Beneficial effects of curcumin and capsaicin on cyclophosphamide-induced premature ovarian failure in a rat model

The experiments were performed according to the animal ethics guidelines of the Inonu University Institutional Animals Ethics Committee (2016/A-87). In total, 40 healthy adult female Wistar albino rats (aged 3–4 months) obtained from The Experimental Animal Institute (Malatya, Turkey) were used in the experiment. Animals were housed in sterilized polypropylene rat cages under a 12/12-h light/dark cycle at an ambient temperature of 21 °C. Food and water were provided ad libitum. Rats were randomly divided into two groups to establish the chemotherapy-induced POF rat model: the control (n = 10) and POF groups (n = 30). POF was induced in the rats by intraperitoneal injection of 200 mg/kg CYC (Endoxan, EIP ECZACIBASI, Istanbul, Turkey) on day 1 and then 8 mg/kg/day for the following 14 days. After 14 days, to determine the effects of CRC and CPS on CYC-induced POF, the POF group was randomly divided into three subgroups (n = 10 per subgroup): POF, POF + CRC (100 mg/kg), and POF + CPS (0.5 mg/kg). The doses of CRC and CPS were chosen based on previous studies [12, 13]. The control group was given no treatment. The animals were euthanized without pain or distress using increasing anesthesia doses (intraperitoneal lethal doses of pentobarbital; Bioveta Inc., Ankara, Turkey) after the treatment course, and the ovaries were removed for histopathological analyses. Blood samples were collected under anesthesia from the left ventricle using an injector. Tissues samples were stored at − 45 °C until analyzed.

Tissues were homogenized using a Teflon glass homogenizer in 150 mM KCl (pH 7.4) at a 1:10 (w/v) dilution of the whole homogenate. The homogenates were centrifuged at 18,000×g and 4 °C for 30 min to determine the malonaldehyde (MDA) and reduced glutathione (GSH) concentrations and the superoxide dismutase (SOD) and catalase (CAT) activities or at 25,000×g for 50 min to determine the glutathione peroxidase (GPx) activity.

E2, FSH, luteinizing hormone (LH), and AMH were quantitatively estimated in rat serum samples using enzyme-linked immunosorbent assay (ELISA) kits (catalog numbers: SL0268Ra, SL0297Ra, SL1093Ra, and SL0504Ra, respectively; Sunlong Biotech Co., Ltd., Zhejiang, China).

For light microscopic evaluation, ovarian tissues were fixed in 10% formalin and embedded in paraffin wax. Paraffin wax-embedded specimens were cut into 5 μm thick sections, mounted on slides, and stained with hematoxylin and eosin (H-E). The tissue samples were examined using a Leica DFC280 light microscope and a Leica Q-Win Image Analysis system (Leica Micros Imaging Solutions Ltd., Cambridge, UK). Histopathological examination of the tissue damage was performed regarding each parameter, such as hemorrhage around the corpus luteum, vascular congestion in the ovarian stroma, hemorrhage, prominent mononuclear cell infiltration beneath the germinative epithelium and follicular atresia. At least five microscopic regions were examined to score the specimens semiquantitatively. Each sample was scored for each criterion using a scale ranging from 0 to 3 (0, none; 1, mild; 2, moderate; 3, severe). Total scores were calculated according to these parameters.

Antioxidant (SOD, CAT, GPx, and GSH) and oxidant parameters (MDA) in rat ovaries are presented in Table 1. MDA levels were significantly decreased, whereas GSH levels and SOD activity were significantly increased, in ovarian tissues in the POF + CRC and POF + CPS groups compared with the POF group. GPx levels and CAT activities were similar among the groups.

In the control group, ovarian tissues showed a normal histological appearance. The secondary follicles (Fig. 1a), multilaminary primary follicles (Fig. 1b and c), primordial follicle (Fig. 1c), and corpus luteum exhibited normal appearances. In the POF group, we observed hemorrhage around the corpus luteum (Fig. 2a), vascular congestion in the ovarian stroma (Fig. 2a), and hemorrhage and prominent mononuclear cell infiltration beneath the germinative epithelium (Fig. 2b). In addition, some atretic ovarian follicles (Fig. 2c) were found in the POF group. We showed that all histological parameters were significantly improved after administration of CPS and CRC. We observed normal multilaminar primary follicles, secondary follicles (white arrow) (Fig. 3a and b), and corpus luteum structures (Fig. 3c) in the POF + CPS group. Decreased vascular congestion (Fig. 4a), fewer atretic follicle (Fig. 4a and b), and normal Graffian follicles, unilaminary primary follicles, multilaminary primary follicles (Fig. 4a and b), secondary follicles, and corpus luteum (Fig. 4c) were detected in the POF + CRC group compared with the POF group. Histopathologic scores for all four groups are demonstrated in Table 2. A significant increment in vascular congestion, hemorrhage, mononuclear cell infiltration and follicular atresia was observed in the POF group compared with the other groups. All histological parameters were significantly improved with the administration of CRC and CPS. However, there were no significant differences between POF + CRC and POF + CPS groups regarding the histopathological scores.

Serum FSH and LH levels were significantly reduced in rats treated with CRC and CPS compared with the POF group. However, E2 and AMH levels were significantly increased in rats treated with CRC and CPS compared with the POF group. However, there was no significant difference in serum hormone levels between the POF + CRC and POF + CPS groups. The effects of CRC and CPS on the ovarian reserve markers are shown in Table 3.