In the present study, the ameliorative effects of Chn/Al-AH in EV-induced POCS rats were investigated for the first time. To induce the PCOS model in rats, Estrogen and drugs with estrogenic effects are commonly used, as these compounds prompt morphological effects similar to those seen in PCOS [25]. Similar to the findings of Farhadi-Azar et al. [26], Our study indicated that EV is efficient enough to induce PCOS in rats.
Furthermore, this study revealed a greater weight gain in the PCOS group than in the control group, which is consistent with earlier studies [27]. However, all treated animals with Chn/Al-AH showed reduced body weight. Ovarian biopsy and biochemical parameters are commonly used for PCOS diagnosis. In this study, we considered both histological and biochemical evaluations. Abnormal changes in sex hormone levels, such as elevated estrogen, testosterone, and LH, and decreased FSH and progesterone levels, are major diagnostic criteria for PCOS [28]. In our study, the induction of PCOS significantly increased serum levels of estrogen and testosterone while decreasing serum progesterone levels. These findings are consistent with previous studies [27, 29]. A significant decrease in estrogen levels was observed after treatment with Chn/Al-AH. In parallel, the administration of Chn/Al-AH significantly increased progesterone levels in the treatment groups. These results are in line with previous studies [27, 29]. Furthermore, we observed a significant decrease in serum testosterone levels after treatment with all doses of Chn/Al-AH, which aligns with previous studies [29, 30]. We did not observe significant changes in LH and FSH levels in the groups receiving Chn/Al-AH compared to the PCOS group.
An increased LH-to-FSH ratio is a characteristic of PCOS [31]. In our study, the LH/FSH ratio in the PCOS group was statistically higher than in the control group. Chn/Al-AH significantly reduced the LH/FSH ratio in the treatment groups when compared to the PCOS group. These results are consistent with the previous studies [30, 32].
Histomorphologically, the presence of cystic follicles along with the absence of corpus luteum represent an indicator of ovarian dysfunction and anovulation. It could be correlated with an unusual rise in serum levels of androgens, indicating PCOS [33]. Subsequently, it causes to decrease or lack of progesterone which leads to endometrial overgrowth and hyperplasia [34].
Focusing on our results, EV-induced PCOS showed an increased number of cystic follicles, absence/or very few corpus luteum along with fewer antral or Graffian follicles [35, 36], which is considered as the cessation of folliculogenesis. Chn/Al-AH increased corpus luteum and follicles at different stages of development and decreased cystic follicles, indicating the recovery of the normal histology and finally ovulation in PCOS rats, aligned with the results of previous studies [36–39]. According to the findings of this study, Chn/Al-AH at 10 mg and 15 mg doses had better-normalizing effects on ovarian morphology.
The presence of endometrial hyperplasia in the uteri is considered a histological indicator of PCOS [40]. Our results indicated that abnormal alteration of uterine morphology including hyperplasia, decreased number of endometrial glands, the appearance of cystic glands, and thickening of the lining epithelium were obvious in the PCOS group consistent with previous studies [41, 42]. In this study, two types of endometrial glands were detected in the uterine sections according to Gunin et al. [43] including normal and cystic glands. Photomicrographs of the control group showed normal endometrial glands represented as simple tubular glands with an oval, round, or elongated shape and narrow lumen. In the PCOS group, cystic glands with a large size and round shape were observed.
Treatment with 10 and 15mg of Chn/Al-AH revealed the relative resuming of normal uterine structure. The epithelium thickness decreased and the number of normal endometrial glands increased after treatment. These findings agree with the results of previous studies, in which various treatments including Silibinin [42], Melatonin and Metformin [44], Vitamin D [45], Metformin [41], dodder total flavone [46], and Ageratum conyzoides [36] were used.
The study by Adelakun et al. demonstrated a correlation between higher levels of TG, TC, and LDL on one hand, and lower levels of HDL on the other hand, with anovulation in PCOS [36]. The findings of this study revealed a significant increase in serum concentrations of TC, LDL, TG, and VLDL, along with a significant decrease in HDL levels in the EV-induced PCOS rats. Treatment with 10 mg and 15 mg doses of Chn/Al-AH significantly reversed the abnormal lipid profiles observed in the PCOS group. These results are consistent with previous findings, such as those from studies using Sumac [47], Genistein for LDL [37], Polyherbal syrup [29], Ginger [48], Ageratum conyzoides [36], and Agaricus subrufescens for TG and TC [49].
Given the crucial role of pro-inflammatory factors in determining the inflammatory state in PCOS, this study assessed serum levels of TNF-α, IL-6, and IL-18. Previous studies have reported elevated levels of TNF-α and IL-6 in PCOS [36, 50]. Several studies have shown significant reductions in the levels of these factors after treatment with compounds such as Gallic Acid [51], hawthorn leaf flavonoids [52], Quercetin [53], resveratrol for TNF-α [32], saffron [54], and Ageratum conyzoides [36].
In the present study, increased levels of TNF-α, IL-6, and IL-18 were observed in the PCOS group, likely attributed to the effectiveness of EV in inducing PCOS in rats. Chn/Al-AH-treated groups at doses of 10 mg and 15 mg demonstrated a significant reduction in TNF-α and IL-6 levels when compared to the PCOS group. These findings are consistent with the previously mentioned studies. However, it is noteworthy that our results regarding IL-18 did not exhibit significant changes after treatment with the Chn/Al-AH.
It has been demonstrated that increased miR-222 expression is linked to PCOS and may play a significant role in the development of this condition [55].
The results obtained from this study showed a noteworthy increase in miRNA-222 expression in the PCOS group when compared to the control group, consistent with the earlier studies [12].
A significant decrease in miR-222 expression was observed after treatment with all doses of the Chn/Al-AH in comparison to the PCOS group. These outcomes concur with the findings of Huang et al. and Almalki et al., in which the expression levels of miRNA-222 exhibited significant decreases following treatment [12, 56]. It has been well-documented that the upregulation of miRNA-222 in PCOS negatively regulates estrogen receptor alpha [57]. Gianpiero Di Leva et al. reported a reciprocal negative regulatory loop between miRNA-222 and the ESR1 gene, wherein miRNA-222 represses ESR1[58].
In this study, neither the control group nor the treatment groups displayed significant differences in the expression of ESR1 when compared to the PCOS group. It may be due to that miRNA-222 might indirectly facilitate ESR1/estrogen signaling by directly inhibiting BECLIN1. The interaction between ESR1 and BECLIN1 could potentially influence the functions of both BECLIN1 and ESR1 as downstream targets of miRNA-222. It is conceivable that a reciprocal regulatory mechanism between BECLIN1 and ESR1 might yield unfavorable outcomes, including the negative modulation of ESR1 [59]. Furthermore, endometrial hyperplasia seems to be dependent on ESR1 activation [60]. Our treatments were not successful enough to completely improve the uterine tissue changes and resume the normal uterine tissue architecture including the absence of hyperplasia. It may be due to the insufficient time duration of our treatment. This area remains an important subject for future investigations to address the effect of long-term treatment with Chn/Al-AH on endometrial hyperplasia and its relation with the expression of ESR1.
Given the widespread and unbridled use of natural herbs and edible products by the general population, it has become necessary to assess their safety on body functions including the female reproductive system. For the first time, the findings of this study indicate that Chn/Al-AH improves the ovary function impairments in the PCOS, as evidenced by a significant decrease in cystic follicles, LH/FSH ratio, serum levels of TNF-α, IL-6, triglyceride, cholesterol, LDL, estrogen, and testosterone and miRNA-222 expression and also a significant increase in corpus luteum, serum levels of progesterone, HDL, and ESR1 expression (Fig. 9). In summary, Chn/Al-AH can achieve a balance in histological, hormonal, lipid profile, inflammatory and genetic aspect of PCOS.
However, it is important to note that this study has its limitations. There was no group treated with a routine drug commonly used to treat PCOS, such as metformin. Including such a group in future studies would allow for a direct comparison between the effects of Chn/Al-AH and conventional treatments.