Protective effects of berberine in a rat model of polycystic ovary syndrome mediated via the PI3K/AKT pathway

Abstract

Background: Ber, a Chinese herbal monomer has been reported to exhibit an array of pharmacological activities related to the lowering of blood glucose and the treatment of polycystic ovarian syndrome (PCOS). In the present study, we aimed to elucidate the effect of berberine (Ber) on a rat model of PCOS mediated via the PI3K/AKT signaling pathway.

Methods: A PCOS animal model was induced with the administration of letrozole, and animals were then randomized into untreated or Ber and metformin hydrochloride treated groups. After administration, fasting blood glucose, HOMA-IR, fasting insulin (FINS) values, and the serum hormone levels were measured in PCOS rats. The ovarian tissues were stained with hematoxylin and eosin and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) for pathological and apoptosis examination. Moreover, the effect of Ber on the proliferation and apoptosis of granulosa cells was detected by CCK-8 assays and flow cytometry. The influence of Ber on granulosa cells was confirmed by blockade of the PI3K/AKT pathway. In addition, the modulatory effect of the blockade of the PI3K/AKT pathway on the expression of related proteins was demonstrated via western blotting.

Results: We found that Ber was able to restore the serum hormone levels and improve IR in a PCOS rat model. The morphological lesions and apoptosis of the ovary were also restored by the Ber treatment. Blockade of the PI3K/AKT pathway attenuated the influences of Ber on the proliferation and apoptosis of granulosa cells.

Conclusion: The beneficial effects of Ber on PCOS included alterations of the serum hormone levels, recovery of morphological lesions in the ovary, improvement of insulin resistance, and cell viability and inhibition of apoptosis, which were all mediated through the PI3K/AKT pathway.

Keywords: PI3K/AKT pathway; berberine; insulin resistance; polycystic ovarian syndrome.

Figure 1

Effects of Ber and MH on the level of FPG, FINS, and HOMA‐IR in PCOS rats. (a), The levels of FPG, FINS, and HOMA‐IR in PCOS rats before administration. (b), The level of FPG, FINS, and HOMA‐IR in PCOS rats after administration for 4 weeks ($\overline{\chi }$ ± s, n = 10), FPG, fasting blood glucose; H‐Ber, high concentration of berberine; HOMA‐IR, homeostasis model assessment of insulin resistance; L‐Ber, low concentration of berberine; MH, metformin hydrochloride; PCOS, polycystic ovarian syndrome. ^▲ p < 0.05, ^▲▲ p < 0.01 vs. control group, ^★ p<0.05, ^★★ p<0.01 vs. PCOS group

Figure 2

Comparison of hormone level detected by ELISA in the serum of rats ($\overline{\chi }$ ± s, n = 10), ELISA, enzyme‐linked immunosorbent assay; H‐Ber, high concentration of berberine; L‐Ber, low concentration of berberine; MH, metformin hydrochloride. ^▲ p < 0.05, ^▲▲ p < 0.01 vs. control group, ^★ p<0.05, ^★★ p<0.01 vs. PCOS group

Figure 3

The vaginal smears of rats in the control and polycystic ovarian syndrome (PCOS). (a), The representative rat's vaginal smears from the control group in diestrus. (b), The representative rat's vaginal smears from the control group in estrus. (c), The representative rat's vaginal smears from the control group in metestrus. (d), The representative rat's vaginal smears from the control group in proestrus. (e), The representative rat's vaginal smears from the PCOS group predominantly exhibited leukocytes, the main cell type observed during the diestrus stage (original magnification, ×200)

Figure 4

The morphological changes of the rats' ovarian tissues were evaluated by hematoxylin and eosin (H&E) staining. (a), representative microphotographs of H&E staining, original magnification × 100 ($\overline{\chi }$ ± s，n = 6); (b) Semiquantitative assessment of the histological lesions ($\overline{\chi }$ ± s，n = 6), H‐Ber, high concentration of berberine; L‐Ber, low concentration of berberine; MH, metformin hydrochloride; PCOS, polycystic ovarian syndrome. ^▲▲ p < 0.01 vs. control group, ^★★ p<0.01 vs. PCOS group

Figure 5

Effect of Ber and MH post‐treatment on the apoptosis of ovarian tissue cells. (a), Representative TUNEL staining images, original magnification ×400. (b) Semiquantitative assessment of the cell apoptosis, ($\overline{\chi }$ ± s，n = 3), H‐Ber, high concentration of berberine; L‐Ber, low concentration of berberine; MH, metformin hydrochloride; PCOS, polycystic ovarian syndrome. ^▲▲ p < 0.01 vs. control group, ^★★ p<0.01 vs. PCOS group

Figure 6

Identification of rat ovarian granulosa cells by hematoxylin and eosin (H&E) staining. (a) Morphology characteristic of ovarian granulosa cells in the control group (a) and the PCOS group (b) (original magnification, ×100). H&E staining images of ovarian granulosa cells in the control group (c) and PCOS group (d) (original magnification, ×200)

Figure 7

Effects of Ber treatment on the growth of ovarian granulosa cells in vitro. (a) Ovarian granulosa cells viability following treated with 0 (control), 0.1, or 0.2 mg/mL of Ber for 12, 24, 48, or 72 h. (b) Ovarian granulosa cells viability following treatment with the simultaneous addition of either LY294002 or MK‐2206 in combination with 0.2 mg/mL Ber, ($\overline{\chi }$ ± s，n = 3), H‐Ber, high concentration of berberine; L‐Ber, low concentration of berberine; PCOS, polycystic ovarian syndrome. ^▲ p < 0.05, ^▲▲ p < 0.01 vs. control group, ^★ p<0.05, ^★★ p<0.01 vs. PCOS group, ^# p<0.05, ^## p<0.01 vs. PCOS+H‐Ber group

Figure 8

Effect of Ber on cell apoptosis after lead acetate induced injury in rat ovarian granulosa cells. (a and c), flow cytometry detection of apoptosis revealed that Ber significantly induced ovarian granulosa cell apoptosis. (b and ) flow cytometry detection of the anti‐apoptotic effects of Ber were blocked in combination with either 20 mM LY294002 or 20 mM MK‐2206. ($\overline{\chi }$ ± s，n = 3), H‐Ber, high concentration of berberine; L‐Ber: Low concentration of berberine; PCOS, polycystic ovarian syndrome. ^▲ p < 0.05, ^▲▲ p < 0.01 vs. control group, ^★ p<0.05, ^★★ p<0.01 vs. PCOS group, ^# p<0.05, ^## p<0.01 vs. PCOS+H‐Ber group

Figure 9

Effects of Ber in combination with either LY294002 or MK‐2206 on the protein expression of selected genes associated with cell apoptosis and regulation of the PI3K/Akt pathway. ($\overline{\chi }$ ± s，n = 3), H‐Ber, high concentration of berberine; PCOS, polycystic ovarian syndrome, L‐Ber: low concentration of berberine, ^▲ p < 0.05, ^▲▲ p < 0.01 vs. PCOS group, ^★ p<0.05, ^★★ p<0.01 vs. PCOS+H‐Ber group