Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210028, Jiangsu, China; Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, 210028, Jiangsu, China.
J Ethnopharmacol. 2025 Jan 10;336:118724. doi: 10.1016/j.jep.2024.118724. Epub 2024 Aug 23.
Wenshen Xiaozheng Tang (WXT), a traditional Chinese medicine (TCM) decoction, is effective for treating endometriosis. However, the effect of WXT on endometrium-derived mesenchymal stem cells (eMSCs) which play a key role in the fibrogenesis of endometriosis requires further elucidation.
The aim of this study was to clarify the potential mechanism of WXT in improving fibrosis in endometriosis by investigating the regulation of WXT on differentiation and paracrine of eMSCs.
The nude mice with endometriosis were randomly divided into model group, WXT group and mifepristone group. After 21 days of treatment, the lesion volume was calculated. Fibrosis in the lesions was evaluated by Masson staining and expression of fibrotic proteins. The differentiation of eMSCs in vivo was explored using a fate-tracking experiment. To further clarify the regulation of WXT on eMSCs, primary eMSCs from the ectopic lesions of endometriosis patients were isolated and characterized. The effect of WXT on the proliferation and differentiation of ectopic eMSCs was examined. To evaluate the role of WXT on the paracrine activity of ectopic eMSCs, the conditioned medium (CM) from ectopic eMSCs pretreated with WXT was collected and applied to treat ectopic endometrial stromal cells (ESCs), after which the expression of fibrotic proteins in ectopic ESCs was assessed. In addition, transcriptome sequencing was used to investigate the regulatory mechanism of WXT on ectopic eMSCs, and western blot and ELISA were employed to determine the key mediator.
WXT impeded the growth of ectopic lesions in nude mice with endometriosis and reduced collagen deposition and the expression of fibrotic proteins fibronectin, collagen I, α-SMA and CTGF in the endometriotic lesions. The fate-tracking experiment showed that WXT prevented human eMSCs from differentiating into myofibroblasts in the nude mice. We successfully isolated eMSCs from the lesions of patients with endometriosis and demonstrated that WXT suppressed proliferation and myofibroblast differentiation of ectopic eMSCs. Moreover, the expression of α-SMA, collagen I, fibronectin and CTGF in ectopic ESCs was significantly down-regulated by the CM of ectopic MSCs pretreated with WXT. Combining the results of RNA sequencing, western blot and ELISA, we found that WXT not only reduced thrombospondin 4 expression in ectopic eMSCs, but also decreased thrombospondin 4 secretion from ectopic eMSCs. Thrombospondin 4 concentration-dependently upregulated the expression of collagen I, fibronectin, α-SMA and CTGF in ectopic ESCs, indicating that thrombospondin 4 was a key mediator of WXT in inhibiting the fibrotic process in endometriosis.
WXT improved fibrosis in endometriosis by regulating differentiation and paracrine signaling of eMSCs. Thrombospondin 4, whose release from ectopic eMSCs is inhibited by WXT, may be a potential target for the treatment of endometriosis.
温肾消症汤（WXT）是一种中药方剂，对子宫内膜异位症的治疗有效。然而，WXT 对在子宫内膜异位症纤维化中起关键作用的子宫内膜来源间充质干细胞（eMSCs）的影响需要进一步阐明。
本研究旨在通过研究 WXT 对 eMSCs 分化和旁分泌的调节作用，阐明 WXT 改善子宫内膜异位症纤维化的潜在机制。
将子宫内膜异位症裸鼠模型随机分为模型组、WXT 组和米非司酮组。治疗 21 天后，计算病变体积。通过 Masson 染色和纤维化蛋白的表达评估病变中的纤维化。通过命运追踪实验探索 eMSCs 在体内的分化。为了进一步阐明 WXT 对 eMSCs 的调节作用，从子宫内膜异位症异位病灶中分离并鉴定原发性 eMSCs。检测 WXT 对异位 eMSCs 增殖和分化的影响。为了评估 WXT 对异位 eMSCs 旁分泌活性的作用，收集 WXT 预处理后的异位 eMSCs 的条件培养基（CM）并用于处理异位子宫内膜基质细胞（ESCs），然后评估异位 ESCs 中纤维化蛋白的表达。此外，采用转录组测序研究 WXT 对异位 eMSCs 的调控机制，采用 Western blot 和 ELISA 测定关键介质。
WXT 抑制子宫内膜异位症裸鼠模型中异位病变的生长，减少胶原沉积和纤维连接蛋白、I 型胶原、α-SMA 和 CTGF 在子宫内膜异位症病变中的表达。命运追踪实验表明，WXT 阻止人 eMSCs 在裸鼠中分化为肌成纤维细胞。我们成功地从子宫内膜异位症患者的病灶中分离出 eMSCs，并证明 WXT 抑制了异位 eMSCs 的增殖和肌成纤维细胞分化。此外，WXT 预处理后的异位 MSC 的 CM 显著下调了异位 ESCs 中 α-SMA、I 型胶原、纤维连接蛋白和 CTGF 的表达。结合 RNA 测序、Western blot 和 ELISA 的结果，我们发现 WXT 不仅降低了异位 eMSCs 中血栓素 4 的表达，而且还降低了异位 eMSCs 中血栓素 4 的分泌。血栓素 4 浓度依赖性地上调了异位 ESCs 中 I 型胶原、纤维连接蛋白、α-SMA 和 CTGF 的表达，表明血栓素 4 是 WXT 抑制子宫内膜异位症纤维化过程中的关键介质。
WXT 通过调节 eMSCs 的分化和旁分泌信号改善子宫内膜异位症的纤维化。WXT 抑制异位 eMSCs 释放的血栓素 4 可能是子宫内膜异位症治疗的潜在靶点。
