Synergistic effects of AgNPs and zileuton on PCOS via ferroptosis and inflammation mitigation

Abstract

Background: The most prevalent endocrine disorder affecting women is PCOS. Programmed death of ovarian cells has yet to be elucidated. Ferroptosis is a kind of iron-dependent necrosis featured by significantly Fe⁺²-dependent lipid peroxidation. The ongoing study aimed to reinforce fertility by combining therapy with AgNPs and (Zileuton) in PCOS rats' model.

Methods: The study included 75 adult female rats divided into 5 groups; control, PCOS, PCOS treated with AgNPs, PCOS treated with Zileuton, and PCOS group treated with AgNPs and Zileuton. The study investigated the anti-ferroptotic, anti-inflammatory, antioxidant, antiapoptotic, histopathological and immunohistochemical examinations of COX-2 and VEGF.

Results: The combination of AgNPs and Zileuton showed significant reduction of inflammatory mediators (IL-6, TNF-α, NFk-B) compared with diseased group (P-value < 0.05), regression of ferroptosis marks (Panx1 and TLR4 expression, Fe⁺² levels) compared with diseased group (P-value < 0.05), depression of apoptotic marker caspase 3 level compared with diseased animals (P-value < 0.05), depression of MDA level, elevation of HO-1, GPx4 activity, and reduction of Cox2 and VEGF as compared with the diseased, AgNPs or zileuton-treated groups (P-value < 0.05).

Conclusion: The study showed that the combination of AgNPs and zileuton guards against, inflammation, apoptosis, and ferroptosis in PCO.

Keywords: AgNPs; COX-2; Ferroptosis; PCOS; Panx1; TLR4; VEGF; Zileuton.

Figure 1.

Relative Panx1 mRNA expression. Data are expressed as mean ± SD. Panx1: pannexin 1. ^a p < 0.05 vs. control group I, ^b p < 0.05 vs. PCO group II, ^c p < 0.05 vs. AgNPs- treated group III, ^d p < 0.05 vs. Zileuton- treated group IV, ^e p < 0.05 vs. AgNPs + Zileuton co- treated group V.

Figure 2.

Relative TLR4 mRNA expression. Data are expressed as mean ± SD. TLR4: toll-like receptor 4. ^a p < 0.05 vs. control group I, ^b p < 0.05 vs. PCO group II, ^c p < 0.05 vs. AgNPs- treated group III, ^d p < 0.05 vs. Zileuton- treated group IV, ^e p < 0.05 vs. AgNPs + Zileuton co- treated group V.

Figure 3.

Photomicrographs of H&E-stained sections of ovaries: Normal control: ovarian follicles in various stages of maturation: primary follicles (PF), multilaminar follicles (MF) and secondary follicles (Sf) which are seen with apparent Oocytes (O) surrounded by corona radiata and follicular antrum (FA). The thick zona granulosa layer (double head arrow) and corpus luteum (CL) are obvious. (a. H&E × 100; scale bar:200 μm, b. and c. H&E × 200; scale bar:100 μm).

Figure 4.

Photomicrographs of H&E-stained sections of ovaries: In the PCOS model group, all sections show the development of follicular cysts (FC), with absent oocyte and thin granulosa cells (double head arrow) with some congested blood capillaries (BV). Also, multiple vacuolations (stars) are noticed with areas of hemorrhage (arrow head) and cellular infiltration (yellow arrow) (a. H&E × 40; scale bar:500μm, b., c. and d. H&E × 100; scale bar:200μm).

Figure 5.

Photomicrographs of H&E-stained ovarian sections from PCO treated with AgNPs group demonstrate some cystic follicles (FC), However, mature follicle with thick granulosa (double head arrow), theca cell layer (yellow arrow), an apparent oocyte (O) surrounded by corona radiata (blue arrow), and follicular antrum (FA) are clearly seen (Figure 5a). In PCO treated with the Zileuton group: mature follicles with an oocyte (O) surrounded by corona radiata (blue arrow), follicular antrum (FA) and corpus luteum (CL) are obvious. Although, some follicular cysts (FC) are present (Figure 5b). In the combined treated group (PCO treated with AgNPs + Zileuton): the Mature Graffian follicle is clearly seen with an apparent oocyte (O) and thick granulosa layer (double head arrow). Also, multilaminar follicles (MF) and corpus luteum (CL) are seen. No follicular cysts are detected (Figure 5c) (H&E × 100; scale bar:200 μm)

Figure 6.

Photomicrographs of COX-2 immunohistochemically stained ovarian sections: (A) In the control group, COX-2 immunoreactivity is limited to stroma with no expression in the follicles. (B) The PCOs sections demonstrate strong immunoreactivity to COX2 in granulosa and theca layers & follicular antrum. (C &D): In PCO treated with AgNPs and PCO treated with Zileuton groups, the immunostaining of COX-2 is shown in the granulosa and theca layers & antrum is less intense than in the PCOS group but still higher than control. (E) COX-2 immunostaining in the last combined treated group presents no expression in the follicles and is limited to stroma in a way similar to the sections of ovaries of the control group. (COX-2; scale bar:200 μm)

Figure 7.

Photomicrographs of VEGF immunohistochemically stained ovarian sections: (A): In the control group, the expression of VEGF is limited to the stroma and granulosa layer. (B): The PCOs show strong immunoreactivity to VEGF in granulosa and theca layers & follicular antrum, and diffuse expression in ovarian stroma. (C&D): In PCO treated with AgNPs and In PCO treated with Zileuton groups, VEGF shows less expression of VEGF in the follicular antrum than PCO group but is still apparently higher than in the control. (E): In the last combined treated group the immunostaining of VEGF is seen in the stroma and very little expression in the follicles similar to the control group. (VEGF; scale bar:200μm)

Figure 8.

(A) Represents Cox2 optical density. Data are expressed as mean ± SD. ^a p < 0.05 vs. control group I, ^b p < 0.05 vs. PCO group II, ^c p < 0.05 vs. AgNPs- treated group III, ^d p < 0.05 vs. Zileuton- treated group IV, ^e p < 0.05 vs. AgNPs + Zileuton co- treated group V. (B) Represents VEGR area percentage. Data are expressed as mean ± SD. ^a p < 0.05 vs. control group I, ^b p < 0.05 vs. PCO group II, ^c p < 0.05 vs. AgNPs- treated group III, ^d p < 0.05 vs. Zileuton- treated group IV, ^e p < 0.05 vs. AgNPs + Zileuton co- treated group V.