Amaranthus spinosus Linn. Extract as an Innovative Strategy to Regulate Biomarkers for Ovarian Hyperthecosis via Circular RNA (hsa-circ-0001577): Evidence From Biochemical, Metabolomics, Histological, and Phytochemical Profiling

Abstract

Amaranthus species, including A. spinosus Linn, are well-known vegetables whose leaves, shoots, fragile stems, and grains are commonly utilized as herbs in soups or sauces, aside from traditional uses to treat a wide range of illnesses. Ovarian hyperthecosis is a common syndrome associated with metabolomics and endocrinology that lowers female fertility. The investigation of novel biomarkers and targeted therapies for the detection and treatment of ovarian hyperthecosis is of interest. Types of noncoding RNAs known as circular RNAs (circRNAs) have covalently closed cyclic structures, are widely distributed, and exhibit expression patterns that are particular to different stages of development. Ovarian hyperthecosis was induced in rats via dehydroepiandrosterone (DHEA) followed by 1 month of treatment with 50 and 100 mg/kg of the A. spinosus EtOH extract. Further, oxidative stress biomarkers including GSH and MDA were investigated in addition to hormonal biomarkers, such as Luteinizing hormone and testosterone hormone, a metabolomics approach modeled using orthogonal partial least squares discriminant analysis (OPLS-DA), and circRNA (hsa-circ-0001577). Furthermore, UHPLC-ESI-Orbitrap-MS analysis was used for metabolites profiling to identify active agents in the plant extract. Results revealed a significant improvement in these biomarkers in the DHEA group treated with A. spinosus, especially at high doses, and further confirmed via histopathological assays. Multivariate data analyses of serum metabolome indicated significant variations in serum profiles among normal, disease, and treated groups. Variable importance in the projection (VIP) values guided the selection of differentiated metabolites, revealing significant changes in metabolite concentrations. UHPLC-ESI-Orbitrap-MS analysis identified 72 bioactive metabolites belonging to phenolics, triterpenoidal saponins, and pyridines In conclusion, A. spinosus could be a management approach for ovarian hyperthecosis therapy via regulating circRNA (hsa-circ-0001577), disturbed hormonal balance, and metabolomics biomarkers based assays.

Keywords: Amaranthus spinosus; circular RNAs; metabolomics; ovarian hyperthecosis; phytochemical profile.

FIGURE 1

Effect of treatment with Amaranthus spinosus EtOH extract (50 and 100 mg/kg, orally, once daily, for 4 weeks) on circRNA (hsa‐circ‐0001577) level. Values are expressed as mean ± SEM, n = 6. Data were analyzed using one‐way ANOVA followed by Tukey's post hoc test, with different letters indicating significant differences at p < 0.05).

FIGURE 2

Effect of Amaranthus spinosus EtOH extract (50 and 100 mg/kg, orally, once daily, for 4 weeks) on GSH level (A) and MDA activity (B). Values are expressed as mean ± SE, n = 6. Data were analyzed using one‐way ANOVA followed by Tukey's post hoc test, with different letters indicating significant differences at p < 0.05.

FIGURE 3

Effect of Amaranthus spinosus EtOH extract (50 and 100 mg/kg, orally, once daily, for 4 weeks) on LH (A), testosterone (B), and FSH (C) levels. Values are expressed as mean ± SE, n = 6. Data were analyzed using one‐way ANOVA followed by Tukey's post hoc test, with different letters indicating significant differences at p < 0.05.

FIGURE 4

Photomicrographs of: Control untreated group (G1): Rat ovary showing normal histological structure of ovarian follicles (arrows), DHEA group (G2): Rat ovary showing multiple ovarian follicular cysts (arrows), DHEA group (G2): Rat ovary showing multiple follicular cysts (arrows), Amaranthus spinosus EtOH (50 mg/kg) group (G3): Rat ovary showing follicular cysts (arrows), and A. spinosus EtOH (100 mg/kg) group (G4): Rat ovary showing only one follicular cyst (arrows); (H&E ×100).

FIGURE 5

Score scatter plot of OPLS‐DA models (A) all four groups (control, ovarian hyperthecosis‐diseased, and dose‐based Amaranthus spinosus ‐treated groups), (B) control and ovarian hyperthecosis‐diseased groups, and (C) ovarian hyperthecosis‐diseased and dose‐based A. spinosus ‐treated groups.

FIGURE 6

Dendrogram‐heatmap displaying serum metabolic profiles of control, diseased, and after Amaranthus spinosus treatment groups.

FIGURE 7

Key metabolic pathways revealed by enrichment analysis of differential metabolites in rats' sera. The node size indicates the pathway impact value.

FIGURE 8

Flow charts illustrating key metabolic pathways (to the left) and box plots (to the right) demonstrating normalized serum levels of differential metabolites in control (red box), diseased (green box), and treated (blue box) groups associated with each enriched pathway: (A) tryptophan metabolism, (B) nicotinate and nicotinamide metabolism, and (C) arginine biosynthesis pathway.