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.

Indole-3-methanol alleviates cisplatin-induced ovarian damage by inhibiting ovarian fibrosis through the TGF-β1/Smad pathway

“…To put it another way, I3C was able to slow the progression of cisplatin-induced ovarian brosis. The TGF-β1/Smad signaling pathway is important in ovarian brosis and has been extensively researched, particularly in polycystic ovary syndrome (Takahashi et al, 2017;Wang et al, 2018;Zhou et al, 2022). Once activated, ligand binding results in the formation of the receptor heterodimeric complex TGFβRII/TGFβRI, which then recruits and activates Smad2 and Smad3, phosphorylates the Smad2/3 complex with Smad4, and translocates to the nucleus to activate downstream target genes (Moustakas and Heldin, 2009).…”