Therapeutic Effects of Silibinin Against Polycystic Ovary Syndrome Induced by Letrozole in Rats via Its Potential Anti-Inflammatory and Anti-Oxidant Activities

Abstract

Background: Current therapies for polycystic ovary syndrome (PCOS) are accompanied by unwanted effects. Silibinin; a flavonolignan has pleiotropic activities and favorable safety profile.

Purpose: To investigate the efficacy of silibinin on estrous cyclicity, inflammation, oxidative stress and ovarian morphology in letrozole-induced PCOS in rats.

Methods: Forty-eight female Wistar albino rats were divided into 2 sets. Rats of the first set (n = 12), assigned as a negative control (NC) received only the vehicle, rats of the second set (n = 36), assigned as PCOS rats, were given letrozole 1mg/Kg orally for 21 days. On day 21, six rats from the first set and six rats from the second set were euthanized for confirmation of PCOS-induction. The remaining animals from the first set assigned as group 1, those in the second set (n = 30) were equally divided into 5 groups and treated daily for 19 days as follows: group 2 (positive control) received only the vehicle, group 3 treated with metformin 300mg/Kg orally, groups 4 and 5 treated respectively with 100 and 200 mg/Kg silibinin intraperitoneally (IP), and group 6 treated with a combination of metformin 300mg/Kg orally and silibinin 100mg/Kg IP. On day 40, blood samples were examined for luteinizing hormone (LH), testosterone (TS) and estradiol (EST) levels, the anti-inflammatory and antioxidant parameters, ovarian and uterine morphology.

Results: Silibinin alone or in combination with metformin was found to be effective in restoring the regularity of estrous cycle by ameliorating the abnormal alterations of LH, TS, EST, tumor necrosis factor (TNF)-α, and oxidative status and by resuming the appearance of corpora lutea and decreasing or even total absence of cystic follicles in the ovaries.

Conclusion: Silibinin was effective in restoring estrous regularities and alleviating hormonal and histomorphological abnormalities of the ovarian and uterine tissues, this could be due to its anti-androgenic, anti-inflammatory and antioxidant properties.

Keywords: PCOS; estrous cycle; inflammation; silybin; testosterone.

Figure 1

Body weight on day 21 in all allocated groups. Ordinary one-way ANOVA test was used. *p<0.05, **p<001 ***<0.0001 significantly different compare to the negative control group (NC); ns: non-significant different compared with the negative control.

Figure 2

(A–F) Estrous cycle changes in one most representative rat in each group during PCOS induction and treatment period. As reported by Myondo et al (A) NC; negative control; (B) PC; positive control, received only distilled water after PCOS-induction; (C) Met 300mg/Kg received metformin 300mg/Kg after PCOS-induction; (D–F) received silibinin 100, 200mg/Kg and 100mg/Kg silibinin with 300mg/Kg metformin respectively after PCOS-induction.

Figure 3

Effect of silibinin alone and in combination with metformin on body weight during the study period. *Indicates significantly different versus baseline (control). Non-identical letters (A–C) indicate significant difference between different groups in different time points. Values represented as means ±SEM, number of animals per group =6. Two-way ANOVA with multiple comparison corrected by Tukey’s test was used.

Figure 4

Effect of silibinin alone and in combination with metformine at the end of the study (Day 40) on (A) Testosterone levels, (B) Luteinizing hormone, (C) Estradiol hormone. *p<0.05, **p<0.001 is statistically significant compared with the positive control (PC) group, ns; non-significant. Ordinary one-way ANOVA with Tukey’s multiple comparison was used.

Figure 5

Effect of silibinin alone and in combination with metformin at the end of the study (Day 40) on (A) Serum total antioxidant capacity and (B) Tumor necrosis factor (TNF-α). Ordinary one-way ANOVA with Tukey’s multiple comparison was used. *p<0.05, **p<0.001 statistically significant compared to positive control (PC) group. ns: non-significant different compared with positive control.

Figure 6

H&E-stained ovarian sections of negative control (NC) and PCOS rats on day 21. (A) NC rat showing a corpus luteum (CL) and ovarian follicles at different growth phases including primary (P), growing (G) and vesicular (V) follicles, X40. (B) higher magnification of A (X100). (C) NC rat showing multiple corpora lutea (CL) and ovarian follicles at different growth phases including primary (P), vesicular (V) and Graafian follicles (GF), X40. (D) higher magnification (X100) of C. (E) PCOS rats with absence of both ovarian follicles and corpora lutea and presence of multiple, variable-sized cystic follicles (CF), X40. (F) higher magnification of E (X100).

Figure 7

Ovarian sections of the positive control (PC) and treatment groups rats on day 40. (A) PC group showing persistence of cystic follicles and absence of corpora lutea. (B and C) Groups 3 (treatment control, Met 300 mg/Kg) and 6 (combination of Sil 100 mg/Kg and Met 300 mg/Kg) exhibiting absence of cystic follicles, resuming the occurrence of multiple corpora lutea (CL) and presence of antral follicles at different growth phases including primary (P), growing (G) and vesicular (V) follicles and disappearance of the cortical stromal inflammation. (D and E) Groups 4 and 5 (Sil 100 mg/Kg and Sil 200 mg/Kg BW respectively) showing few cystic follicles (CF), resuming the occurrence of corpora lutea (CL) and appearance of antral follicles at different growth phases and disappearance of the cortical stromal inflammation H&E, X100.

Figure 8

Uterine sections of negative control (NC) and PCOS rats on day 21. (A and B) NC rats showing normal uterine morphology represented by a normal looking endometrium with simple columnar epithelial lining and straight endometrial glands in the lamina propria. (C and D) PCOS rats exhibiting marked endometrial hyperplasia with profound, irregular endometrial folds toward the uterine lumen, cystic dilatation (black arrows) and convolution (blue arrows) of endometrial glands and rapid proliferation of endometrial epithelial cells as indicated by epithelial thickening and presence of many normal-looking mitotic figures (green arrows). H and E, (A and C) X100, (B and D) X400.

Figure 9

Uterine sections of positive control (PC) and treatment rats on day 40. (A) PC group showing marked endometrial hyperplasia with profound endometrial folds toward the uterine lumen and cystic dilatation (black arrows) and convolution (blue arrows) of the endometrial glands. (B and C) Groups 3 (treatment control, Met 300 mg/Kg) and 6 (combination of Sil 100 mg/Kg and Met 300 mg/Kg) showing approximately normal uterine morphology. (D and E) Groups 4 and 5 (Sil 100 mg/Kg and Sil 200 mg/Kg BW respectively) showing mild endometrial hyperplasia represented by short endometrial folds toward the uterine lumen. H and E, X200.