Rhamnocitrin Attenuates Ovarian Fibrosis in Rats with Letrozole-Induced Experimental Polycystic Ovary Syndrome

Abstract

Polycystic ovary syndrome (PCOS) is a common endocrine-related cause of infertility in women and has an unknown etiology. Studies have shown that rhamnocitrin (Rha) exhibits positive effects on the reproductive system. This study investigated Rha's antifibrotic effects on PCOS rats and revealed its underlying mechanisms. Female SD rats were randomized into 4 groups (n = 8, each); the control group received tea oil by intraperitoneal injection and 1% w/v CMC by oral gavage; the PCOS group received letrozole (1 mg/kg); the PCOS+Rha group received letrozole and Rha (5 mg/kg); the PCOS+Met group received letrozole and Met (265 mg/kg) for 21 days. At the study end, Rha treatment restored letrozole-induced alterations in the relative ovarian weights, body weight, and relative weights of uterine and visceral adipose tissues. Histological observation showed that Rha ameliorates ovarian structure and fibrosis in PCOS. Administration of Rha reduced letrozole-induced metabolic dysfunction by ameliorating the levels of TC, TG, and HDL-C in the PCOS rats. Rha treatment also modulated the serum levels of sex hormones, which decreased T, E2, and LH and increased FSH in PCOS rats. In addition, Rha treatment modulated insulin resistance and increased gene expression of antioxidant enzymes (Cat, Sod2, Gpx3, Mgst1, Prdx3, Gsta4, Gsr, and Sod1) in the ovaries of the PCOS rats. Finally, Rha treatment appeared to increase the activity of PPAR-γ and inhibit the TGF-β1/Smad pathway in the ovaries of the PCOS rats. Our findings suggest that Rha significantly ameliorated metabolic disturbances and ovarian fibrosis in the PCOS rats. Rha perhaps is an effective compound for preventing ovarian fibrosis in the future.

Figure 1

Picture of each estrous cycle in rats. Cellular characteristics of the estrous cycle were determined by Papanicolaou staining assays (×100).

Figure 2

Effects of rhamnocitrin (Rha) on ovarian, uterine, and periuterine adipocyte morphology. (a) Photographs of the morphology of the ovaries and uterus. (b) HE staining of ovarian tissue (scale bar = 500 μm; black box defines the area to be amplified, high magnification scale bar = 20 μm). (c) Masson's trichrome staining of ovarian tissue (scale bar = 200 μm; black box defines the area to be amplified, high magnification scale bar = 100 μm).

Figure 3

Effect of rhamnocitrin (Rha) on body weight (BW) and visceral adiposity. (a) Initial BW, (b) final BW, (c) visceral adiposity, (d) H&E staining of periuterine adipocytes (scale bar = 200 μm), and (e) adipocytes area. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. the PCOS group; ^◆P < 0.05 and ^◆◆P < 0.01, the PCOS+Rha group vs. the PCOS+Met group. Values are mean ± SD (n = 8).

Figure 4

Effects of rhamnocitrin (Rha) on the blood levels of (a) FBG, (b) HMOA-IR, (c) and TyG; (d) postload glycemic change; and (e) AUC of OGTT. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. the PCOS group; ^◆P < 0.05 and ^◆◆P < 0.01, the PCOS+Rha group vs. the PCOS+Met group. Values are mean ± SD (n = 8).

Figure 5

Effects of rhamnocitrin (Rha) on the serum levels of (a) T, (b) LH, (c) FSH, (d) E2, and (e) the LH/FSH ratio. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. the PCOS group; ^◆P < 0.05 and ^◆◆P < 0.01, the PCOS+Rha group vs. the PCOS+Met group. Values are mean ± SD (n = 8).

Figure 6

Effects of rhamnocitrin (Rha) on the serum levels of (a) TC, (b) TG, (c) HDL-C, (d) the TC/HDL-C ratio, and (e) the TG/HDL-C ratio. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. the PCOS group. Values are mean ± SD (n = 8).

Figure 7

Effects of rhamnocitrin (Rha) on the serum levels of (a) MDA and (b) SOD and (c, d) the mRNA levels of antioxidant enzymes (Cat, Sod2, Gpx3, Mgst1, Gsta4, Gsr, Sod1, and Prdx3). Values are mean ± SD. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. the PCOS group; ^◆P < 0.05 and ^◆◆P < 0.01, the PCOS+Rha group vs. the PCOS+Met group.

Figure 8

Effect of rhamnocitrin (Rha) on the protein levels of (a) p-Smad2, (b) p-Smad3, (c) Smad4, and (d) Smad7 in the ovary. Values are mean ± SD. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. the PCOS group; ^◆P < 0.05 and ^◆◆P < 0.01, the PCOS+Rha group vs. the PCOS+Met group.

Figure 9

Effect of rhamnocitrin (Rha) on the protein levels of (a) TGF-β1, (b) TGF-βR1, (c) PPAR-γ, (d) collagen I, (e) α-SMA, and (f) CTGF in the ovary. Values are mean ± SD. ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. the PCOS group; ^◆P < 0.05 and ^◆◆P < 0.01, the PCOS+Rha group vs. the PCOS+Met group.

Figure 10

Effect of rhamnocitrin (Rha) on the protein expression of TGF-β1, Smad2, Smad3, Smad4, and Smad7 in the ovary. Representative immunohistochemistry images of the TGF-β1, Smad2, Smad3, Smad4, and Smad7 proteins in the ovary are shown with quantification (scale bar = 100 μm; black box defines the area to be amplified, high magnification scale bar = 50 μm). ^∗P < 0.05 and ^∗∗P < 0.01 vs. the control group; ^#P < 0.05 and ^##P < 0.01 vs. the PCOS group. Values are mean ± SD (n = 8).