has been suggested as a monitoring indicator of female ovarian reserve function and treatment in women with PCOS. Therefore, we examined the effect of AGEs on AMH expression in KGN cells. The results showed that the expression of AMH was up-regulate in a dose- and time-dependent manner of AGEs (Fig. 2A and B). Consistent with our findings, the immunofluorescence and immunocytochemistry results showed that, AGEs could increase the expression of AMH in KGN cells compared with the control group (Fig. 2C and D).
We next assessed the effects whether AGEs affect the apoptosis and proliferation in KGN cells through AMH by interfering with the expression of AMH with siRNA. The results showed that AMH protein levels were lower in the siRNA group than in the control group (Fig. 2E). Furthermore, the ratio of apoptosis-related protein Bax/Bcl2 was significantly decreased after inhibition of AMH expression, while the expression of proliferation-related protein PCNA was upregulated (Fig. 2F and G), indicating that AGEs may promote the apoptosis and inhibit the proliferation of KGN cells by increasing the expression of AMH.
AGEs facilitate the expression of AMH through PI3K/AKT/SF1 pathway in KGN cells.
We investigated the potential signaling pathway of AGEs promoting AMH expression. We have examined the change of SF1, PI3K and Akt expression after AGE treatment. It was found that the mRNA and protein expression levels of SF1 were increased in a concentration- and time-dependent manner compared with the control in KGN cells (Fig. 3A and B). Immunofluorescence results further confirmed that AGEs (100 µg/ml, 24 hours) up-regulated the expression of SF1 in KGN cells (Fig. 3C). Simultaneously, the results showed that AGEs facilitated the phosphorylation of PI3K and Akt in KGN cells (Fig. 3E and F).
To further study whether AGEs promoted the expression of AMH by via PI3K/Ak/SF1 pathway, we used PI3K inhibitors (Wortmannin 100nM) and cells were transfected with a SF1 siRNA. The results showed that AMH expression was significantly down-regulated after knockdown the SF1 (Fig. 3D). The results showed that the expression levels of AMH and SF1 were down-regulated in Wortmannin and AGEs co-treatment group comparison with the AGEs alone treatment group (Fig. 3J). Concurrently, the Bax/Bcl2 ratio was decreased and PCNA levels were increased in the AGEs alone group (Fig. 3G-I). Taken together, these findings provide evidence of the involvement of PI3K/Akt/SF1 pathway in AGE mediated stimulation of AMH expression in KGN cells.
High AGEs diet s promote the development of PCOS in rats.
Female rats had 4–5 days of estrous cycle, comprising preoestrus, estrum, metaoestrus and diestrus phases. Vaginal smears were monitored for 7 consecutive days and analyzed microscopically to assess the effects of AGEs treatment on the estrous cycle. The results showed that both AGEs group and letrozole-induced PCOS model group showed estrous cycle disorder. The PCOS model group stayed in the diestrus phases, and the AGEs group did not have a normal estrous cycle (Fig. 4A).
Weight gain was significantly increased in both the AGEs and PCOS groups as compared to the control group. However, the AGEs group had not change in ovary/body weight index and the ovaries size (Fig. 4B and C). The histological features in the control ovaries were normal, whereas the ovaries were morphologically disorganized, with an increased number of follicular cysts, a thinner granulosa cell layer and a lack of corpus luteum in the PCOS and AGEs groups (Fig. 4D). Masson staining showed a significantly higher degree of ovarian cortical fibrosis in the AGEs and PCOS groups (Fig. 4E). These results indicate that phenotypes induced by AGEs were similar morphological changes to PCOS in ovarian tissue, such as polycystic changes in the ovaries, inhibited normal ovulation in rats, and increased ovarian cortical fibrosis.
AGEs-induced elevated AMH promote the development of PCOS in rats.
We further explore the effects of AGEs on expression of AMH and the progression of PCOS in ovarian granulosa cells of rats. The results showed that AMH expression was upregulated in the ovaries of both AGEs and PCOS groups compared with the control group (P < 0.05). Meanwhile, the upregulation of AMH expression was significantly higher in the letrozole and AGEs co-treatment group (P < 0.05) (Fig. 5A and B). The results indicated that high AGEs up-regulated the expression of AMH in the ovary of PCOS and non-PCOS rats. TUNEL assay and Western blot assays revealed that the combined group of AGEs and PCOS had more increased apoptosis in ovarian granulosa cells, markedly increased Bax/Bcl2 ratio and decreased PCNA protein expression (P < 0.05) (Fig. 5C and D). These findings implicate that high AGEs diet further exacerbates the level of apoptosis and inhibits cell proliferation (P < 0.05) (Fig. 5E and F). Consistent with the cells, we observed a significant increase of SF1 protein levels and phosphorylation levels of PI3K and Akt proteins in the tissues of rats in the AGEs group (P < 0.05). (Fig. 5G and H). These results thus suggest that AGEs may be contribute to PCOS via activating SF1/PI3K/Akt signaling pathway.