摘要
目的分析枸杞多糖(lycium barbarum polysaccharides,LBP)对过氧化氢刺激引起的人子宫内膜间质细胞(endometrial stromal cells,ESCs)凋亡的影响以及对同源物磷酸脂酶基因(phosphatase and tensin homologue deleted on chromosome 10,PTEN)/蛋白激酶(protein kinase,AKT)信号通路蛋白的激活情况。方法体外培养传代人ESCs,实验设计对照组、LBP组、过氧化氢组以及治疗组共4组。对照组:常规培养液;LBP组:常规培养液+100 mg/L的LBP;过氧化氢组:常规培养液+100μmol/L的过氧化氢;治疗组:提前加入100 mg/L的LBP作用24 h,再加入100μmol/L的过氧化氢,观察细胞在形态方面的改变。细胞活力检测试剂盒(cell counting kit-8,CCK-8)分析各组细胞的存活率,流式细胞技术测量细胞的凋亡率,比色法测量4组细胞中丙二醛(malondiald ehyde,MDA)的含量和超氧化物歧化酶(superoxide dismutase,SOD)的活力,蛋白免疫印迹分析细胞中B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 Associated X,Bax)、PTEN、AKT以及磷酸化的AKT(p-protein kinase,p-AKT)的含量。结果LBP组和对照组相比,细胞形态、存活率、凋亡率以及细胞中的Bcl-2、Bax、PTEN、p-AKT和AKT比较,差异均无统计学意义(均P>0.05)。过氧化氢组和正常组比较,细胞呈圆形,脱落多而贴壁少;存活率[(51.37±3.28)%]降低,而凋亡率[(53.42±2.37)%]升高;MDA含量[(4.56±0.70)mmol/L]上升而SOD活力[(9.39±0.72)U/L]下降,胞质中Bcl-2(0.148±0.020)和p-AKT(0.457±0.053)含量降低,Bax(1.076±0.036)和PTEN(0.538±0.049)含量升高(均P<0.05)。治疗组和过氧化氢组比较,细胞伸展且有短突起,脱落少而贴壁多;存活率[(87.72±4.52)%]升高,而凋亡率[(25.02±2.76)%]降低;MDA含量[(2.14±0.47)mmol/L]下降而SOD活力[(16.60±1.25)U/L]上升,胞质中Bcl-2(0.852±0.051)和p-AKT(0.942±0.095)含量上升,Bax(0.202±0.014)和PTEN(0.283±0.022)含量下降(均P<0.05)。结论LBP能明显拮抗过氧化氢刺激导致的细胞存活率降低及凋亡率升高,降低氧化应激产物,这与激活PTEN/AKT信号通路密切相关。
Objective To explore the effects of Lycium barbarum polysaccharides(LBP)on apoptosis of human endometrial stromal cells(ESCs)induced by hydrogen peroxide and on activation of phosphatase and tensin homologue deleted on chromosome 10(PTEN)/protein kinase(ATK)signaling pathway proteins.Methods Human ESCs were cultured in vitro and arranged into four groups:control group,LBP group,hydrogen peroxide group and treatment group.Different solutions were added to the four groups respectively:control group with conventional cell culture medium,LBP group with conventional cell culture medium+100 mg/L LBP,hydrogen peroxide group with conventional cell culture medium+100 mg/L hydrogen peroxide,and treatment group was treated as follows:100 mg/L LBP was added in advance for 24 hours,and then 100μmol/L hydrogen peroxide was added.The morphological changes of the cells were observed.The cell counting kit-8(CCK-8)was used to measure the cell survival rate.The apoptosis rate was measured by flow cytometry.The content of malondialdehyde(MDA)and activities of superoxide dismutase(SOD)were measured by colorimetry.The contents of B-cell lymphoma-2(Bcl-2),Bcl-2 Associated X(Bax),PTEN,AKT and pprotein kinase(p-AKT)were analyzed by Western blot.Results There were not statistically significant differences in the cell morphology,survival rate,apoptosis rate and contents of Bcl-2,Bax,PTEN,p-AKT and AKT between LBP group and control group(all P>0.05).Compared with normal group,the cells in the hydrogen peroxide group were round,shedding more and adhering less,the survival rate decreased[(51.37±3.28)%]and the apoptosis rate increased[(53.42±2.37)%],MDA content increased[(4.56±0.70)mmol/L]and SOD activity decreased[(9.39±0.72)U/L],the contents of Bcl-2(0.148±0.020)and p-AKT(0.457±0.053)in cytoplasm decreased,while the contents of Bax(1.076±0.036)and PTEN(0.538±0.049)increased(all P<0.05).Compared with hydrogen peroxide group,the cells in treatment group extended and had short processes,less shedding and more adherence,the survival rate increased[(87.72±4.52)%]and the apoptosis rate decreased[(25.02±2.76)%],MDA content decreased[(2.14±0.47)mmol/L]and SOD activity[(16.60±1.25)U/L]increased,the contents of Bcl-2(0.852±0.051)and p-AKT(0.942±0.095)in cytoplasm increased,while the contents of Bax(0.202±0.014)and PTEN(0.283±0.022)decreased(all P<0.05).Conclusion Lycium barbarum polysaccharide can significantly antagonize the decreased cell survival rate and increased apoptosis rate induced by hydrogen peroxide,and reduce oxidative stress products,which is closely related to the activation of PTEN/AKT signaling pathway.
作者
张伟伟
赵芸娟
王燕
单铁英
任俊利
汤丽丽
ZHANG Wei-wei;ZHAO Yun-juan;WANG Yan;SHAN Tie-ying;REN Jun-li;TANG Li-li(Department of Obstetrics and Gynecology,Handan Maternal and Child Health Hospital,Handan Hebei,056002,China;Department of Gynecology,Physical Examination Center of Handan Infectious Disease Hospital,Handan,Hebei,056002,China;Clinical Laboratory,Physical Examination Center of Handan Infectious Disease Hospital,Handan,Hebei,056002,China;Medical College,Hebei University of Engineering,Handan,Hebei,056002,China;Department of Pediatrics,Handan Central Hospital,Handan,Hebei,056002,China;Department of Gynecology,Handan Hospital of Traditional Chinese Medicine,Handan,Hebei,056002,China)
出处
《职业与健康》
CAS
2022年第5期616-620,共5页
Occupation and Health
