Efeitos protetores in vitro e in vivo da aloe vera contra danos ovarianos induzidos por Doxorrubicina em camundongos fêmeas

Abstract

Doxorubicin (DOX) is used to treat several types of cancer, but it causes ovarian toxicity by increasing the formation of reactive oxygen species (ROS), causing oxidative stress and an exacerbated inflammatory response. Thus, the use of natural compounds with antioxidant and anti-inflammatory properties against the toxic side effects induced by antineoplastic drugs has been highlighted. This study aimed to investigate the action of Aloe vera extract against the deleterious effects of DOX on follicular survival and growth in female mice. For this purpose, female Swiss mice with a regular estrous cycle in phases 1 and 2 were used. For phase 1 the animals (n = 37) had their ovaries collected and cultured individually in a 24-well plate at 37.5° C, in 5% CO2, for 6 days. The ovaries were cultured in DMEM+ alone; in medium supplemented with DOX (0.3 μg/mL); or in this case in DMEM+ supplemented with DOX (0.3 μg/mL) combined with Aloe vera (5%, 10%, 25% or 50%). After culture, the ovaries were collected and fixed for analysis. In phase 2, the animals (n = 48) were divided into six groups (8 per group): positive control (NAC+DOX), which received pretreatment with N-acetylcysteine ​​orally (PO) and, 1 hour later, a single intraperitoneal (IP) dose of DOX; negative control (SAL+DOX), with PO pretreatment with saline followed, 1 hour later, by DOX (IP); three groups pretreated with Aloe vera extract (0.1, 1.0 or 10.0 mg/kg, PO) and, 1 hour later, subjected to a dose of DOX (IP) (AV0.1+DOX, AV1.0+DOX and AV10.0+DOX); and control (SAL+SAL), which received saline solution by PO and IP. DOX was administered in a single dose and Aloe vera was administered on three consecutive days; 24 hours after the last application (on the fourth day), the ovaries were collected, thus, the experiment lasted four days. The ovaries collected in phases 1 and 2 were intended for histological analysis (morphology, growth, activation, extracellular matrix (ECM) configuration and stromal density), immunohistochemistry (TNF-α) and evaluation of mRNA levels for superoxide dismutase (SOD), catalase (CAT), nuclear factor-erythroid 2-related factor 2 (NRF2) and tumor necrosis factor-α (TNF-α) by real-time PCR. Statistical analysis was performed using GraphPad Prism and differences were considered significant when P < 0.05. Phase 1 results showed that ovaries cultured with DOX alone showed a reduction in the percentage of normal follicles and in the density of stromal cells. However, the addition of Aloe vera extract to the medium not only attenuated these structural damages but also increased collagen deposition. Furthermore, ovaries cultured with DOX and Aloe vera (10 and 25%) showed a reduction in TNF-α immunostaining and an increase in mRNA expression for antioxidant enzymes (SOD, CAT and NRF2). The results of phase 2 showed that 0.1 and 1.0 mg/kg of Aloe vera preserved both ovarian follicles and stromal density against DOX-induced damage. In addition, 0.1 and 1.0 mg/kg of Aloe vera reduced TNF-α immunostaining and increased the percentage of collagen fibers. Furthermore, the 1.0 mg/kg dose of Aloe vera increased the mRNA expression for antioxidant enzymes (SOD, CAT and NRF2), while 0.1 mg/kg of Aloe vera maintained these expression levels similar to the saline group. In conclusion, the results obtained indicate that Aloe vera extract, both in vitro and in vivo, demonstrated a protective effect on the ovaries of female mice against damage induced by DOX. This effect was evidenced by increased antioxidant response, reduced inflammation, preservation of the stroma and maintenance of follicular morphology.