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Abstract：
UNASSIGNED: This study aims to systematically evaluate the protective role of quercetin (QCT), a naturally occurring flavonoid, against oxidative damage in human endometrial stromal cells (HESCs) induced by hydrogen peroxide (H2O2). Oxidative stress, such as that induced by H2O2, is known to contribute significantly to cellular damage and has been implicated in various reproductive health issues. The study is focused on investigating how QCT interacts with specific molecular pathways to mitigate this damage. Special attention was given to the p38 MAPK/NOX4 signaling pathway, which is crucial to the regulation of oxidative stress responses in cellular systems. By elucidating these mechanisms, the study seeks to confirm the potential of QCT not only as a protective agent against oxidative stress but also as a therapeutic agent that could be integrated in treatments of conditions characterized by heightened oxidative stress in endometrial cells.
UNASSIGNED: I n vitro cultures of HESCs were treated with QCT at different concentrations (0, 10, 20, and 40 μmol/L) for 24 h to verify the non-toxic effects of QCT on normal endometrial cells. Subsequently, 250 μmol/L H2O2 was used to incubate the cells for 12 h to establish an H2O2-induced HESCs injury model. HESCs were pretreated with QCT for 24 h, which was followed by stimulation with H2O2. Then, CCK-8 assay was performed to examine the cell viability and to screen for the effective intervention concentration. HESCs were divided into 3 groups, the control group, the H2O2 model group, and the H2O2+QCT group. Intracellular levels of reactive oxygen species (ROS) were precisely quantified using the DCFH-DA fluorescence assay, a method known for its accuracy in detecting and quantifying oxidative changes within the cell. The mitochondrial membrane potential was determined by JC-1 staining. Annexin Ⅴ/PI double staining and flow cytometry were performed to determine the effect of QCT on H2O2-induced apoptosis of HESCs. Furthermore, to delve deeper into the cellular mechanisms underlying the observed effects, Western blot analysis was conducted to measure the expression levels of the critical proteins involved in oxidative stress response, including NADPH oxidase 4 (NOX4), p38 mitogen-activated protein kinase (p38 MAPK), and phosphorylated p38 MAPK (p-p38 MAPK). This analysis helps increase understanding of the specific intracellular signaling pathways affected by QCT treatment, giving special attention to its potential for modulation of the p38 MAPK/NOX4 pathway, which plays a significant role in cellular defense mechanisms against oxidative stress.
UNASSIGNED: In this study, we started off by assessing the toxicity of QCT on normal endometrial cells. Our findings revealed that QCT at various concentrations (0, 10, 20, and 40 μmol/L) did not exhibit any cytotoxic effects, which laid the foundation for further investigation into its protective roles. In the H2O2-induced HESCs injury model, a significant reduction in cell viability was observed, which was linked to the generation of ROS and the resultant oxidative damage. However, pretreatment with QCT (10 μmol/L and 20 μmol/L) significantly enhanced cell viability after 24 h (P<0.05), with the 20 μmol/L concentration showing the most substantial effect. This suggests that QCT can effectively reverse the cellular damage caused by H2O2. Furthermore, the apoptosis assays demonstrated a significant increase in the apoptosis rates in the H2O2 model group compared to those in the control group (P<0.01). However, co-treatment with QCT significantly reversed this trend (P<0.05), indicating QCT\'s potential protective role in mitigating cell apoptosis. ROS assays showed that, compared to that in the control group, the average fluorescence intensity of ROS in the H2O2 model group significantly increased (P<0.01). QCT treatment significantly reduced the ROS fluorescence intensity in the H2O2+QCT group compared to the that in the H2O2 model group, suggesting an effective alleviation of oxidative damage (P<0.05). JC-1 staining for mitochondrial membrane potential changes revealed that compared to that in the control, the proportion of cells with decreased mitochondrial membrane potential significantly increased in the H2O2 model group (P<0.01). However, this proportion was significantly reduced in the QCT-treated group compared to that of the H2O2 model group (P<0.05). Finally, Western blot analysis indicated that the expression levels of NOX4 and p-p38 MAPK proteins were elevated in the H2O2 model group compared to those of the control group (P<0.05). Following QCT treatment, these protein levels significantly decreased compared to those of the H2O2 model group (P<0.05). These results suggest that QCT may exert its protective effects against oxidative stress by modulating the p38 MAPK/NOX4 signaling pathway.
UNASSIGNED: QCT has demonstrated significant protective effects against H2O2-induced oxidative damage in HESCs. This protection is primarily achieved through the effective reduction of ROS accumulation and the inhibition of critical signaling pathways involved in the oxidative stress response, notably the p38 MAPK/NOX4 pathway. The results of this study reveal that QCT\'s ability to modulate these pathways plays a key role in alleviating cellular damage associated with oxidative stress conditions. This indicates not only its potential as a protective agent against cellular oxidative stress, but also highlights its potential for therapeutic applications in treating conditions characterized by increased oxidative stress in the endometrium, thereby offering the prospect of enhancing reproductive health. Future studies should explore the long-term effects of QCT and its clinical efficacy in vivo, thereby providing a clear path toward its integration into therapeutic protocols.
UNASSIGNED: I n vitro cultures of HESCs were treated with QCT at different concentrations (0, 10, 20, and 40 μmol/L) for 24 h to verify the non-toxic effects of QCT on normal endometrial cells. Subsequently, 250 μmol/L H2O2 was used to incubate the cells for 12 h to establish an H2O2-induced HESCs injury model. HESCs were pretreated with QCT for 24 h, which was followed by stimulation with H2O2. Then, CCK-8 assay was performed to examine the cell viability and to screen for the effective intervention concentration. HESCs were divided into 3 groups, the control group, the H2O2 model group, and the H2O2+QCT group. Intracellular levels of reactive oxygen species (ROS) were precisely quantified using the DCFH-DA fluorescence assay, a method known for its accuracy in detecting and quantifying oxidative changes within the cell. The mitochondrial membrane potential was determined by JC-1 staining. Annexin Ⅴ/PI double staining and flow cytometry were performed to determine the effect of QCT on H2O2-induced apoptosis of HESCs. Furthermore, to delve deeper into the cellular mechanisms underlying the observed effects, Western blot analysis was conducted to measure the expression levels of the critical proteins involved in oxidative stress response, including NADPH oxidase 4 (NOX4), p38 mitogen-activated protein kinase (p38 MAPK), and phosphorylated p38 MAPK (p-p38 MAPK). This analysis helps increase understanding of the specific intracellular signaling pathways affected by QCT treatment, giving special attention to its potential for modulation of the p38 MAPK/NOX4 pathway, which plays a significant role in cellular defense mechanisms against oxidative stress.
UNASSIGNED: In this study, we started off by assessing the toxicity of QCT on normal endometrial cells. Our findings revealed that QCT at various concentrations (0, 10, 20, and 40 μmol/L) did not exhibit any cytotoxic effects, which laid the foundation for further investigation into its protective roles. In the H2O2-induced HESCs injury model, a significant reduction in cell viability was observed, which was linked to the generation of ROS and the resultant oxidative damage. However, pretreatment with QCT (10 μmol/L and 20 μmol/L) significantly enhanced cell viability after 24 h (P<0.05), with the 20 μmol/L concentration showing the most substantial effect. This suggests that QCT can effectively reverse the cellular damage caused by H2O2. Furthermore, the apoptosis assays demonstrated a significant increase in the apoptosis rates in the H2O2 model group compared to those in the control group (P<0.01). However, co-treatment with QCT significantly reversed this trend (P<0.05), indicating QCT\'s potential protective role in mitigating cell apoptosis. ROS assays showed that, compared to that in the control group, the average fluorescence intensity of ROS in the H2O2 model group significantly increased (P<0.01). QCT treatment significantly reduced the ROS fluorescence intensity in the H2O2+QCT group compared to the that in the H2O2 model group, suggesting an effective alleviation of oxidative damage (P<0.05). JC-1 staining for mitochondrial membrane potential changes revealed that compared to that in the control, the proportion of cells with decreased mitochondrial membrane potential significantly increased in the H2O2 model group (P<0.01). However, this proportion was significantly reduced in the QCT-treated group compared to that of the H2O2 model group (P<0.05). Finally, Western blot analysis indicated that the expression levels of NOX4 and p-p38 MAPK proteins were elevated in the H2O2 model group compared to those of the control group (P<0.05). Following QCT treatment, these protein levels significantly decreased compared to those of the H2O2 model group (P<0.05). These results suggest that QCT may exert its protective effects against oxidative stress by modulating the p38 MAPK/NOX4 signaling pathway.
UNASSIGNED: QCT has demonstrated significant protective effects against H2O2-induced oxidative damage in HESCs. This protection is primarily achieved through the effective reduction of ROS accumulation and the inhibition of critical signaling pathways involved in the oxidative stress response, notably the p38 MAPK/NOX4 pathway. The results of this study reveal that QCT\'s ability to modulate these pathways plays a key role in alleviating cellular damage associated with oxidative stress conditions. This indicates not only its potential as a protective agent against cellular oxidative stress, but also highlights its potential for therapeutic applications in treating conditions characterized by increased oxidative stress in the endometrium, thereby offering the prospect of enhancing reproductive health. Future studies should explore the long-term effects of QCT and its clinical efficacy in vivo, thereby providing a clear path toward its integration into therapeutic protocols.
摘要：
■本研究旨在系统地评估槲皮素（QCT）的保护作用，一种天然存在的类黄酮，抗过氧化氢(H2O2)诱导的人子宫内膜基质细胞(HESCs)氧化损伤。氧化应激,例如由H2O2诱导的,已知对细胞损伤有显著贡献,并且已经牵涉到各种生殖健康问题。该研究的重点是研究QCT如何与特定的分子途径相互作用以减轻这种损害。特别注意p38MAPK/NOX4信号通路，这对于调节细胞系统中的氧化应激反应至关重要。通过阐明这些机制，本研究试图证实QCT不仅是一种抗氧化应激的保护剂,而且是一种治疗药物,可用于治疗以子宫内膜细胞氧化应激增强为特征的疾病.
■用不同浓度(0、10、20和40μmol/L)的QCT处理HESCs体外培养24h，以验证QCT对正常子宫内膜细胞的无毒性作用。随后，用250μmol/LH2O2孵育细胞12h,建立H2O2诱导的HESCs损伤模型。HESCs用QCT预处理24h，然后用H2O2刺激。然后，进行CCK-8测定以检查细胞活力并筛选有效的干预浓度。HESCs分为3组,对照组,H2O2模型组，和H2O2+QCT组。使用DCFH-DA荧光测定法精确定量细胞内活性氧（ROS）的水平，一种以检测和定量细胞内氧化变化的准确性而闻名的方法。通过JC-1染色测定线粒体膜电位。采用膜联蛋白Ⅴ/PI双染色和流式细胞术检测QCT对H2O2诱导的HESCs凋亡的影响。此外，为了更深入地研究观察到的效应背后的细胞机制，进行蛋白质印迹分析以测量参与氧化应激反应的关键蛋白的表达水平。包括NADPH氧化酶4（NOX4），p38丝裂原活化蛋白激酶(p38MAPK),和磷酸化p38MAPK（p-p38MAPK）。这种分析有助于增加对QCT治疗所影响的特定细胞内信号通路的理解。特别注意其调节p38MAPK/NOX4通路的潜力，在抗氧化应激的细胞防御机制中起着重要作用。
■在这项研究中，我们从评估QCT对正常子宫内膜细胞的毒性开始.我们的发现表明，QCT在各种浓度（0，10，20和40μmol/L）没有表现出任何细胞毒性作用，为进一步研究其保护作用奠定了基础。在H2O2诱导的HESCs损伤模型中,观察到细胞活力显着降低，这与ROS的产生和由此产生的氧化损伤有关。然而,QCT(10μmol/L和20μmol/L)预处理后24h细胞活力显著提高(P<0.05),20μmol/L浓度显示出最显著的效果。这表明QCT可以有效逆转H2O2引起的细胞损伤。此外，细胞凋亡实验表明，与对照组相比，H2O2模型组的细胞凋亡率显着增加（P<0.01）。然而,联合QCT治疗显著逆转了这一趋势(P<0.05)，表明QCT在减轻细胞凋亡方面具有潜在的保护作用。ROS检测表明,与对照组相比，H2O2模型组ROS平均荧光强度明显升高(P<0.01)。QCT治疗后H2O2+QCT组的ROS荧光强度明显低于H2O2模型组,提示氧化损伤的有效缓解(P<0.05)。线粒体膜电位变化的JC-1染色显示，与对照组相比，H2O2模型组线粒体膜电位下降的细胞比例明显增加(P<0.01)。然而,与H2O2模型组相比，QCT治疗组的这一比例显着降低（P<0.05）。最后,Westernblot分析显示,模型组大鼠的NOX4和p-p38MAPK蛋白表达水平较对照组升高(P<0.05)。QCT治疗后,与H2O2模型组相比，这些蛋白水平显着降低（P<0.05）。这些结果表明，QCT可能通过调节p38MAPK/NOX4信号通路发挥其对氧化应激的保护作用。
■QCT已证明对H2O2诱导的HESCs氧化损伤具有显著的保护作用。这种保护主要通过有效减少ROS积累和抑制参与氧化应激反应的关键信号通路来实现。尤其是p38MAPK/NOX4通路。这项研究的结果表明，QCT调节这些途径的能力在减轻与氧化应激条件相关的细胞损伤中起着关键作用。这不仅表明其作为抗细胞氧化应激的保护剂的潜力，但也强调了其在治疗以子宫内膜氧化应激增加为特征的疾病中的治疗应用潜力，从而提供了增强生殖健康的前景。未来的研究应探讨QCT的长期影响及其在体内的临床疗效，从而为其整合到治疗方案中提供了明确的途径。
■用不同浓度(0、10、20和40μmol/L)的QCT处理HESCs体外培养24h，以验证QCT对正常子宫内膜细胞的无毒性作用。随后，用250μmol/LH2O2孵育细胞12h,建立H2O2诱导的HESCs损伤模型。HESCs用QCT预处理24h，然后用H2O2刺激。然后，进行CCK-8测定以检查细胞活力并筛选有效的干预浓度。HESCs分为3组,对照组,H2O2模型组，和H2O2+QCT组。使用DCFH-DA荧光测定法精确定量细胞内活性氧（ROS）的水平，一种以检测和定量细胞内氧化变化的准确性而闻名的方法。通过JC-1染色测定线粒体膜电位。采用膜联蛋白Ⅴ/PI双染色和流式细胞术检测QCT对H2O2诱导的HESCs凋亡的影响。此外，为了更深入地研究观察到的效应背后的细胞机制，进行蛋白质印迹分析以测量参与氧化应激反应的关键蛋白的表达水平。包括NADPH氧化酶4（NOX4），p38丝裂原活化蛋白激酶(p38MAPK),和磷酸化p38MAPK（p-p38MAPK）。这种分析有助于增加对QCT治疗所影响的特定细胞内信号通路的理解。特别注意其调节p38MAPK/NOX4通路的潜力，在抗氧化应激的细胞防御机制中起着重要作用。
■在这项研究中，我们从评估QCT对正常子宫内膜细胞的毒性开始.我们的发现表明，QCT在各种浓度（0，10，20和40μmol/L）没有表现出任何细胞毒性作用，为进一步研究其保护作用奠定了基础。在H2O2诱导的HESCs损伤模型中,观察到细胞活力显着降低，这与ROS的产生和由此产生的氧化损伤有关。然而,QCT(10μmol/L和20μmol/L)预处理后24h细胞活力显著提高(P<0.05),20μmol/L浓度显示出最显著的效果。这表明QCT可以有效逆转H2O2引起的细胞损伤。此外，细胞凋亡实验表明，与对照组相比，H2O2模型组的细胞凋亡率显着增加（P<0.01）。然而,联合QCT治疗显著逆转了这一趋势(P<0.05)，表明QCT在减轻细胞凋亡方面具有潜在的保护作用。ROS检测表明,与对照组相比，H2O2模型组ROS平均荧光强度明显升高(P<0.01)。QCT治疗后H2O2+QCT组的ROS荧光强度明显低于H2O2模型组,提示氧化损伤的有效缓解(P<0.05)。线粒体膜电位变化的JC-1染色显示，与对照组相比，H2O2模型组线粒体膜电位下降的细胞比例明显增加(P<0.01)。然而,与H2O2模型组相比，QCT治疗组的这一比例显着降低（P<0.05）。最后,Westernblot分析显示,模型组大鼠的NOX4和p-p38MAPK蛋白表达水平较对照组升高(P<0.05)。QCT治疗后,与H2O2模型组相比，这些蛋白水平显着降低（P<0.05）。这些结果表明，QCT可能通过调节p38MAPK/NOX4信号通路发挥其对氧化应激的保护作用。
■QCT已证明对H2O2诱导的HESCs氧化损伤具有显著的保护作用。这种保护主要通过有效减少ROS积累和抑制参与氧化应激反应的关键信号通路来实现。尤其是p38MAPK/NOX4通路。这项研究的结果表明，QCT调节这些途径的能力在减轻与氧化应激条件相关的细胞损伤中起着关键作用。这不仅表明其作为抗细胞氧化应激的保护剂的潜力，但也强调了其在治疗以子宫内膜氧化应激增加为特征的疾病中的治疗应用潜力，从而提供了增强生殖健康的前景。未来的研究应探讨QCT的长期影响及其在体内的临床疗效，从而为其整合到治疗方案中提供了明确的途径。
