Empagliflozin Protects against Haloperidol Experimentally-Induced Ovarian Toxicity

Abstract

The present experiment aimed to identify the potential protective role of empagliflozin (EMPA) on haloperidol (HAL)-induced ovarian damage in female rats because of its anti-inflammatory, antioxidant, and antiapoptotic effects. EMPA was administered in the presence and absence of HAL. Thirty-two adult female albino rats were divided into four groups. Control group, EMPA group: received EMPA (10 mg/kg/day) p.o., HAL group: received HAL (2 mg/kg/day) p.o., HAL + EMPA group: HAL (2 mg/kg/day) combined with EMPA for 28 days. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and anti-mullerian hormone (AMH) levels were measured. Ovarian oxidative stress parameters, besides inflammatory and apoptotic biomarkers, and ovarian Sirtuin-1 (Sirt-1) were evaluated. Ovarian histopathological examination and heat shock protein 70 (Hsp70) immunohistochemical study were performed. HAL significantly increased serum levels of FSH, LH, and ovarian inflammatory, apoptotic, and oxidative stress biomarkers and decreased serum AMH levels and Sirt-1 expression. Histopathological findings of ovarian damage and high Hsp70 immunoexpression were detected. EMPA significantly normalized the distributed hormonal levels, oxidative stress, inflammatory, and apoptotic biomarkers with a prompt improvement in the histopathological picture and a decrease in Hsp70 immunoexpression. Accordingly, EMPA protected against HAL-induced ovarian toxicity by modulating the Sirt-1/Hsp70/TNF-α/caspase-3 signaling pathway.

Keywords: Hsp70; Sirt-1; caspase-3; empagliflozin; haloperidol; ovarian tissue.

Figure 1

Effect of EMPA on Sirt-1 Western blotting in HAL-induced ovarian toxicity in rats. Results represent the mean ± SEM (8 rats/group). ^a Significant (p < 0.05) difference from the control group. ^b Significant (p < 0.05) difference from the EMPA group. ^c Significant (p < 0.05) difference from HAL. [EMPA = empagliflozin; HAL = haloperidol; Sirt-1= Sirtuin 1].

Figure 2

Photomicrographs of sections in the ovary: (A1) control and (B1) EMPA groups, respectively, showed normal ovarian structure with inner vascularized medulla (M) and outer cortex (C) with numerous growing follicles formed of oocytes with the surrounding granulosa cells (arrows) and corpus luteum (CL). (A2,B2) are higher magnification for the growing follicles from control and EMPA groups, respectively showing oocytes with prominent nucleus (O) surrounded by layers of granulosa cells (G) and outer theca cells (T). Notice the follicular antrum (An). (C1,C2) of HAL group showed the ovarian medulla with marked congested blood vessels (BV). The cortex appears with hardly seen follicles. Large cystic follicle (CF) and degenerated follicle with detached degenerated granulosa cells (G) (inset) are noticed. The cystic follicle lined by thin attenuated flat granulosa cells (arrows) with absent oocyte. (D1,D2) of HAL + EMPA group showed ovarian structure appears more or less normal with normal medulla (M) and cortex (C) with many growing follicles (GF) (arrows) and corpus luteum (CL). (D2) is higher magnification for a growing follicle showing normal oocytes(O) surrounded by granulosa cells (G) H&E; (A1,B1,C1,D1) X100 scale bar 100 µ (A2,B2,C2,D2) X100 scale bar 50µ. (E) The mean number of follicles in the studied groups expressed as mean ± S.E.M (n = 8). ^a is significantly different from control, ^b is significantly different from EMPA and ^c is significantly different from HAL. (p < 0.05) using one-way ANOVA followed by Tukey–Kramer post hoc test.

Figure 3

Photomicrographs of rat ovarian tissues immunostained for HSP70. The control group (A,B) and group 2 (C,D) showing negative immune staining, group-3 (E–G) showing HSP70 positive immune-expression (red arrows) either in the granulosa cells, interstitial cells and endothelial lining of blood vessel blood vessels. Group 4 (H) showing scarcely immune-positive cells (red arrows) of the granulosa and interstitial cells. Growing follicles at different stages (black arrows); corpus luteum (CL), granulosa cells (GC), interstitial cells (I), cystic follicle (CF) and blood vessels (BV). Immunohistochemistry, counterstained with H; (A,C,E,H) × 100 scale bar 100µ and (B,D,F,G,I) × 400. (J) The mean number of Hsp70 immune-positive cells in the studied groups expressed as mean ± S.E.M (n = 8). ^a is significantly different from control, ^b is significantly different from EMPA group and ^c is significantly different from HAL group at p < 0.05 using one-way ANOVA followed by Tukey–Kramer post hoc test.