Syzygium cumini Fruit Extract and Quercetin Ameliorate Cadmium-Induced Ovarian Apoptosis in Rats Via miRNA- 204 - 5p-Mediated Bcl- 2 Upregulation and Bax/Caspase 9/Caspase 3 Downregulation

Abstract

The correlation between exposure to environmental pollutants and adverse impacts on fertility has been documented. Cadmium (Cd) is one of the most toxic elements that impairs female reproductive capacity. Syzygium cumini is a plant that contains polyphenols with strong antioxidant properties. Our study aimed to investigate the possible protective effects of Syzygium cumini fruit extract (SCFE) and quercetin (QU) against Cd-induced ovarian toxicity and apoptosis in rats assisted by measuring oxidative stress markers, hormonal profile, intrinsic apoptotic pathway, miRNA-204-5p, and histopathology. For that, 45 adult female rats were divided randomly into five groups; control, cadmium chloride (CdCl₂), CdCl₂ + QU, CdCl₂ + SCFE200, and CdCl₂ + SCFE400, and the experiment lasted 35 days. Cadmium exposure induced irregular estrus cycle, and oxidative challenges, as evidenced by increased malondialdehyde and decreased antioxidant indicators that include superoxide dismutase, catalase and reduced glutathione, and hormonal imbalances, including reduced follicle-stimulating hormone, luteinizing hormone, anti-mullarian hormone, estrogen, and progesterone levels. Additionally, Cd triggered ovarian apoptosis through upregulating miRNA- 204 - 5p expression that in turn downregulated Bcl- 2 and upregulated the members of intrinsic mitochondrial apoptotic pathway including Bax and caspases 3, 9. The histopathological findings, morphometric and lesion scoring in ovarian, and uterine tissues confirmed the negative impacts of Cd. Interestingly, treatment with QU or SCFE, especially at the higher dose (400 mg/kg), significantly ameliorated these Cd-induced adverse effects, suggesting their potential as protective agents against ovarian toxicity.

Keywords: Syzygium; Apoptosis; Cadmium; MicroRNAs; Quercetin.

Fig. 1

LC/MS total ion chromatogram (A positive mode, B negative mode) of the SCFE

Fig. 2

Effects of Syzygium cumini fruit extract and quercetin administration on the body weight (A), ovarian weight (B), gonadosomatic index (C), and estrus regularity (D) in control and treated rats. g, gram; GSI, gonadosomatic index; QU, quercetin; SCFE, Syzygium cumini fruit extract. Values are represented as mean ± SEM (standard error of the mean). ns, means no significant difference (P > 0.05) between groups. In D, data are presented as a number of rats with regular and irregular estrus cycles (out of 9 female rats). Fisher’s exact test was applied at P < 0.05. *Different from the control group (P < 0.05)

Fig. 3

The Ovarian mRNA relative expression levels of Bax, Bcl- 2, miRNA- 204 - 5p, and caspase 3 and caspase 9 in control and treated rats. QU, quercetin; SCFE, Syzygium cumini fruit extract. Values are represented as mean ± SEM (standard error of mean). ap < 0.05 versus control group. bp < 0.05 versus CdCl2 group. cp < 0.5 versus CdCl2 + QU group. dp < 0.5 versus CdCl2 + SCFE 200 group. Using one-way ANOVA followed by Tukey’s post hoc test

Fig. 4

A photomicrograph of ovary of adult female albino rats showing A a photomicrograph of control group showed normal ovarian tissue with mature and growing follicles (F) and contained normal oocyte (arrow) as well as normal ovarian blood vessels (V). (H&E) stain × 100. B A photomicrograph of CdCl₂-treated group exhibited growing ovarian follicles (F) that appeared in pre mature state containing degenerated oocytes (arrow). The ovarian blood vessels (V) appeared highly congested. (H&E) stain × 100. C A photomicrograph of CdCl₂ + quercetin-treated group showed growing follicles (F) containing degenerated oocyte (arrow) and congested blood vessels (V) (H&E). Stain × 100. D A photomicrograph of CdCl2 + low dose group of Syzygium cumini fruit extract (SCFE200) revealed few mature and growing follicles (F) containing normal oocyte (arrow) and normal ovarian blood vessels (V). (H&E) stain × 100. E A photomicrograph of CdCl2 + high dose group of syzygium cumini fruit extract (SCFE400) showed many mature and growing follicles (F) containing normal oocyte (arrow) and normal ovarian blood vessels (V). (H&E) stain × 100

Fig. 5

A photomicrograph of uterus of adult female albino rats showing A a photomicrograph of control group showed endometrium covered with simple columnar layer (arrow) over thick submucosal layer and contained numerous active endometrial glands (arrowhead). H&E × 100. B A photomicrograph of CdCl₂-treated group showed degeneration and stratification of uterine epithelium (arrow) as well as inactive collapsed endometrial glands (arrow head). (H&E) stain × 100. C A photomicrograph of the CdCl₂ + quercetin-treated group showed normal endometrial epithelium (arrow) and few less active endometrial glands (arrow head). H&E × 100. D A photomicrograph of CdCl2 + low-dose group of Syzygium cumini fruit extract (SCFE200) showed normal endometrial epithelium (arrow), and the endometrial glands (arrow head) became active. H&E × 100. E A photomicrograph of CdCl2 + high-dose group of Syzygium cumini fruit extract (SCFE400) showed normal endometrial epithelium (arrow) and the endometrial glands (arrow head) became numerous and highly active. H&E × 100