葛根素通过上调Wnt/β-catenin信号通路改善化疗诱导的雌性生殖干细胞的氧化应激损伤和凋亡  

Puerarin Ameliorates Chemotherapy-Induced Oxidative Stress Injury and Apoptosis of Female Germline Stem Cells through Up-Regulation of Wnt/β-catenin Signaling Pathway

作　　者：迪丽努尔·安外尔 热孜宛古丽·约麦尔 陆萍[1] DILINUER Anwaier;REZIWANGULI Yuemaier;LU Ping(Dept.of Gynecology,People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China;Center for Research and Education,center of People’s Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China)

机构地区：[1]新疆维吾尔自治区人民医院妇科,乌鲁木齐830001 [2]新疆维吾尔自治区人民医院科研教育中心,乌鲁木齐830001

出　　处：《中国医院用药评价与分析》2023年第4期420-426,430,共8页Evaluation and Analysis of Drug-use in Hospitals of China

基　　金：新疆维吾尔自治区自然科学基金项目(No.2022D01C104)。

摘　　要：目的:探讨葛根素(PUE)对化疗诱导的雌性生殖干细胞(FGSCs)氧化应激损伤和凋亡的影响及相关机制。方法:分离并鉴定乳鼠卵巢表层上皮的原代FGSCs,采用细胞计数试剂盒-8(CCK-8)法检测PUE对FGSCs增殖活力的影响。将FGSCs分为对照组(Control)、白消安(BU)组、BU+PUE组和BU+PUE+DKK-1组;采用CCK-8法检测各组FGSCs的增殖活性;采用2,7-二氯荧光素法与试剂盒检测各组FGSCs中活性氧(ROS)、丙二醛(MDA)含量,谷胱甘肽过氧化物酶(GPx)、超氧化物歧化酶2(SOD2)活性;采用线粒体膜电位检测JC-1染色法检测各组FGSCs中线粒体膜电位水平;采用细胞凋亡检测试剂盒检测各组FGSCs的凋亡率;采用蛋白质印迹法检测各组FGSCs中生殖干性相关蛋白小鼠Vasa同源体(Mvh)、八聚体结合转录因子4(Oct4)及Wnt/β-catenin信号通路中β-catenin蛋白和凋亡相关蛋白B细胞淋巴瘤2(Bcl-2)、活化的半胱氨酸天冬氨酸蛋白酶3(cleaved caspase-3)的表达水平。结果:成功分离了符合条件的FGSCs,且PUE能促进细胞的增殖活力。与Control组相比,BU组、BU+PUE组和BU+PUE+DKK-1组细胞中ROS、MDA和cleaved caspase-3的表达水平均明显升高,差异均有统计学意义(P<0.05),细胞的增殖活力,线粒体膜电位,细胞中GPx、SOD2、Mvh、Oct4、β-catenin及Bcl-2的表达水平均明显降低,差异均有统计学意义(P<0.05)。与BU组相比,BU+PUE组细胞中ROS、MDA和cleaved caspase-3的表达水平均明显降低,差异均有统计学意义(P<0.05),细胞的增殖活力,线粒体膜电位,GPx、SOD2、Mvh、Oct4、β-catenin及Bcl-2的表达水平却明显升高,差异均有统计学意义(P<0.05),BU+PUE+DKK-1组与BU组的差异无统计学意义(P>0.05)。结论:PUE可通过Wnt/β-catenin途径减轻BU诱导的氧化应激损伤和细胞凋亡,并恢复FGSCs的增殖活性及干性。OBJECTIVE:To probe into the effect and mechanism of puerarin(PUE)on chemotherapy-induced oxidative damage and apoptosis of female germ stem cells(FGSCs).METHODS:Primary FGSCs were isolated and identified from the superficial ovarian epithelium of suckling mice,CCK-8 method was used to detect the effect of PUE on the proliferative activity of FGSCs.FGSCs were divided into Control group,busulfan(BU)group,BU+PUE group and BU+PUE+DKK-1 group;CCK-8 method was used to detect the proliferative activity in each group of FGSCs;DCFH-DA method with kit was used to detect reactive oxygen species(ROS),malondialdehyde(MDA)content,glutathione peroxidase(GPx)and superoxide dismutase 2(SOD2)activities in each group of FGSCs;JC-1 staining method was used to detect the mitochondrial membrane potential levels in each group of FGSCs;Annexin V-FITC/PI method was used to detect the apoptosis rates in each group of FGSCs;Western blotting was used to detect the reproductive stemness-related proteins Mvh and Oct4,the expression levels ofβ-catenin protein and apoptosis-related proteins B-cell lymphoma 2(Bcl-2)and cleaved caspase-3 in the Wnt/β-catenin signaling pathway.RESULTS:Eligible FGSCs were successfully isolated,and PUE promoted the proliferative viability of the cells.Compared with the Control group,the expression of ROS,MDA and cleaved caspase-3 were significantly higher in the BU group,BU+PUE group and BU+PUE+DKK-1 group,with statistically significant differences(P<0.05),and the cell proliferative viability,mitochondrial membrane potential,GPx,SOD2,Mvh,Oct4,β-catenin and Bcl-2 expression levels were significantly lower,with statistically significant differences(P<0.05).Compared with the BU group,the expression of ROS,MDA and cleaved caspase-3 were significantly lower in the BU+PUE group,with statistically significant differences(P<0.05),but the cell proliferation viability,mitochondrial membrane potential,GPx,SOD2,Mvh,Oct4,β-catenin and Bcl-2 expression levels were significantly higher,with statistically significant difference

关 键 词：葛根素 雌性生殖干细胞 氧化应激 凋亡 

分 类 号：R96[医药卫生—药理学]