Plumbagin inhibits proliferation and promotes apoptosis of ovarian granulosa cells in polycystic ovary syndrome by inactivating PI3K/Akt/mTOR pathway

Abstract

Polycystic ovary syndrome (PCOS) is recognized as a general endocrine disease and reproductive disorder. Although evidence indicates that PCOS has a complex etiology and genetic basis, the pathogenic mechanisms and signal pathway in PCOS remain unclear. In this study, the normal structure of follicle and corpus luteum were observed, and no cyst nor hyperemia was observed under the light microscopic study with hematoxylin and eosin (H&E) staining. Eestosterone and progesterone were evaluated by radioimmunoassay in rat serum. The alterations of proliferative ability and cell cycle distribution of each group were assessed by Cell Counting Kit-8 (CCK8) assay and flow cytometry. The protein expression of p-mTOR/mTOR, p-PI3K/PI3K, p-AKT/AKT, and GAPDH were analyzed by western blotting. Both doses of PLB could benefit the ovarian morphology and polycystic property. PLBinduced a suppress effect on the proliferation of rat ovarian granulosa cells. In addition, PLB also induced concentration-dependent apoptosis in rat ovarian granulosa cells. The rat ovarian granulosa cells treated with PLB that the expression levels of p-AKT, p-mTOR, and p-PI3K were significantly decreased in a concentration-dependent manner. PLB not only plays a critical role in attenuating the pathology and polycystic property changes in the ovary but can also induce rat ovarian granulosa cell apoptosis through the PI3K/Akt/mTOR signal pathway. This study showed the innovative role of PLB in the pathogenesis of PCOS and provides a new therapeutic modality for the treatment of PCOS.

Keywords: PI3K/Akt/mTOR signal pathway; apoptosis; ovarian granulosa cells; plumbagin; polycystic ovary syndrome.

Figure 1.

The effect of PLB on ovarian in PCOS rats. (A) The effect of PLB on ovarian histopathology in normal and PCOS rats, determined by H&E staining. (B) The progesterone expression level in normal and PCOS rats, determined by radioimmunoassay. (C) The testosterone expression level in normal and PCOS rats, determined by radioimmunoassay. Statistical analysis was performed according to Material and Methods (Error Bar: Mean ± SD, **p < .01 compared to control group, and ##p < .01 compared to PCOS group).

Figure 2.

PLB inhibits the proliferation of rat ovarian granulosa cells in PCOS. (A) The relative cell viability level in rat ovarian granulosa cells treated with PLB at 0, 1, 5 and 10 µM, determined by CCK8 assay. (B) Cell cycle distribution of rat ovarian granulosa cells treated with PLB at different concentration was detected by flow cytometry. (C) Bar graphs showing the ratio of cell cycle in rat ovarian granulosa cells treated with PLB at 0, 1, 5 and 10 µM for 24 h. Statistical analysis was performed according to Material and Methods (Error Bar: Mean ± SD, **p < .01).

Figure 3.

PLB induces the apoptosis of rat ovarian granulosa cells in PCOS. Apoptosis of rat ovarian granulosa cells was measured by flow cytometric analysis. The bar graphs showing the ratio of apoptosis cells in each groups. Statistical analysis was performed according to Material and Methods (Error Bar: Mean ± SD, ** p < .01).

Figure 4.

PLB induces the apoptosis of rat ovarian granulosa cells in PCOS via PI3K/Akt/mTOR signal pathway. Immunoblotting for PI3K, Akt and mTOR after different concentration of PLB treatment in rat ovarian granulosa cells. The bar graphs showing the relative protein level of PI3K, Akt and mTOR in each groups. Statistical analysis was performed according to Material and Methods (Error Bar: Mean ± SD, ** p < .01).