槲皮素通过抑制p38 MAPK/NOX4信号通路减轻H_(2)O_(2)诱导的人子宫内膜基质细胞氧化应激损伤

Quercetin Alleviates H_(2)O_(2)-Induced Oxidative Stress Damage to Human Endometrial Stromal Cells by Inhibiting the p38 MAPK/NOX4 Signaling Pathway

目的 探讨槲皮素对过氧化氢(hydrogen peroxide, H_(2)O_(2))诱导人子宫内膜基质细胞(human endometrial stromal cells, HESCs)损伤的保护作用及其可能的作用机制。方法 体外培养HESCs,加入不同浓度的槲皮素(0、10、20和40μmol/L)作用24 h,验证给予不同剂量的槲皮素对正常子宫内膜细胞没有毒性,随后采用250μmol/L H_(2)O_(2)孵育细胞12 h,建立H_(2)O_(2)诱导的HESCs损伤模型。槲皮素预处理24 h,H_(2)O_(2)刺激HESCs后,CCK-8检测细胞活力,筛选有效的干预剂量,随后将HESCs分为空白组、H_(2)O_(2)模型组、H_(2)O_(2)+槲皮素组,采用DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species, ROS)水平;AnnexinⅤ/PI双染,流式细胞术检测槲皮素对H_(2)O_(2)诱导HESCs凋亡的影响;JC-1染色法检测细胞线粒体膜电位;Western blot检测相关蛋白NADPH氧化酶4(NADPH oxidase 4, NOX4)、p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase, p38 MAPK)及磷酸化p38 MAPK(p-p38 MAPK)表达。结果 根据CCK-8实验结果,选择槲皮素20μmol/L为有效干预剂量。ROS检测显示,与空白组相比,H_(2)O_(2)模型组ROS的平均荧光强度升高(P<0.01),而与H_(2)O_(2)模型组相比槲皮素处理下调了ROS的平均荧光强度,减轻了氧化损伤(P<0.05)。细胞凋亡检测结果显示,H_(2)O_(2)模型组细胞凋亡率较空白组增加(P<0.01),而与槲皮素共同作用则逆转了细胞凋亡率的增加(P<0.05)。JC-1染色检测线粒体膜电位变化情况显示,与空白组相比,H_(2)O_(2)诱导所致的线粒体膜电位降低的细胞比例增加(P<0.01),而槲皮素处理后线粒体膜电位降低的细胞比例低于H_(2)O_(2)模型组(P<0.05)。Western blot结果显示,与空白组相比,H_(2)O_(2)模型组NOX4蛋白、p-p38 MAPK蛋白相对表达量升高(P<0.05);而加入槲皮素后,与H_(2)O_(2)模型组相比,NOX4蛋白、p-p38 MAPK蛋白相对表达量降低(P<0.05)。结论 槲皮素预处理对H_(2)O_(2)诱导的HESCs氧化损伤具有保护作用,其机制可能是减少ROS过量累积以及抑制p38 MAPK/NOX4信号通路。

Objective 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(H_(2)O_(2)).Oxidative stress,such as that induced by H_(2)O_(2),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.Methods In vitro cultures of HESCs were treated with QCT at different concentrations(0,10,20,and 40μmol/L)for 24 h to verify the non-toxic effects of QCT on normal endometrial cells.Subsequently,250μmol/L H_(2)O_(2) was used to incubate the cells for 12 h to establish an H_(2)O_(2)-induced HESCs injury model.HESCs were pretreated with QCT for 24 h,which was followed by stimulation with H_(2)O_(2).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 H_(2)O_(2) model group,and the H_(2)O_(2)+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.AnnexinⅤ/PI double staining and flow cytometry were performed to determine the effect of QCT on H_(2)O_(2)-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.Results 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 40μmol/L)did not exhibit any cytotoxic effects,which laid the foundation for further investigation into its protective roles.In the H_(2)O_(2)-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(10μmol/L and 20μmol/L)significantly enhanced cell viability after 24 h(P<0.05),with the 20μmol/L concentration showing the most substantial effect.This suggests that QCT can effectively reverse the cellular damage caused by H_(2)O_(2).Furthermore,the apoptosis assays demonstrated a significant increase in the apoptosis rates in the H_(2)O_(2) 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 H_(2)O_(2) model group significantly increased(P<0.01).QCT treatment significantly reduced the ROS fluorescence intensity in the H_(2)O_(2)+QCT group compared to the that in the H_(2)O_(2) 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 H_(2)O_(2) model group(P<0.01).However,this proportion was significantly reduced in the QCT-treated group compared to that of the H_(2)O_(2) 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 H_(2)O_(2) 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 H_(2)O_(2) 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.Conclusion QCT has demonstrated significant protective effects against H_(2)O_(2)-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.