The successful establishment of a PCOS rat model with various metabolic and reproductive phenotypes similar to those of human PCOS has been well documented with the use of Letrozole [20]. In line with the findings of previous investigations, our results also revealed an abnormal estrous cycle, hormonal imbalance, metabolic alterations, inflammation and ovulatory dysfunction following LET treatment for 21 consecutive days [30, 31]. The present investigation suggested the positive impact of ARE on alleviating the diverse symptoms associated with LET-induced PCOS. The most bioactive secondary metabolites found in ARE previously demonstrated a wide range of therapeutic potential in maintaining metabolic, neuroendocrine and reproductive homeostasis.
Hyperandrogenism and steroidogenic abnormalities in PCOS negatively affect the HPG axis and gonadotropin release [3]. Similarly, the LET-induced PCOS group exhibited hyperandrogenism, as indicated by increased serum testosterone levels (Fig. 4). This excess androgen accumulation eventually disrupted the function of the HPG axis, leading to overactivation of LH and suppression of FSH [32]. Additionally, increased concentrations of ovarian 3βHSD in response to excess LH secretion resulted in steroidogenic disruption and hormonal imbalance in PCOS individuals [31]. Alterations in circulating sex steroids contributed to an abnormal estrous cycle with a prolonged diestrus phase in LET-induced PCOS (Fig. 2).
The neuroendocrine actions of ARE decreased LH hypersecretion and maintained the proper gonadotrophic (LH:FSH) ratio in the PCOS group by modulating the HPG-axis and GnRH pulsatile secretion. The most prominent neutraceuticle of ARE, sarsasapogenin (416.51 m/z) previously displayed HPG-axis regulatory properties via the Kiss1/GPR54 system, which subsequently balanced gonadotropes [33]. Moreover, ARE administration alleviated excess androgen production by limiting the concentration of the ovarian steroidogenic enzyme 3βHSD, which further improved hormone fluctuations and typical estrous patterns. A previous investigation suggested that apigenin inhibits 3βHSD through interference with the steroidogenic pathway, resulting in indirect suppression of testosterone production [34]. Moreover, isoflavones and quercetin both exhibit testosterone lowering activity and estrous cycle regularity in letrozole-induced PCOS animals [35, 36]. Additionally, both the phytotherapeutic genistein and the isoflavones of ARE have been shown to antagonize AR expression by regulating the transcriptional activation and nuclear translocation of AR [37]. Thus ARE supplementation mitigated hyperandrogenism-mediated excessive AR signalling in ovarian tissue (Fig. 9), improving the GC layer and ovulatory functions.
Previous evidence has indicated that excess androgen in women with PCOS often disturbs systemic metabolism by enhancing fat deposition and insulin resistance [2]. The LET-induced PCOS group exhibited a spike in fasting glucose and triglyceride levels, which contributed to a high TyG index (Fig. 3). The TyG index is a reliable indicator for assessing insulin resistance in the present study [38]. A hyperglycemic state increases free testosterone in the bloodstream by decreasing SHBG production while insulin resistance triggers theca cells to produce more androgens [39]. Insulin resistance is strongly associated with hyperleptinemia, which plays a crucial role in the development of obesity by affecting peripheral tissues [40].The LET-treated PCOS group exhibited greater leptin levels and increased body weight, suggesting an association with increasing adiposity in the present study (Fig. 5; Table 2).
Metabolic disruptions associated with PCOS were effectively reversed following ARE therapy, which improved hyperandrogenism mediated insulin resistance, leading to enhanced glucose and lipid metabolism. The antihyperglycemic effect of ARE significantly reduced blood glucose levels and increased insulin secretion, as confirmed by the decrease in the TyG index (Fig. 3). Quercetin regulates glucose by activating AMPK/insulin-independent pathways, enhancing GLUT-4 receptors on the cell membrane to facilitate glucose uptake [41]. The suppression of the key enzymes involved in gluconeogenesis and the protection of β-cells by quercetin [42], contributed to the antidiabetic effects of ARE. ARE administration also decreased triglyceride and cholesterol levels (Fig. 3) by regulating androgen-driven lipid homeostasis. The hypolipidemic efficacy of ARE was likely due to the presence of β-sitosterol, which was previously shown to lower blood cholesterol levels by impeding its absorption in the intestines [43]. γ -Linolenic acid in ARE was previously shown to decrease leptin levels by upregulating PPAR-γ signalling [44]. Thus ARE supplementation reduced PCOS-induced central adiposity with hyperleptinemia by promoting lipolysis and decreasing lipogenesis (Fig. 5).
Although free radicals are essential for regulating ovarian physiological processes, excessive ROS production can increase the risk of ovarian dysfunction by altering intraovarian redox [45]. Previously, an LET-induced PCOS model showed the involvement of oxidative stress and an inflammatory state in the formation of cystic follicles [31]. The reduced expression of ovarian antioxidative enzymes (SOD, CAT and GPx) in the present study suggested a greater rate of oxidative stress in the LET-treated group (Fig. 6). This oxidative stress further stimulates the activation of proinflammatory mediators that may lead to hyperandrogenism and IR [46]. Additionally, hyperinsulinemia promotes granulosa cell (GC) apoptosis, which ultimately results in follicular atresia and ovulatory disorders [8]. Our results also demonstrated increased expression of proinflammatory (NFκB, TNFα and IL-6) and proapoptotic (P53 and Bax) mediators but reduced expression of antiapoptotic (BCL2) markers following LET administration (Fig. 7).
The potent antioxidative action of ARE enhances the expression of endogenous antioxidant enzymes and neutralizes the excessive intraovarian ROS production triggered by metabolic alterations in the PCOS group. Moreover, apigenin significantly decreases total oxidative stress (TOS) levels and increases total antioxidant capacity (TAC) in the ovarian tissues of individuals with PCOS [47]. Moreover, quercetin was also proven to enhance SOD, CAT and GPx activities by inhibiting NADPH oxidase, safeguarding cells from oxidative damage-induced lipid peroxidation [36]. Moreover, the ARE also subsequently inhibited the OS-linked redox-sensitive activation of ovarian inflammatory and apoptotic signalling in PCOS rats which might result in improved cellular homeostasis. The presence of sarsasapogenin was previously reported to suppress LPS-induced acute tissue inflammation by halting IKK/NF-kB/JNK mediated inflammatory signalling [48]. Sarsasapogenin decreases site-specific phosphorylation of IKK and JNK while increasing IκB-α protein levels, collectively contributing to the inactivation of NFκB transcription [48, 49]. However, ferulic acid also impeded NFkB activation by reducing proinflammatory mediators (TNF-α and IL-6) and hindering the STAT1/PIAS1 downstream signalling pathway [50]. Therefore, ARE suppressed NFkB-TNF-α-IL-6 mediated ovarian tissue inflammation, which further improved the follicular microenvironment and minimized ovarian ailments in PCOS rats. Moreover, ARE attenuated OS-mediated ovarian cellular apoptosis by suppressing proapoptotic BAX and P53 expression and enhancing antiapoptotic BCL2 expression (Fig. 7). Thus ARE could play a critical role in normalizing the follicular maturation process and improving oocyte quality. In the ARE, gallic acid regulates the expression of the apoptotic markers BAX and BCL2, modulating PI3K/AKT signalling [51]. In addition, ARE also controlled the overactivation of CCND1 and prevented abnormal cell cycle phase transition. The presence of cinnamic acid in ARE limits excessive cellular proliferation by inhibiting Cyclin D1 [52].
PCOS individuals often experience abnormal angiogenesis, characterized by increased stromal vascularisation and increased levels of proangiogenic markers [9]. Deregulation of ovarian vascularization in LET-induced PCOS was strongly modified following ARE supplementation by preventing the overproduction of the proangiogenic factor VEGF-B (Fig. 5). Activation of the VEGF gene occurs when androgens bind to AR binding sites located within its promoter region [53]. The phenolic compound gallic acid has been shown to demonstrate an antiangiogenic effect on ovarian cells through the modulation of PTEN/AKT/HIF-1α/ VEGF signalling [54]. ARE-mediated restoration of appropriate angiogenic siganalling during the ovarian cycle may further facilitate ovulation and corpus luteum formation by regulating folliculogenesis.
Consistent with earlier investigations, our results revealed the presence of multiple cystic follicles with degenerative granulosa layers, increased follicular atresia and reduced secretory glands with endometrial walls in the histomorphology of the ovaries and uterus of the PCOS group [30, 31]. The hyposecretion of FSH impairs the follicular maturation process and development of the corpus luteum, leading to decreased secretion of progesterone in PCOS group [55]. The rejuvenating action of ARE restored gonadal weight and morphology in PCOS rats through the regulation of hormonal and inflammatory signalling. This further enhanced the proliferation of healthy follicles with proportionate thecal vascularization and reduced cyst formation and follicular atresia. The restoration of the follicular ovulatory process following ARE therapy was confirmed by the appearance of secondary and tertiary follicles with thickened granulosa layers and developing oocytes in the PCOS group (Fig. 8A). Moreover, ARE maintained luteinization regularity and upregulated the growth of the corpus luteum which further increased progesterone levels (Figs. 4 and 8A). The corpus luteum is acknowledged for its vital role in progesterone secretion, a key factor in governing reproductive cycles and preparing the uterus for potential conception [56]. The phytoprogestin-like property of kaempferol thickens the uterine lining by regulating the expression of progesterone-associated genes [57]. In addition, β-sitosterol also regulates endometrial receptivity in individuals with PCOS via harmonized reproductive hormonal levels [58]. Thus, ARE renovated uterine morphology by maintaining uterine weight, endometrial width and the number of secretory glands in PCOS group (Fig. 8B).
Taken together, the present investigation provides the fresh evidence that compared with the standard drug metformin, ARE at a dosage of 250 mg/kg bw is better able to reverse endrocrinal dysregulation in PCOS. In addition, the oxidative stress, angiogenic and metabolic regulatory properties of ARE strongly support the use of A. racemosus as an alternative supplementary therapeutic approach for addressing PCOS and associated fertility issues. Nevertheless, additional exploration is necessary to assess the prolonged usage of this herb and its efficacy compared with the other PCOS drugs.