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.
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.
复制标题
Rhamnocitrin 通过调节 Wisp2 介导的 PPARγ/NF-κB/TGF-β1/Smad2/3 信号通路的激活来减少卵巢颗粒细胞的纤维化
DOI：
10.21037/atm-22-2496
复制
发表时间：
2022-07
影响因子：
--
通讯作者：
Song, Jia-Le
中科院分区：
文献类型：
--
作者：
Zhou, Yan-Yuan;He, Chun-Hua;Lan, Huan;Dong, Zhe-Wen;Wu, Ya-Qi;Song, Jia-Le
关键词：
多囊卵巢综合征（PCOS）是女性无排卵性不孕症最常见的病因。鼠李柠檬素（Rha）具有抗炎和抗氧化作用。WNT1诱导信号通路蛋白2（Wisp2）和核因子-κB（NF -κB）参与许多疾病的纤维化过程。我们旨在阐明Rha在多囊卵巢综合征纤维化中的作用及其潜在机制。
用鼠李柠檬素处理经脱氢表雄酮（DHEA）孵育的卵巢颗粒细胞KGN。采用细胞计数试剂盒 - 8（CCK - 8）和5 - 乙炔基 - 2’ - 脱氧尿苷（EdU）染色检测细胞增殖。通过酶联免疫吸附测定（ELISA）检测上清液中Wisp2和转化生长因子 - β1（TGF - β1）的水平。通过免疫荧光（IF）观察α - 平滑肌肌动蛋白（α - SMA）蛋白。采用蛋白质印迹法测定纤维化因子的水平。通过共聚焦显微镜观察p65核转位。我们在经DHEA或Rha处理的细胞中使用Wisp2过表达和敲低来验证Wisp2的功能。通过免疫共沉淀试验、荧光素酶报告基因试验和染色质免疫沉淀（ChIP）评估Wisp2与NF - κB以及Wisp2与过氧化物酶体增殖物激活受体γ（PPARγ）之间的相互作用。
结果表明，Rha提高了DHEA处理细胞降低的增殖能力。此外，Rha逆转了上清液中Wisp2的降低和TGF - β1的升高。DHEA处理使结缔组织生长因子（CTGF）、α - SMA、Ⅰ型胶原蛋白（Collagen I）、TGF - β1、磷酸化 - Smad2（p - Smad2）和磷酸化 - Smad3（p - Smad3）蛋白上调，而使Wisp2、沉默信息调节因子1（Sirt1）和PPARγ下调，这些作用被Rha逆转。同时，DHEA上调磷酸化 - IκBα（p - IκBα）和磷酸化 - p65（p - p65）并促进p65核转位，这些作用被Rha抑制。Rha的这些作用被Wisp2敲低所拮抗，并被Wisp2过表达所模拟。我们证实了Wisp2与NF - κB以及Wisp2与PPARγ之间的蛋白质相互作用。
Wisp2介导的PPARγ/NF - κB/TGF - β1/Smad2/3信号通路有助于Rha改善卵巢颗粒细胞纤维化，提示Rha可作为治疗多囊卵巢综合征的一种新型药物。
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. 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). 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. 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.
登录
查看更多内容
影响因子：
4.1
作者：
Grünberg JR;Elvin J;Paul A;Hedjazifar S;Hammarstedt A;Smith U
通讯作者：
Smith U
影响因子：
5.4
作者：
Fang, Shih-Hua;Rao, Yerra Koteswara;Tzeng, Yew-Min
通讯作者：
Tzeng, Yew-Min
影响因子：
2.2
作者：
Jiang, H.;Zhan, W. Q.;Jiang, S. X.
通讯作者：
Jiang, S. X.
影响因子：
6.1
作者：
Makled, Mirhan N.;El-Kashef, Dalia H.
通讯作者：
El-Kashef, Dalia H.
影响因子：
3.2
作者：
Sun, Bei;Xia, Qiumei;Gao, Zhiyong
通讯作者：
Gao, Zhiyong