Fig 1.
Experimental Plan for Assessing P. dioscoridis’s Impact in a PCOS Rat Model.
The figure describes a two-step experimental procedure that involves a biological experiment after P. dioscoridis characterization. Treatments were administered to five experimental groups: Control (0.5% CMC-Na), P. dioscoridis (100 mg/kg), letrozole (1 mg/kg), letrozole + metformin (300 mg/kg) and letrozole + P. dioscoridis. Towards the end of the study, behavioral tests including the forced swim test (FST), elevated plus maze (EPM), and light-dark box (LDB) were carried out. Daily vaginal smears, brain tissue PCR, ovary histology, serum ELISA, and dopamine receptor analysis were conducted. The timeline emphasizes sacrifice points, behavioral evaluations, and treatment duration.
Table 1.
PCR gene expression for dopamine receptor 2, MAO-A and DOPA decarboxylase.
Table 2.
Annotated metabolites in P. dioscoridis EtOH extract via UPLC-MS-MS.
Fig 2.
Representative UPLC-MS-MS chromatogram of P. dioscoridis ethanolic extract performed in (A) negative and (B) positive ionization mode.
Fig 3.
Vaginal smear of normal control group showing the four stages of the estrous cycle.
(A) Proestrous phase containing predominantly small nucleated epithelial cells (black arrow). (B) Estrous phase characterized by anucleated keratinized epithelial cells (green arrow). (C) Metestrous phase: in this phase, leukocytes (blue arrow), small nucleated epithelial cells, and anucleated keratinized epithelial cells are present. (D) Diestrous phase mainly comprises of leukocytes (blue arrow).
Fig 4.
Vaginal smear at different stages of estrous cycle after PCOS induction and treatment.
(A) PCOS group with diestrous phase. (B) Metformin treated group with metestrous phase. (C) P. dioscoridis treated group with estrous phase.
Fig 5.
Effect of metformin and P. dioscoridis on anxiety and depressive like behaviors in letrozole-induced PCOS.. Data are represented by Means
± SE. All data were analyzed using ANOVA followed by Bonferroni post hoc test. a (significant versus CTRL group), b (significant versus P. dioscoridis group), and c (significant versus PCOS).
Fig 6.
Effect of metformin and P. dioscoridis on sex hormonal profile and brain dopamine level in letrozole-induced PCOS.
Data are represented by Means ± SE. All data were analyzed using ANOVA followed by Bonferroni post hoc test. a (significant versus CTRL group), b (significant versus P. dioscoridis group), c (significant versus PCOS), and d (significant versus metformin+ PCOS group).
Table 3.
Pearson correlation between brain dopamine levels and behavioral analysis.
Table 4.
Pearson correlation between brain dopamine levels and hormonal results.
Fig 7.
A photomicrograph of sections of the ovary in the control and P. dioscoridis groups [H&E x100 (A, F), H&E x200 (B-E)].
A) The control group [H&E x 100] shows normal architecture of the ovary with superficial cortex (C) and deep medulla (M). The cortex has various ovarian follicles. The medulla has loose connective tissue rich in blood vessels (BV). Multilaminar primary follicle (MF), secondary follicle (SF), and corpus luteum (CL) are seen in the cortex. B) A higher magnification of the previous section [H&E x 200] shows unilaminar (UF) and multilaminar (MF) primary follicles. UF is formed of a single layer of cuboidal follicular cells surrounding an oocyte. MF is formed of several rows of cuboidal granulosa cells enclosing an oocyte. A corpus luteum (CL) with small pale theca lutein cells (TL), and large acidophilic granulosa lutein cells (GL) is visible in the section. Blood vessels (BV) are clearly seen. C) A secondary follicle (SF) [H&E x 200] with antral (A) cavity and granulosa (G) cells surrounding an oocyte (O). A layer of theca folliculi (TF) envelopes the secondary follicle. D) Mature Graffian follicle (GF) [H&E x 200] has large antrum (A) contains liquor folliculi surrounded with membrana granulosa (G), theca interna (TI), and theca externa (TE). An oocyte (O) surrounded with zona pellucida (ZP) and corona radiate (CR) is suspended in the antrum. E) A corpus luteum (CL) [H&E x 200] with large granulosa lutein (GL) cells and small theca lutein (TL) cells enclosed within connective tissue capsule (CT), and has a core of CT and is richly vascularized (BV) is seen in the cortex. F) The P. dioscoridis group shows normal structure of the ovary similar to that of the control group [H&E x 100].
Fig 8.
A comparison among study groups regarding: A) Granulosa thickness (µm), B) Theca thickness (µm), C) Number of antral follicles, D) Number of corpora lutea, E) Number of cystic follicles, F) Number of atretic follicles.
Data are analyzed by one-way ANOVA and Bonferroni post hoc test at P < 0.05 and presented as mean ±SE. [a] versus the control group, [b] versus the P. dioscoridis group, [c] versus the PCOS group, and [d] versus the PCOS + metformin group.
Fig 9.
A photomicrograph of sections of the ovary in the PCOS, PCOS
+ metformin, and PCOS + P. dioscoridis groups [H&E x100]. A-B). The PCOS group shows large cystic follicles (CF) in the ovarian cortex with thin granulosa (G) layer and thick theca externa (TE) layer compared to the control group. Many atretic (degenerated) ovarian follicles (AF) are seen in the cortex. C) PCOS + metformin group shows fewer and smaller cystic follicles (CF) in the ovarian cortex compared to that observed in PCOS group. A secondary follicle (SF) and an atretic follicle (AF) also could be detected. D-E) PCOS + P. dioscoridis group shows many corpora lutea (CL) in the ovarian cortex. Multilaminar primary follicle (MF) and secondary follicle (SF) are also present. Few cystic follicles (CF) could be detected in the cortex.
Fig 10.
A photomicrograph of sections of the ovary in different groups [Masson’s trichrome x100].
A-B) The control and P. dioscoridis groups respectively showing greenish collagen fibers (arrow) in the stroma of the cortex (C), medulla (M), the theca externa (arrow) ensheathing the ovarian follicles, the connective tissue of the corpus luteum (CL). C) The PCOS group shows increased collagen fibers (arrow) deposition in the stroma of the cortex and medulla and the theca exterrna (TE) encircling the cystic follicles (CF). D) PCOS + metformin group shows collagen fibers (arrow) deposition in the stroma of cortex and medulla, ensheathing cystic follicles (CF) and blood vessels (BV) nearly similar to PCOS group. E) PCOS + P. dioscoridis group shows decreased collagen fibers deposition in the ovary compared to PCOS group. F) Chart represents color area percentage of greenish collagen fibers in different groups. Data are analyzed by one-way ANOVA and Bonferroni post hoc test at P < 0.05 and presented as mean ±SE. [a] versus the control group, [b] versus the P. dioscoridis group, and [c] versus the PCOS group.
Fig 11.
A photomicrograph of sections of the ovary in different groups [ki67 immunostaining x200].
A) The control group shows ki67 immunoreaction in the granulosa (G) cells of different ovarian follicles, the unilaminar primary (UF), the multilaminar primary (MF), and the secondary (SF) follicle. The reaction appears as a dark brown nuclear staining. B) The P. dioscoridis group shows ki67 immunoreaction in the granulosa (G) cells of ovarian follicles similar to control group. C) The PCOS group shows decreased ki67 positive cells compared to control group. D) PCOS + metformin group shows decreased ki67 positive cells compared to control group. It is nearly similar to PCOS group. E) PCOS + P. dioscoridis group shows increased ki67 positive cells compared to PCOS group. F) Chart represents color area percentage of ki67 immunoreaction in different groups. Data are analyzed by one-way ANOVA and Bonferroni post hoc test at P < 0.05 and presented as mean ±SE. [a] versus the control group, [b] versus the P. dioscoridis group, [c] versus the PCOS group, and [d] versus the PCOS + metformin group.
Table 5.
Effect of metformin and P. dioscoridis ethanolic extract on dopamine receptor 2, DOPA decarboxylase and MAO-A gene expressions in letrozole-induced PCOS.
Fig 12.
Hypothesized P. dioscoridis Mechanism in PCOS.
The pathophysiology of PCOS may be counteracted by P. dioscoridis, as shown in the figure. In addition to hormonal imbalances (decreased estrogen/progesterone, increased LH/FSH/testosterone), PCOS is characterized by neurochemical dysregulation with decreased dopamine and dopamine receptor activity and ovarian abnormalities like cystic follicles. These alterations are linked to depression, anxiety, and disturbances of the HPG axis. P. dioscoridis restores hormone levels, enhances ovarian structure, reduces neurobehavioral disorders, and may restore dopamine levels by upregulating DOPA decarboxylase and blocking MAO-A.