摘要
目的探究熊果酸(ursolic acid,UA)对异位子宫内膜基质细胞(ectopic endometrial stromal cells,EESCs)增殖、迁移和铁死亡的影响及其作用机制。方法构建子宫内膜异位症小鼠模型,并分离原代EESCs,使用不同浓度(0、2.5、5、10、20、40、50、80、100、200 μmol/L)的熊果酸处理细胞;并将细胞分为Control组(正常培养)、2.5 μmol/L UA组(2.5 μmol/L熊果酸处理)、5.0 μmol/L UA组(5.0 μmol/L熊果酸处理)、10.0 μmol/L UA组(10 μmol/L熊果酸处理)、和UA+DUSP19组(10 μmol/L熊果酸+50 μmol/L的JAK2/STAT3信号通路激活剂DUSP19处理)。使用CCK-8法检测细胞存活率;使用平板克隆法检测细胞增殖;使用Transwell小室实验检测细胞迁移能力;使用试剂盒检测各组细胞Fe2+水平以及丙二醛(MDA)、活性氧(ROS)和超氧化物歧化酶(superoxide dismutase,SOD)含量;使用蛋白免疫印迹检测各组细胞Ki67、PCNA、CyclinD1、p-JAK2、p-STAT3、JAK2、STAT3蛋白表达水平。结果 Control、2.5 μmol/L UA、5.0 μmol/L UA、10.0 μmol/L UA组克隆数分别为:(152.22±15.47)、(121.22±11.54)、(92.00±5.54)、(66.44±6.88)个;Ki67蛋白表达量分别为:1.08±0.10、0.73±0.07、0.61±0.06、0.45±0.02;PCNA蛋白表达量分别为:0.85±0.07、0.64±0.05、0.41±0.03、0.31±0.05;CyclinD1蛋白表达量分别为:0.98±0.11、0.65±0.06、0.51±0.05、0.42±0.07;迁移数目分别为(92.78±6.27)、(62.22±2.20)、(50.22±4.59)、(39.11±4.33)个;Fe2+水平分别为:(1.06±0.07)、(1.21±0.11)、(1.33±0.08)和(1.47±0.09)μmol/g;MDA含量分别为:(0.48±0.06)、(0.65±0.07)、(0.85±0.08)和(1.03±0.11)μmol/g;ROS含量分别为(19.85±1.21)%、(24.83±2.79)%、(29.04±1.86)%、(33.87±2.45)%;SOD含量分别为:(36.41±3.56)、(31.03±2.81)、(25.63±2.84)和(19.62±1.67)U/mg;p-JAK2蛋白表达量分别为:0.85±0.10、0.75±0.06、0.53±0.05、0.31±0.03;p-STAT3蛋白表达量分别为:1.08±0.11、0.79±0.06、0.63±0.07、0.42±0.03。UA组和UA+DUSP19组的p-JAK2蛋白含量分别为:0.38±0.05、0.75±0.08;p-STAT3蛋白表达量分别为:0.46±0.04、0.80±0.03;细胞存活率分别为:(52.55±2.44)%、(82.18±4.72)%;Fe2+水平分别为:(1.57±0.06)和(1.21±0.13)μmol/g。以上指标,Control组与2.5 μmol/L UA组、5.0 μmol/L UA组、10.0 μmol/L UA组之间差异均具有统计学意义(P<0.05);2.5 μmol/L UA组、5.0 μmol/L UA组、10.0 μmol/L UA组之间差异均具有统计学意义(P<0.05);UA组和UA+DUSP19组之间p-JAK2、p-STAT3、细胞存活率和Fe2+水平差异均具有统计学意义(P<0.05)。结论熊果酸可通过调控JAK2/STAT3信号通路抑制EESCs细胞增殖、迁移,并诱导铁死亡,进而发挥对子宫内膜异位症的保护作用。
Objective To investigate the effects of ursolic acid(UA)on proliferation,migration and iron death of ectopic endometrial stromal cells(EESCs)and its mechanism.Methods Mouse model of endometriosis was established and the primary EESCs were isolated.The cells were treated with UA at different concentrations(0,2.5,5,10,20,40,50,80,100,200μmol/L).The cells were divided into Control group(normal culture),2.5μmol/L UA group(2.5μmol/L UA treatment),5.0μmol/L UA group(5.0μmol/L UA treatment),10.0μmol/L UA group(10μmol/L UA treatment),and UA+DUSP19 group(10μmol/L UA+50μmol/L JAK2/STAT3 signal pathway activator DUSP19 treatment).Cell survival rate was detected by CCK-8 method.Cell proliferation was detected by plate cloning method.Transwell chamber assay was used to detect cell migration.The levels of Fe2+and the contents of malondialdehyde(MDA),reactive oxygen species(ROS)and superoxide dismutase(SOD)were detected by kit.Protein expression levels of Ki67,PCNA,CyclinD1,p-JAK2,p-STAT3,JAK2 and STAT3 were detected by western blot.Results The number of clones in Control,2.5μmol/L UA,5.0μmol/L UA and 10.0μmol/L UA groups were as follows:152.22±15.47,121.22±11.54,92.00±5.54,66.44±6.88;Ki67 protein expression was 1.08±0.10,0.73±0.07,0.61±0.06,0.45±0.02,respectively;The expression of PCNA protein was 0.85±0.07,0.64±0.05,0.41±0.03,0.31±0.05,respectively;CyclinD1 protein expression levels were 0.98±0.11,0.65±0.06,0.51±0.05,0.42±0.07,respectively.The migration numbers were 92.78±6.27,62.22±2.20,50.22±4.59 and 39.11±4.33,respectively;Fe2+levels were(1.06±0.07)μmol/g,(1.21±0.11)μmol/g,(1.33±0.08)μmol/g,(1.47±0.09)μmol/g,respectively;MDA content was(0.48±0.06)μmol/g,(0.65±0.07)μmol/g,(0.85±0.08)μmol/g,(1.03±0.11)μmol/g,respectively;ROS contents were(19.85±1.21)%,(24.83±2.79)%,(29.04±1.86)%,(33.87±2.45)%,respectively;SOD content were(36.41±3.56)U/mg,(31.03±2.81)U/mg,(25.63±2.84)U/mg,(19.62±1.67)U/mg,respectively;p-JAK2 protein expression was 0.85±0.10,0.75±0.06,0.53±0.05,0.31±0.03,respectively;p-STAT3 protein expression was 1.08±0.11,0.79±0.06,0.63±0.07,0.42±0.03,respectively.The p-JAK2 protein content in UA group and UA+DUSP19 group was 0.38±0.05 and 0.75±0.08,respectively;p-STAT3 protein expression was 0.46±0.04 and 0.80±0.03,respectively;The cell survival rates were(52.55±2.44)%and(82.18±4.72)%,respectively;Fe2+levels were(1.57±0.06)μmol/g and(1.21±0.13)μmol/g,respectively.The differences in the above indicators between the Control group and the 2.5μmol/L UA group,5.0μmol/L UA group and 10.0μmol/L UA group were statistically significant(P<0.05).There were statistically significant differences among 2.5μmol/L UA group,5.0μmol/L UA group and 10.0μmol/L UA group(P<0.05).There were statistically significant differences in p-JAK2,p-STAT3,cell survival rate and Fe2+levels between UA group and UA+DUSP19 group(P<0.05).Conclusion Ursolic acid can inhibit the proliferation and migration of EESCs cells and induce iron death by regulating JAK2/STAT3 signaling pathway,thus playing a protective role in endometriosis.
作者
王聪
门贵香
邵帅
马丽娜
Wang Cong;Men Guixiang;Shao Shuai;Ma Lina(Department of Obstetrics,Linyi People’s Hospital,Linyi 276000,China)
出处
《中华内分泌外科杂志》
CAS
2023年第3期317-322,共6页
Chinese Journal of Endocrine Surgery
基金
山东省医药卫生科技发展计划项目(202205021115)。