Rhamnocitrin decreases fibrosis of ovarian granulosa cells by regulating the activation of the PPARγ/NF-κB/TGF-β1/Smad2/3 signaling pathway mediated by Wisp2

Abstract

Background: Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility in women. Rhamnocitrin (Rha) has anti-inflammatory and antioxidant actions. The WNT1-inducible-signaling pathway protein 2 (Wisp2) and nuclear factor (NF)-κB are involved in fibrosis in many diseases. We aimed to elucidate the role of Rha in fibrosis of PCOS and the underlying mechanisms.

Methods: Dehydroepiandrosterone (DHEA)-incubated ovarian granulosa KGN cells were treated by Rha. Cell proliferation was detected with cell counting kit-8 (CCK-8) and 5-ethynyul-2'-deoxyuridine (EdU) staining. The levels of Wisp2 and transforming growth factor-β1 (TGF-β1) in supernatant were measured by enzyme-linked immunosorbent assay (ELISA). We observed α-smooth muscle actin (α-SMA) protein by immunofluorescence (IF). The levels of fibrosis factors were determined using Western blot. We observed p65 nuclear translocation with confocal microscopy. We used Wisp2 overexpression and knockdown in cells treated with DHEA or Rha to validate Wisp2 function. Interaction between Wisp2 and NF-κB, as well as Wisp2 and PPARγ, were assessed by co-immunoprecipitation assay, luciferase reporter assay and chromatin immunoprecipitation (ChIP).

Results: The results showed that Rha elevated the reduced proliferation of DHEA-treated cells. In addition, Rha reversed the decreased Wisp2 and the increased TGF-β1 in supernatant. The proteins CTGF, α-SMA, Collagen I, TGF-β1, p-Smad2, and p-Smad3 were up-regulated while Wisp2, Sirt1, and PPARγ were down-regulated by DHEA treatment, which were reversed by Rha. Meanwhile, DHEA up-regulated p-IKBa and p-p65 and promoted p65 nuclear translocation, which were inhibited by Rha. These effects of Rha were antagonized by Wisp2 knockdown and were mimicked by Wisp2 overexpression. We confirmed the protein interaction between Wisp2 and NF-κB, along with Wisp2 and PPARγ.

Conclusions: Wisp2-mediated PPARγ/NF-κB/TGF-β1/Smad2/3 signaling contributes to Rha-improved ovarian granulosa cells fibrosis, suggesting Rha as a novel agent for the treatment of PCOS.

Keywords: PPARγ/nuclear factor κB (NF-κB)/TGF-β1/Smad2/3 signaling; Rhamnocitrin (Rha); WNT1-inducible-signaling pathway protein 2 (Wisp2); fibrosis; polycystic ovary syndrome (PCOS).

Figure 1

Rha restored cell proliferation of DHEA-treated KGN cells. (A) TGF-β1, α-SMA, and Collagen I levels in KGN cells followed by treatment with DHEA at various concentrations (10 nM, 100 nM, 1 µM, 10 µM, 100 µM) assessed using Western blot. (B) Cell viability was measured with CCK-8 after DHEA and different dosage of Rha treatment. (C) KGN cell proliferation was assessed via EdU assay after DHEA and different dosage of Rha treatment. n=3. Data are exhibited as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 vs. control group; ^#P<0.05, ^##P<0.01 vs. DHEA group. TGF-β1, transforming growth factor-β1; DHEA, dehydroepiandrosterone; CCK-8, cell counting kit-8; EdU, 5-ethynyul-2’-deoxyuridine; SD, standard deviation; OD, optical density.

Figure 2

Rha regulated the fibrosis of KGN cells. Following DHEA and different dosage of Rha treatment, (A) Wisp2 and TGF-β1 expressions validated by ELISA; (B,C) the expressions of CTGF, TGF-β1, α-SMA, Collagen I, Sirt1, Wisp2, PPARγ, p-Smad2, Smad2, p-Smad3 and Smad3 were analyzed by Western blot. (D) α-SMA level was analyzed by IF staining. n=3. Data are exhibited as mean ± SD. ***P<0.001 vs. control group; ^##P<0.01, ^###P<0.001 vs. DHEA group. ELISA, enzyme-linked immunosorbent assay; IF, immunofluorescence; SD, standard deviation; DHEA, dehydroepiandrosterone.

Figure 3

Rha regulated cell proliferation of KGN cells through modulating Wisp2 expression. KGN cells were treated by DHEA and H-Rha, along with Wisp2 or sh-Wisp2 transfection. (A) Wisp2 expression in KGN was examined by qRT-PCR; (B) KGN cell viability measured by CCK-8; (C) cell proliferation was detected by EdU incorporation assay. n=3. Data are exhibited as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 vs. control group; ^##P<0.01, ^###P<0.001 vs. DHEA group; ^&&P<0.01, ^&&&P<0.001 vs. DHEA + H-Rha group. qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, cell counting kit-8; EdU, 5-ethynyul-2’-deoxyuridine; SD, standard deviation; DHEA, dehydroepiandrosterone; OD, optical density.

Figure 4

Rha regulated the fibrosis of KGN cells through modulating Wisp2 level. KGN cells were treated by DHEA and H-Rha, along with Wisp2 or sh-Wisp2 transfection. (A) Levels of Wisp2 and TGF-β1 were detected by ELISA. (B,C) Expressions of CTGF, TGF-β1, α-SMA, Collagen I, Sirt1, Wisp2, PPARγ, p65, p-p65, IKBa, p-IKBa, p-Smad2, Smad2, p-Smad3 and Smad3 were confirmed by Western blot. (D) α-SMA expression detected by IF staining. n=3. Data are exhibited as mean ± SD. ***P<0.001 vs. control group; ^##P<0.01, ^###P<0.001 vs. DHEA group; ^&&&P<0.001 vs. DHEA + H-Rha group. TGF-β1, transforming growth factor-β1; DHEA, dehydroepiandrosterone; IF, immunofluorescence; SD, standard deviation.

Figure 5

The interaction between Wisp2, NF-κB and PPARγ. (A) Wisp2 and NF-κB interaction was confirmed in KGN cells by Co-IP. (B) Wisp2 modulated the binding of NF-κB and downstream genes by Chip-qPCR. (C) Co-localization of p65 in KGN cells, determined by immunofluorescence detection and confocal photography. (D) Luciferase activity of KGN cells transfected with PPARγ WT, PPARγ MUT, Wisp2 overexpressed plasmid, vector, or blank plasmid, alone or in combination. n=3. Data are exhibited as mean ± SD. *P<0.05 vs. Vector + PPARγ-WT. ^#P<0.05 vs. Vector + PPARγ-WT and Wisp2 + PPARγ-WT. Co-IP, co-immunoprecipitation; ChIP-qPCR, chromatin immunoprecipitation polymerase chain reaction; SD, standard deviation.