女性的子宫内膜具有独特的周期性脱落与再生能力，这一过程对维持子宫内膜健康和正常的生殖功能至关重要。子宫内膜的再生依赖于子宫内膜间质干细胞的激活和分化，确保损伤的组织能够高效修复。在这一过程中，活性氧（Reactive Oxygen Species, ROS）作为一种重要的信号分子，在调控细胞行为和维持组织功能中起关键作用。适量的 ROS 水平在信号传导、细胞增殖及干细胞激活中发挥重要作用，尤其在静止干细胞激活中充当第二信使。然而，过量的ROS 可能引发细胞氧化损伤，阻碍组织修复；而过低的 ROS 则可能无法充分激活信号通路，影响修复效率。已有相关研究说明生理水平的 ROS 在伤口愈合中的重要作用，但其在子宫内膜间质干细胞脱静止及子宫内膜修复中的具体作用机制仍不明确。
目的：本研究旨在探讨 ROS 调控子宫内膜间质干细胞静止与激活状态的作用机制，并通过抗氧化剂干预实验验证生理量的 ROS 清除对子宫内膜生理修复的影响。
方法：建立小鼠月经样模型，使用广谱抗氧化剂 N-乙酰半胱氨酸（N-acetylcysteine，NAC）和线粒体靶向抗氧化剂米托蒽醌甲磺酸盐（Mitoquinonemesylate，MitoQ）进行干预，通过 HE 染色、免疫组织化学、免疫荧光及流式细胞术等手段评估抗氧化剂清除 ROS 对子宫内膜修复的影响；分选模型小鼠子宫内膜干细胞，并检测不同干预条件下干细胞激活相关标志物的表达及 G0 期细胞比例变化。
结果：经过对照组、抗氧化剂组和线粒体靶向抗氧化剂组的不同处理，小鼠子宫在孕酮撤退 24 h、36 h 和 48 h 时的大体观察、组织形态学分析、DHE 染色等结果显示，成功建立了抗氧化剂干预的小鼠月经样模型。
免疫组化结果表明，清除 ROS 后的小鼠子宫内膜在子宫内膜基质、血管和腺体的修复过程中出现明显延迟，与正常生理修复进程相比存在显著差异。免疫荧光共定位和流式共染实验进一步揭示，当 ROS 被清除至低于生理水平时，在孕酮撤退 36 h 子宫内膜干细胞的脱静止过程受到显著抑制。在抗氧化剂处理的体外细胞模型中，ROS 清除后，细胞 qPCR 检测显示静止期标志物的表达显著增加；同时，人子宫内膜间充质干细胞的 G0 期比例显著上升，表明处于休眠状态的干细胞比例明显增加。进一步的 Western Blot 实验显示，抗氧化剂处理组中 p-AMPK 表达降低，提示 p-AMPK 通路可能参与了干细胞脱静止过程的调控。
这些结果为子宫内膜修复中 ROS 的调控机制提供了重要依据，并为优化相关治疗策略奠定了理论基础。
结论：本研究成功建立了抗氧化剂干预的小鼠月经样模型，明确了 ROS 在子宫内膜修复及干细胞激活中的关键作用。通过免疫组化、免疫荧光共定位及流式细胞术的综合分析，发现过量清除 ROS 会延迟子宫内膜的修复进程，干扰干细胞的脱静止过程，尤其在孕酮撤退 36 h 后表现尤为显著，适量的 ROS 有助于恢复干细胞的激活功能，从而维持正常的修复进程。
本研究不仅深化了对活性氧（ROS）作为生理性修复关键因子在子宫内膜修复与干细胞功能调控中作用机制的理解，为设计更安全有效的抗氧化治疗方案提供了理论依据，并强调了治疗过程中应避免干扰女性月经周期的重要性。
此外，本研究通过揭示 ROS 的精准调控机制，为子宫内膜损伤或疾病中的干细胞疗法提供了新的干预策略，从而为子宫内膜修复异常相关疾病提供了潜在的创新药物靶点和治疗策略。

The human endometrium exhibits a unique capacity for cyclical shedding and regeneration, which is essential for maintaining endometrial health and normal reproductive function. This regenerative process relies on the activation and differentiation of endometrial stromal stem cells (EnSCs), ensuring efficient repair of damaged tissue. Reactive oxygen species (ROS), as crucial signaling molecules, play a key role in regulating cellular behavior and maintaining tissue function. Physiological
levels of ROS are involved in signal transduction, cell proliferation, and particularly in the activation of quiescent stem cells, where ROS act as second messengers. However,
excessive ROS can lead to oxidative damage and impair tissue repair, whereas insufficient ROS may fail to trigger adequate signaling, thus compromising repair efficiency. Although previous studies have highlighted the importance of physiological ROS in wound healing, their specific role in EnSC activation from quiescence and endometrial regeneration remains unclear.
Objective:
This study aims to elucidate the mechanisms by which ROS regulate the transition of EnSCs from quiescence to activation, and to assess the impact of antioxidant-mediated ROS scavenging on physiological endometrial repair.
Methods:
A mouse menstruation-like model was established. Two types of antioxidants were used for intervention: the broad-spectrum antioxidant N-acetylcysteine (NAC) and the mitochondria-targeted antioxidant mitoquinone mesylate (MitoQ). The effects of ROS clearance on endometrial repair were evaluated using hematoxylin-eosin (HE) staining,immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry. EnSCs
were isolated from model mice, and the expression of stem cell activation markers and the proportion of G0 phase cells under different treatment conditions were analyzed.
Results:
Following treatment with either NAC or MitoQ, gross morphology, histological analysis, and dihydroethidium (DHE) staining at 24, 36, and 48 hours post-progesterone withdrawal confirmed the successful establishment of the antioxidant-intervened menstruation-like mouse model. IHC results showed that ROS clearance led to significant delays in the repair of stromal, vascular, and glandular components of the
endometrium compared to physiological repair. IF co-localization and flow cytometry revealed that excessive ROS scavenging significantly inhibited EnSC activation from
quiescence, particularly at 36 hours after progesterone withdrawal. In vitro, antioxidant treatment led to increased expression of quiescence markers (by qPCR), and a
significantly higher proportion of human EnSCs remained in the G0 phase, indicating a marked increase in the dormant stem cell population. Furthermore, Western blot
analysis showed decreased expression of phosphorylated AMPK (p-AMPK) in antioxidant-treated groups, suggesting that the p-AMPK pathway may be involved in
regulating stem cell activation. These findings provide compelling evidence for the critical regulatory role of ROS
in endometrial repair and stem cell activation and establish a theoretical foundation for optimizing related therapeutic strategies.
Conclusion:
This study successfully established an antioxidant-intervened menstruation-like mouse model and demonstrated the key role of ROS in endometrial repair and EnSC activation. Through comprehensive analyses using IHC, IF co-localization, and flow
cytometry, we found that excessive ROS scavenging delays endometrial repair and disrupts the activation of quiescent stem cells, with the most pronounced effects observed 36 hours post-progesterone withdrawal. Physiological levels of ROS facilitate stem cell activation and support normal tissue regeneration.
This research deepens our understanding of ROS as essential physiological mediators in endometrial repair and stem cell function. It also offers theoretical support
for designing safer and more effective antioxidant therapies, emphasizing the importance of avoiding disruption to the menstrual cycle during treatment. Moreover, by elucidating the precise regulatory role of ROS, this study proposes new intervention strategies for stem cell-based therapies in endometrial damage or disease, providing potential drug targets and innovative therapeutic approaches for disorders related to abnormal endometrial repair.