四川大学学报（医学版）2024，Vol.55Issue(3)：552-558,7.DOI:10.12182/20240560107
槲皮素通过抑制p38 MAPK/NOX4信号通路减轻H2O2诱导的人子宫内膜基质细胞氧化应激损伤
Quercetin Alleviates H2O2-Induced Oxidative Stress Damage to Human Endometrial Stromal Cells by Inhibiting the p38 MAPK/NOX4 Signaling Pathway
摘要
Abstract
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(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.Methods In 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.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 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.Conclusion 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.关键词
槲皮素/子宫内膜基质细胞/氧化应激/细胞凋亡/活性氧Key words
Quercetin/Endometrial stromal cells/Oxidative stress/Apoptosis/Reactive oxygen species引用本文复制引用
陈秀楠,王瑞琦,单红英,周平,李蓉..槲皮素通过抑制p38 MAPK/NOX4信号通路减轻H2O2诱导的人子宫内膜基质细胞氧化应激损伤[J].四川大学学报（医学版）,2024,55(3):552-558,7.基金项目
国家自然科学基金(No.82288102)资助 （No.82288102）
