Mitigation of letrozole induced polycystic ovarian syndrome associated inflammatory response and endocrinal dysfunction by Vitex negundo seeds

PCOS, a heterogeneous condition associated with chronic inflammation, oxidative stress, and metabolic disturbances deleteriously affects the quality of life and long-term health of affected individuals with the condition [5]. Unfortunately, no satisfactory treatment has been identified for PCOS thus so far. This study aimed to investigate the therapeutic effects of the ethanolic extract of Vitex nigundo seed (VNE) on letrozole induced PCOS. Letrozole, a targeted non-steroidal aromatase inhibitor, suppresses aromatase, leading to elevated testosterone levels and hyperandrogenism, replicating various PCOS phenotypes. Despite letrozole administration, there was a surprising increase in ovarian aromatase. Since only ovarian tissue was quantified and aromatase activity wasn’t directly measured, it’s unclear why aromatase expression remains significantly higher despite the use of an aromatase inhibitor. Though the concentration of aromatase is higher, at that extent the activity of aromatase may not sufficiently be achieved which reflected from the very negligible increase in circulating estradiol level. It is plausible that, this might stem from feedback associated with competitive enzyme inhibition by letrozole. Our findings also reveal a significant increase in testosterone, indicating an impaired aromatization of testosterone to estrogen. The heightened substrate concentration might partially overcome competitive inhibition by letrozole. A subtle, statistically non-significant, overproduction of estrogen might result from this intricate phenomenon which was reported previously in recent study [16]. In this study, letrozole is hypothesized to hinder aromatase activity, inhibiting androgen conversion, resulting in clinically observed elevated testosterone, increased LH, and decreased FSH levels, causing a reproductive hormonal imbalance and multiple cysts development as a result of ovulatory disparities.

Our findings demonstrated the positive impact of VNE on alleviating PCOS symptoms attributed to its potent anti-inflammatory and antioxidant properties along with its regulatory role on reproductive hormones. These results highlight the potential of VNE as a treatment option for PCOS related reproductive and metabolic disorders, addressing the unmet need for effective PCOS management.

Almost 50% of women afflicted with PCOS exhibit overweight or obesity [17]. Excess body weight aggravates insulin resistance followed by metabolic aberration, hyperandrogenism, inflammation and infertility in individuals with PCOS [18]. In current study, we observed the anabolic effect of letrozole on rats with significantly increased relative body weight. However, the hydroethanolic extract of Vitex nigundo seed found to be effective in weight reduction (Table 1). Previous studies are also showing the evidence of similar fashion of weight reducing potential of Vitex nigundo seed [14, 15]. Obesity and body fat percentage have a direct effect on serum estrogen release as a result of increased peripheral aromatizati1on in adipose tissue. This directly impact the LH:FSH ratio [19]. Administration of VNE in the present investigation found with correction of LH:FSH ratio (Fig. 4C). This might be attributed to the direct inhibition of LH production pertinent to the action of cinnamic acid present in Vitex negundo seed extract, which was documented earlier for interaction with estrogen receptor (ER) in the hypothalamus [20]. Molecular docking analysis in the present study further supports the ability of cinnamic acid in modulating ER signalling through limiting the overexpression of ER-α in the ovary (Fig. 9B). Since, the C-terminus part of ER-α contains a ligand-mediated transactivation domain, binding of cinnamic acid to ligand binding domain may limit the overexpression of ER-α followed by altered ER signalling in PCOS. Thus, reestablishing the balance in hypothalamus–pituitary–ovary (HPO) axis leading to reduction in LH followed by the correction of steroidogenic enzymes aromatase as well as 3β-HSD. Cinnamic acid also been found to inhibit the functional activity of 17β-HSD as well as 3β-HSD leading to decreased production of testosterone [21, 22]. Modulating the HPO axis along with regulating steroidogenesis and gonadotropin release, VNE exerts a profound effect on restoring the homeostasis of ovarian hormones, specifically estrogen and progesterone. This restorative mechanism is intricately associated with the elevation of progesterone levels (Table 2), which in turn correlates with augmented numbers of corpus luteum (Fig. 7), indicative of successful ovulation and improved fertility. While, LTZ-induced PCOS rats exhibited increased numbers of follicular cysts with thickened theca cell layer and decreased presence of follicles at different stages (Fig. 7). This morphological change is associated with altered LH:FSH ratio that triggers the proliferation of ovarian theca cells, leading to heightened androgen production, by upregulated steroidogenic enzymes ultimately resulting in hyperandrogenism in PCOS [23]. Diverse patterns of ER expression in various ovarian cells control follicular growth and development. In PCOS, altered expression of ERα and ERβ is linked to abnormal follicular growth and ovulatory dysfunction [24]. Estrogen stimulates thecal androgen production [25]. In PCOS, increased ERα in ovarian thecal cells indicates heightened responsiveness to estrogen, leading to overproduction of androgens. Our histological observation of increased theca cell thickness supports excess androgen production and aligns with elevated ovarian ER-α expression.

The result of the current study depicted in the Fig. 8 displayed an increased immune-expression of NR3C4 or AR protein within the ovarian cells of LTZ treated rats and is in agreement with the observation of others investigations in this time [26, 27]. Upregulation of NR3C4 might be due to the accumulation of endogenous testosterone resulting in impaired folliculogenesis and ovulatory dysfunction [28]. On the other hand, supplementation of VNE showed an antiandrogenic action by suppressing expression of ovarian NR3C4 followed by suppressed serum testosterone release (Table 2). This action of VNE might be ascribed to the possible interaction of plumbagin with NR3C4 indicated by molecular docking analysis in the present study (Fig. 9B). Our study revealed that plumbagin formed hydrogen bonds with Arg752 and Lys808, while also engaging in hydrophobic interactions with Gln711 in the ligand binding domain (LBD). Earlier research highlighted Arg752 and Gln711 as pivotal amino acids for testosterone binding to the LBD of androgen receptor [29]. Analyzing crystal structures of human androgen receptor ligand-binding domains complexed with different agonists has yielded valuable insights into the determinants influencing binding affinity. The interaction between plumbagin and these crucial amino acids (Arg752 and Gln711) likely underpins the observed inhibitory impact on androgen signalling. This interaction could serve as a potential mechanism through which VNE exerts its effects on androgen signalling pathways.

Elevated testosterone levels, acting through androgen receptors in pancreatic cells and hepatocytes, result in hyperinsulinemia and compromised metabolic functions [30]. This sequence of events leads to insulin resistance and the consequent progression of type 2 diabetes mellitus in individuals affected with PCOS. The current study showed that administration of VNE resulted in notable reduction in plasma glucose level in LTZ induced PCOS rats (Fig. 2A). Earlier study also established a hypoglycemic effect of VN in PCOS rats [14]. The hypoglycemic effect could be due to the presence of cinnamic acid and plumbagin that directly influences glucose digestion, absorption and metabolism including increased glucose uptake by restoring GLUT4 translocation to plasma membranes [31, 32]. Assessment of lipid profile in this study revealed hypocholesterolemic effect of VNE. However, no significant change found in triglyceride level either in LTZ treated group or in VNE treated group. The positive control treated only with the VNE showed a hypolipidemic effect which was statistical significance as compared to treated group (Fig. 2B & C). The positive effect on regulation of plasma lipids profile might be due to antioxidant property of active components of VNE [12].

Oxidative stress is another prominent feature of PCOS and is associated with insulin resistance, disrupted steroidogenesis, increased lipid peroxidation, and activation of inflammatory cytokines, leading to impaired reproductive function and metabolism [33]. Oxidative stress hinders the production and release of SHBG by reducing the activity of HNF-4α, which could potentially play a significant role in the development of hyperandrogenism in PCOS [34]. Several studies highlight substantial mitochondrial dysfunction and heightened oxidative stress in PCOS, influencing both reproductive and transgenerational outcomes [35]. In the present study, we examined the expression of MnSOD, localized in mitochondria, emphasizing its direct relevance in addressing PCOS-associated mitochondrial issues. In line with previous reports on LTZ-induced PCOS, the PCOS group in this study demonstrated decreased activity of antioxidant enzymes [36]. However, treatment with VNE successfully protected the ovarian tissue from oxidative stress induced by PCOS (Fig. 3A and B). This might be associated with its active components that could counteract oxidative damage, leading to correction of antioxidant enzyme status in ovarian tissue. This antioxidant action may protect ovarian tissue from oxidative stress thereby preserving follicular health, and improve reproductive outcomes in PCOS.

In the realm of PCOS, oxidative-inflammatory cascades play a prominent factor intricately associated with the pathogenesis and detrimental consequences of the condition [37]. The pivotal role of proinflammatory cytokines has been well-established in regulating ovarian steroidogenesis, follicular maturation, and ovulation, as well as contributing to the development of insulin resistance and hyperandrogenism in PCOS [37]. The interplay between TNF-alpha, IL-6, and NF-ĸB forms a complex signalling pathway that regulates inflammation and immune responses. The current study observed a notably upregulated expression of TNF-α, IL-6 as well as NF-κB in the PCOS group, which was accompanied with metabolic disturbances and hyperandrogenism (Figs. 5 and 6). The efficacy of VNE as an anti-inflammatory agent, as demonstrated earlier [38], was further corroborated by our findings. Present study revealed a significantly repressed expression of TNF-α, IL-6 along with NF-κB in ovarian tissue following VNE supplementation (Figs. 5 and 6). Notably, plumbagin one of the major components found in the seed of VNE (Fig. 1), has been reported to directly inhibiting NF-ĸB activation, subsequently leading to the sequential suppression of TNF-α and other inflammatory cytokines [39]. In addition, Negundin-B, another active compound found in VNE, has been associated with the inhibition of lipoxygenase, an oxidative enzyme [40]. A study found the potential of lipoxygenase inhibitors to reduce TNF-α effectively followed by attenuation of inflammation [41]. Furthermore, present study demonstrates the ability of negundin B to bind with the TNFα-dimer interface that hinders the interaction of TNFα with its receptors (Fig. 9C) which in turn affects the downstream signalling pathways of inflammation.

The interplay between oxidative stress, inflammation, and apoptosis contributes to ovulatory dysfunction in PCOS. Oxidative stress-induced inflammation upregulates the expression of pro-inflammatory cytokines such as TNF-α, promoting apoptosis [42]. Moreover, androgens exhibit proinflammatory properties by the activation of nuclear factor of T-cells (NFAT5), which subsequently triggers the upregulation of TNF-ɑ and IL-1β followed by apoptosis of granulosa cell [43, 44]. Furthermore, prior research indicated the involvement of androgens in facilitating BAX-mediated apoptosis and also negatively impact on the expression of Bcl-2 [45, 46]. Subsequently dysregulated expression of apoptosis-related proteins, including increased BAX and p53, and suppressed Bcl-2 further disrupts the delicate balance between cell survival and death, ultimately leading to impaired ovulation in PCOS [47]. This association is reaffirmed by our present study, which demonstrates a concurrence between elevated androgen levels, heightened NR3C4 expression in a PCOS model, and upregulated expression of p53 and BAX along with downregulated expression of anti-apoptotic Bcl-2. However, VNE administration successfully restored these markers in ovarian tissue, indicating its antiapoptotic role in PCOS (Fig. 6A & B).

VEGF is produced by follicular granulosa and ovarian theca cells in response to gonadotropin stimulation [48]. Additionally, evidence indicates that estrogen primarily through ERα regulates VEGF transcription [49]. Individuals with PCOS exhibit higher concentration of VEGF-B in serum and ovarian follicular fluid, a prominent angiogenic factor, which is positively correlated with insulin insensitivity and inflammation [50]. Elevated VEGF may be the indicator of early changes arising during the progression of PCOS towards hyperplasia and cancer [51]. Present study also evident a similar pattern of VEGF-B level in PCOS group which was brought down following the administration of VNE (Fig. 5). In PCOS, elevated CCND-1 levels, a key regulator of the cell cycle, are associated with inflammation. The concurrent upregulation of ERα expression, along with inflammation, plays a pivotal role in estrogen-induced proliferation by initiating CCND-1 gene transcription and overexpression [52]. This process contributes to abnormal cell proliferation and disturbances in ovarian follicular development, defining the features of PCOS [53]. Present study demonstrates VNE supplementation to alleviate the elevated signalling of CCND-1 in PCOS (Fig. 5). These reduction in VEGF-B and CCND-1 suggests that VNE may exert modulatory effects on key processes such as inflammation-associated angiogenesis and folliculogenesis, potentially through the regulation of cell cycle progression.