Amburana cearensis leaf extract protects against cisplatin-induced ovarian damage through regulation of p-PTEN and p-Akt proteins in mice

Abstract

Objectives: To evaluate the effects of Amburana cearensis leaf extract against cisplatin-induced ovarian toxicity in mice and involvement of p-PTEN and p-Akt proteins.

Materials and methods: A. cearensis ethanolic leaf extract was analyzed by high-performance liquid chromatography (HPLC). Mice were pretreated once daily for 3 days as follows: (1) the control group was pretreated with oral administration (o.p.) of saline solution, followed by intraperitoneal (IP) injection of saline solution. The other groups were pretreated (o.p.) with (2) saline solution (cisplatin group), (3) N-acetylcysteine (positive control), with (4) 50, or (5) 200 mg/kg body weight of A. cearensis extract, followed by injection of 5 mg/kg body weight (IP) of cisplatin. The ovaries were harvested and destined for histological (follicular morphology), immunohistochemistry (apoptosis and cell proliferation), and fluorescence (reactive oxygen species [ROS], glutathione concentrations [GSH], and active mitochondria) analyses. Furthermore, immunoexpression of p-PTEN and p-Akt was evaluated to elucidate a potential mechanism by which A. cearensis extract could prevent cisplatin-induced ovarian damage.

Results: After HPLC analysis, protocatechuic acid was detected in the extract. The pretreatment with N-acetylcysteine or A. cearensis extract maintained the percentage of normal follicles and cell proliferation, reduced apoptosis and ROS concentrations, and increased GSH concentrations and mitochondrial activity compared with cisplatin treatment. Furthermore, pretreatment with A. cearensis extract regulated p-PTEN and p-Akt immunoexpression after cisplatin exposure.

Conclusion: Pretreatment with A. cearensis extract prevented cisplatin-induced ovarian damage through its anti-oxidant actions and by modulating the expression of phosphorylated PTEN and Akt proteins.

Keywords: Anti-oxidants; Antineoplastic protocols; Fertility preservation; Ovarian follicle; Phytotherapy.

Figure 1

Chromatogram of Amburana cearensis ethanolic extract obtained by HPLC (A; “1" indicates the peak referring to protocatechuic acid) and protocatechuic acid standard (B) at 270 nm. UV spectrum of protocatechuic acid in the sample (C) and the protocatechuic acid standard (D)

Figure 2

Histological sections of mouse ovarian fragments in the control group (A, B), exposed to cisplatin alone (C, D), and exposed to N-acetylcysteine (E, F), 50 (G, H) or 200 (I, J) mg/kg Amburana cearensis extract plus cisplatin. O: oocyte; GC: granulosa cells; Scale bars: 50 µm (A, C, E, G and I: 100x; B, D, F, H and J: 400x)

Figure 3

Total follicular survival (percentage of morphologically normal follicles; A), and percentage of morphologically normal follicles in different stages (primordial, primary, and secondary follicles; B) in the control group, mice exposed to cisplatin alone, and exposed to N-acetylcysteine or A. cearensis extract (50 or 200 mg/kg) before cisplatin treatment. *Cisplatin compared with the control group; #N-acetylcysteine or Amburana cearensis extract pretreatment compared with cisplatin only group (P<0.05)

Figure 4

Percentage of PCNA-positive cells (A) in the control group, mice exposed to cisplatin alone, and exposed to N-acetylcysteine or Amburana cearensis extract (50 or 200 mg/kg) before cisplatin treatment. *Cisplatin compared with the control group; #N-acetylcysteine or A. cearensis extract pretreatment compared with cisplatin only group (P<0.05). Immunohistochemical localization of PCNA-positive cells: follicles in the control group (B), treated with cisplatin alone (C), or pretreated with 50 mg/kg A. cearensis extract before cisplatin treatment (D). Reaction control (E). O: oocyte; GC: granulosa cells; Scale bars: 50 µm (400x)

Figure 5

Percentage of apoptosis of follicular cells (A) in the control group mice exposed to cisplatin alone and exposed to N-acetylcysteine or Amburana cearensis extract (50 or 200 mg/kg) before cisplatin treatment. *Cisplatin compared with the control group; #N-acetylcysteine or A. cearensis extract pretreatment compared with cisplatin only group (P<0.05). Immunohistochemical localization of activated caspase-3: follicles in the control group (B), treated with cisplatin alone (C), or pretreated with 50 mg/kg A. cearensis extract before cisplatin treatment (D). Reaction control (E). O: oocyte; GC: granulosa cells; Scale bars: 50 µm (400x)

Figure 6

Detection of intracellular concentrations of reactive oxygen species (ROS), glutathione concentrations (GSH), and mitochondrial activity (A): follicles in the control (a, f, k), treated with cisplatin alone (b, g, l), pretreated with N-acetylcysteine (c, h, m), and pretreated with 50 (d, i, n) or 200 (e, j, o) mg/kg Amburana cearensis extract before cisplatin treatment. Scale bars: 50 μm (100x). Intracellular concentrations of ROS, GSH, and mitochondrial activity (B) in follicles of different groups exposed to cisplatin alone or in association with N-acetylcysteine, 50 or 200 mg/kg A. cearensis extract. *Cisplatin compared with the control group; #N-acetylcysteine or A. cearensis extract pretreatment compared with cisplatin only group (P<0.05)

Figure 7

Immunohistochemical localization for p-PTEN (A, B) and p-Akt (C, D): follicles exposed to cisplatin alone (A, C) or exposed to 50 mg/kg A. cearensis extract before cisplatin treatment (B, D). Reaction controls (E, F). O: oocyte; GC: granulosa cells; Scale bars: 50 µm (400x)