黄芪总黄酮通过ERα/STAT3信号通路调节子宫内膜异位症中子宫内膜细胞增殖和迁移

Regulation of endometrial cell proliferaattiioonn aanndd mmiiggrraattiion in endometriosis by total flavonoids through the ERα/STAT3 signaling pathway

目的研究黄芪总黄酮(TFA)对子宫内膜异位症(EMs)子宫内膜细胞增殖和迁移的影响,并基于ERα/STAT3信号通路探讨其潜在分子机制。方法分离人EMs患者子宫内膜细胞。(1)为初步探究不同剂量TFA对EMs子宫内膜细胞增殖和迁移的影响,将实验分为5组:对照(NC)组、EMs组、TFA高剂量组(TFA-H,TFA2mg/mL干预异位子宫内膜细胞)、TFA中剂量组(TFA-M,TFA1mg/mL干预异位子宫内膜细胞)、TFA低剂量组(TFA-L,TFA0.5mg/mL干预异位子宫内膜细胞)。采用平板克隆形成实验、CCK-8检测细胞增殖能力,细胞划痕实验检测细胞迁移能力,Westernblotting、RT-qPCR检测细胞ERα、STAT3蛋白和mRNA表达水平。(2)为探究ERα/STAT3信号通路对EMs子宫内膜细胞的影响,使用ERα诱导剂Feruti-nin干预EMs子宫内膜细胞,将实验分为两组:NC组,ERα诱导剂(Ferutinin)组。Westernblot、RT-qPCR检测细胞ERα、STAT3蛋白和mRNA表达水平,CCK-8检测细胞增殖能力,Transwell实验检测细胞迁移能力。(3)为进一步探究TFA及ERα/STAT3信号通路在EMs子宫内膜细胞的作用机制,将实验分为4组:NC组、EMs组、TFA-M组、TFA-M+Ferutinin组。Westernblot、RT-qPCR检测细胞ERα、STAT3蛋白和mRNA表达水平,CCK-8检测细胞增殖能力,Transwell实验检测细胞迁移能力。结果(1)与NC组相比,EMs组细胞克隆增殖能力增加(P<0.01);与EMs组相比,TFA-L组(P<0.05)、TFA-M组(P<0.05)和TFA-H组(P<0.01)细胞的克隆增殖能力降低。与NC组相比,EMs组细胞活力增加(P<0.01);与EMs组相比,TFA-L组、TFA-M组和TFA-H组细胞活力降低(P<0.05)。与NC组相比,EMs组细胞相对迁移距离增加(P<0.01);与EMs组相比,TFA-L组(P<0.05)、TFA-M组(P<0.05)和TFA-H组(P<0.01)细胞相对迁移距离减少。与NC组相比,EMs组细胞ERα和STAT3蛋白及mRNA表达水平增加(P<0.01);与EMs组相比,TFA-L组(P<0.05)、TFA-M组(P<0.01)和TFA-H组(P<0.01)细胞的ERα和STAT3蛋白及mRNA表达水平降低。根据以上实验结果,选择TFA-M进行后续实验。(2)与NC组相比,Ferutinin组细胞ERα和STAT3蛋白及mRNA表达水平增加(P<0.01)。与NC组相比,Ferutinin组细胞细胞活力增加(P<0.01)。与NC组相比,Ferutinin组细胞克隆增殖能力增加(P<0.01)。(3)与NC组相比,EMs组细胞ERα和STAT3蛋白及mRNA表达水平增加(P<0.05);与EMs组相比,TFA-M组细胞ERα和STAT3蛋白及mR-NA表达水平减少(P<0.05);与TFA-M组相比,TFA-M+Ferutinin组细胞ERα和STAT3蛋白及mRNA表达水平增加(P<0.05)。与NC组相比,EMs组细胞活力增加(P<0.05);与EMs组相比,TFA-M组细胞活力减少(P<0.05);与TFA-M组相比,TFA-M+Ferutinin组细胞活力增加(P<0.05)。与NC组相比,EMs组细胞克隆增殖能力增加(P<0.05);与EMs组相比,TFA-M组细胞克隆增殖能力降低(P<0.05);与TFA-M组相比,TFA-M+Ferutinin组细胞克隆增殖能力增加(P<0.05)。结论TFA可下调EMs中子宫内膜细胞的增殖和迁移能力,这可能与ERα/STAT3信号通路相关。

Objective To investigate the effects of Total Flavonoids of Astragalus(TFA)on the proliferation and migration of endometrial cells in Endometriosis(EMs)and explore the potential molecular mechanisms based on the ERa/STAT3 signaling pathway.Methods Endometrial cells were isolated from human EMs patients.In order to preliminatively explore the effects of different doses of TFA on the proliferation and migration of EMs endometrial cells,the experiment was divided into 5 groups,Control(NC)group,ectopic(EMs)group,Astragalus flavone high-dose group(TFA-H,2 mg/mLTFA intervention in ectopic endometrial cells),Astragalus flavone medium-dose group(TFA-M,1 mg/mLTFA intervention in ectopic endometrial cells),and Astragalus flavone low-dose group(TFA-L,O.5 mg/mLTFA for ectopic endometrial cells).Plate clone formation assay and CCK-8 assay were used to detect cell proliferation ability,cell scratch assay was used to detect cell migration ability,Western blotting and RT-qPCR were used to detect ERα,STAT3 protein and mRNA expression levels of cells.In order to investigate the effects of ERa/STAT3 signaling pathway on EMs endometrial cells,ERαinducer Ferutinin was used to intervene EMs endometrial cells.They were divided into two groups,NC group and ERαinducer(Ferutinin)group.Western bltting and RT-qPCR were used to detect the expression levels of ERα,STAT3 protein and mRNA,CCK-8 was used to detect cell proliferation ability,and Transwell assay was used to detect cell migration ability.In order to further explore the mechanism of TFA and ERo/STAT3 signaling pathway in EMs endometrial cells,the experiments were divided into four groups:NC group,EMs group,TFA-M group,and TFA-M+Ferutinin group.Western blotting and RT-qPCR were used to detect the expression levels of ERα,STAT3 protein and mRNA,CCK-8 was used to detect cell proliferation ability,and Transwell assay was used to detect cell migration ability Human endometrial cells from EMs patients were isolated.In the preliminary exploration of the impact of different doses of TFA on EMs endometrial cell proliferation and migration,the study was divided into five groups,Control(NC)group,Endometriosis(EMs)group,High-dose TFA group(TFA-H,2 mg/mL TFA intervention in EMs endometrial cells),Medium-dose TFA group(TFA-M,1 mg/mL TFA intervention in EMs endometrial cells),and Low-dose TFA group(TFA-L,O.5 mg/mL TFA intervention in EMs endometrial cells).Clonogenic formation assay,CCK-8 assay,scratch assay,Western blotting,and RT-qPCR were employed to evaluate cell proliferation,migration,and the expression levels of ERαand STAT3 proteins and mRNA.Results(1)Compared to the NC group,the EMs group showed increased clonogenic proliferative ability(P<0.01).Compared to the EMs group,the TFA-L group(P<0.05),TFA-M group(P<0.05),and TFA-H group(P<0.01)exhibited decreased clonogenic proliferative ability.Compared to the NC group,the EMs group demonstrated increased cell viability(P<0.01).Compared to the EMs group,the TFA-L group,TFA-M group,and TFA-H group showed decreased cell viability(P<0.05).Compared to the NC group,the EMs group exhibited an increase in relative migration distance(P<0.01).Compared to the EMs group,the TFA-L group(P<0.05),TFA-M group(P<0.05),and TFA-H group(P<0.01)showed a decrease in relative migration distance.Compared to the NC group,the EMs group showed increased expression levels of ERαand STAT3 proteins and mRNA(P<0.01).Compared to the EMs group,the TFA-L group(P<0.05),TFA-M group(P<0.01),and TFA-H group(P<0.01)exhibited decreased expression levels of ERαand STAT3 proteins and mRNA.Based on these results,TFA-M was selected for subsequent experiments.(2)Compared to the NC group,the Ferutinin group showed increased expression levels of ERαand STAT3 proteins and mRNA(P<0.O1).Compared to the NC group,the Ferutinin group demonstrated increased cell viability(P<O.O1).Compared to the NC group,the Ferutinin group exhibited increased clonogenic proliferative ability(P<0.01).(3)Compared to the NC group,the EMs group showed increased expression levels of ERαand STAT3 proteins and mRNA(P<0.05).Compared to the EMs group,the TFA-M group exhibited decreased expression levels of ERαand STAT3 proteins and mRNA(P<0.05).Compared to the TFA-M group,the TFA-M+Ferutinin group showed increased expression levels of ERαand STAT3 proteins and mRNA(P<0.05).Compared to the NC group,the EMs group demonstrated increased cell viability(P<0.05).Conclusion TFA has the potential to downregulate the proliferation and migration capabilities of endometrial cells in EMs,which may be associated with the ERα/STAT3 signaling pathway.