Department of Gynecology, Hospital of Obstetrics and Gynecology, Fudan University, 419# Fangxie Road, Shanghai, 200011, China.
Department of Pathology, Hospital of Obstetrics and Gynecology, Fudan University, Shanghai, China.
Cell Commun Signal. 2024 Jun 10;22(1):318. doi: 10.1186/s12964-024-01683-x.
Interleukin 33 (IL-33) is a crucial inflammatory factor that functions as an alarm signal in endometriosis (EMs). Epithelial-mesenchymal transition (EMT), a process related to inflammatory signals, intracellular reactive oxygen species (ROS) production, and lipid peroxidation, have been proposed as potential mechanisms that contribute to the development and progression of EMs. IL-33 is highly upregulated in the ectopic milieu. Moreover, ectopic endometrial cells constitutively express interleukin-33 receptor ST2 (IL-33R). However, the role of IL-33/ST2 in the EMT of EMs remains largely unknown. In this study, we aimed to mechanistically determine the role of IL-33/ST2 in EMs-associated fibrosis.
We established a non-lethal oxidative stress model to explore the conditions that trigger IL-33 induction. We performed α-smooth muscle actin (α-SMA) protein detection, cell counting kit-8 (CCK-8) assays, and scratch assays to analyze the impact of IL-33 on primary endometrial stromal cells (ESCs) proliferation and invasion. Clinical samples from patients with or without EMs were subjected to immunohistochemical (IHC) and and immunofluorescence(IF) staining to assess the clinical relevance of IL-33 receptor ST2 and EMT-related proteins. Furthermore, we used the ectopic human endometrial epithelial cell line 12Z and normal human epithelial cell line EEC to evaluate the effects of IL-33 on Wnt/β-catenin signaling. The effect of IL-33 on EMT-associated fibrosis was validated in vivo by intraperitoneal injections of IL-33 and antiST2.
We observed that ectopic milieu, characterized by ROS, TGF-β1, and high level of estrogen, triggers the secretion of IL-33 from ectopic ESCs. Ectopic endometrial lesions exhibited higher level of fibrotic characteristics and ST2 expression than that in the normal endometrium. Exogenous recombinant human (rhIL-33) enhanced ESC migration and survival. Similarly, 12Z cells displayed a higher degree of EMT characteristics with elevated expression of CCN4 and Fra-1, downstream target genes of the WNT/β-catenin pathway, than that observed in EECs. Conversely, blocking IL-33 with neutralizing antibodies, knocking down ST2 or β-catenin with siRNA, and β-catenin dephosphorylation abolished its effects on EMT promotion. In vivo validation demonstrated that IL-33 significantly promotes EMs-related fibrosis through the activation of Wnt/β-catenin signaling.
Our data strongly support the vital role of the IL-33/ST2 pathway in EMs-associated fibrosis and emphasize the importance of the EMT in the pathophysiology of fibrosis. Targeting the IL-33/ST2/Wnt/β-catenin axis may hold promise as a feasible therapeutic approach for controlling fibrosis in EMs.
白细胞介素 33 (IL-33) 是一种重要的炎症因子，在子宫内膜异位症 (EMs) 中充当警报信号。上皮-间充质转化 (EMT) 是一种与炎症信号、细胞内活性氧 (ROS) 产生和脂质过氧化有关的过程，被认为是导致 EMs 发生和发展的潜在机制。IL-33 在异位环境中高度上调。此外，异位子宫内膜细胞持续表达白细胞介素 33 受体 ST2 (IL-33R)。然而，IL-33/ST2 在 EMs 相关纤维化中的作用在很大程度上仍不清楚。在本研究中，我们旨在从机制上确定 IL-33/ST2 在 EMs 相关纤维化中的作用。
我们建立了一种非致死性氧化应激模型，以探索触发 IL-33 诱导的条件。我们进行了 α-平滑肌肌动蛋白 (α-SMA) 蛋白检测、细胞计数试剂盒-8 (CCK-8) 检测和划痕实验，以分析 IL-33 对原代子宫内膜基质细胞 (ESCs) 增殖和侵袭的影响。对患有或不患有 EMs 的患者的临床样本进行免疫组织化学 (IHC) 和免疫荧光 (IF) 染色，以评估 IL-33 受体 ST2 和 EMT 相关蛋白的临床相关性。此外，我们使用异位人子宫内膜上皮细胞系 12Z 和正常人类上皮细胞系 EEC 来评估 IL-33 对 Wnt/β-连环蛋白信号的影响。通过腹腔内注射 IL-33 和抗 ST2 验证了 IL-33 对 EMT 相关纤维化的影响。
我们观察到，ROS、TGF-β1 和高水平雌激素等异位环境会触发异位 ESCs 分泌 IL-33。异位子宫内膜病变表现出比正常子宫内膜更高的纤维化特征和 ST2 表达水平。外源性重组人 (rhIL-33) 增强了 ESC 的迁移和存活。同样，与 EECs 相比，12Z 细胞表现出更高程度的 EMT 特征，其 WNT/β-连环蛋白通路下游靶基因 CCN4 和 Fra-1 的表达水平升高。相反，用中和抗体阻断 IL-33、用 siRNA 敲低 ST2 或 β-连环蛋白以及β-连环蛋白去磷酸化消除了其对 EMT 促进的作用。体内验证表明，IL-33 通过激活 Wnt/β-连环蛋白信号显著促进 EMs 相关纤维化。
我们的数据强烈支持 IL-33/ST2 通路在 EMs 相关纤维化中的重要作用，并强调 EMT 在纤维化病理生理学中的重要性。靶向 IL-33/ST2/Wnt/β-连环蛋白轴可能是控制 EMs 纤维化的一种可行的治疗方法。
