目的:白细胞介素33(IL-33)是一种至关重要的炎症因子,在子宫内膜异位症(EMs)中起警报信号的作用。上皮-间质转化(EMT),与炎症信号相关的过程,细胞内活性氧(ROS)的产生,和脂质过氧化,IL-33在异位环境中高度上调。此外,异位子宫内膜细胞组成型表达白介素-33受体ST2(IL-33R)。然而,IL-33/ST2在EMsEMT中的作用尚不清楚.在这项研究中,我们旨在从机制上确定IL-33/ST2在EMs相关纤维化中的作用.
方法:我们建立了非致死氧化应激模型,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)测定,和划痕试验分析IL-33对原代子宫内膜基质细胞(ESCs)增殖和侵袭的影响。对来自具有或不具有EM的患者的临床样品进行免疫组织化学(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和正常人上皮细胞系EEC来评估IL-33对Wnt/β-catenin信号传导的影响。通过腹膜内注射IL-33和抗ST2在体内验证IL-33对EMT相关纤维化的作用。
结果:我们观察到异位环境,以ROS为特征,TGF-β1和高水平的雌激素,引发异位ESC分泌IL-33。与正常子宫内膜相比,异位内膜病变表现出更高的纤维化特征和ST2表达水平。外源性重组人(rhIL-33)增强ESC迁移和存活。同样,12Z细胞表现出更高程度的EMT特征,CCN4和Fra-1(WNT/β-catenin途径的下游靶基因)的表达升高,比在EEC中观察到的要多。相反,用中和抗体阻断IL-33,用siRNA敲除ST2或β-catenin,β-连环蛋白去磷酸化消除了其对EMT促进的影响。体内验证表明IL-33通过激活Wnt/β-catenin信号传导显著促进EMs相关纤维化。
结论: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-连环蛋白轴可能有望成为控制EM纤维化的可行治疗方法。
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.