长栲利素A通过PI3K/AKT通路抑制脑胶质瘤细胞增殖  

Longikaurin A inhibits glioma proliferation through phosphatidylinositol 3-kinase/protein kinase B pathway

作　　者：孟祥瑞 高一粟 孙关 Meng Xiangrui;Gao Yisu;Sun Guan(Department of Neurosurgery of Yancheng First People's Hospital,Yancheng Clinical College of Xuzhou Medical University,224006 Yancheng,China;Yancheng Medical Research Center of Nanjing University Medical School,224006 Yancheng,China)

机构地区：[1]徐州医科大学盐城临床学院,盐城市第一人民医院神经外科,盐城224006 [2]南京大学医学院盐城医学研究中心,盐城224006

出　　处：《中华神经医学杂志》2023年第5期480-488,共9页Chinese Journal of Neuromedicine

基　　金：江苏省自然科学基金面上项目(BK20211115);江苏省卫健委老年项目(LD2021037)。

摘　　要：目的探讨长栲利素A对脑胶质瘤细胞增殖的影响及其作用机制。方法复苏冻存的人脑胶质瘤细胞株U87、SNB19并予以体外传代培养,应用CCK-8实验检测0、0.5、1.0、1.5、2.0、2.5、3.0、4.0、6.0、8.0μmol/L长栲利素A干预24 h、48 h后细胞活性的变化,应用Edu增殖实验检测0、1、2、3μmol/L长栲利素A干预后Edu阳性细胞数量的变化,应用细胞克隆形成实验检测0、0.1、0.2、0.3μmol/L长栲利素A干预后细胞集落形成数的变化,应用流式细胞术检测0、1、2、3μmol/L长栲利素A干预后细胞周期占比的变化,应用Western blotting检测0、1、2、3μmol/L长栲利素A干预后细胞增殖相关蛋白Ki-67、c-Myc及细胞周期相关蛋白细胞周期素B1(Cyclin B1)、细胞周期素D1(Cyclin D1)、细胞周期蛋白依赖性激酶-1(CDK1)以及磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/AKT)信号通路相关蛋白的表达变化。结果与0μmol/L相比,0.5、1.0、1.5、2.0、2.5、3.0、4.0、6.0、8.0μmol/L长栲利素A干预24 h、48 h后U87、SNB19细胞活性均逐渐降低,且呈剂量依赖性。与0μmol/L长栲利素A组相比,2、3μmol/L长栲利素A组U87、SNB19细胞中Edu阳性细胞数量明显降低,0.1、0.2、0.3μmol/L长栲利素A组U87、SNB19细胞集落形成数明显降低,2、3μmol/L长栲利素A组U87、SNB19细胞中G2/M期细胞占比明显升高,2、3μmol/L长栲利素A组U87、SNB19细胞中Ki-67、c-Myc、Cyclin B1、CDK1、磷酸化PI3K及磷酸化AKT表达明显降低,Cyclin D1表达明显升高,差异均有统计学意义(P<0.05)。结论长栲利素A可抑制脑胶质瘤细胞的增殖并将细胞周期阻滞于G2/M期,且呈剂量依赖性,其作用机制与PI3K/AKT信号通路抑制有关。Objective To investigate the effect and mechanism of longikaurin A(LK-A)on glioma proliferation.Methods Resuscitated frozen human glioma cell lines U87 and SNB19 were in vitro sub-cultured;CCK-8 assay was used to detect the cell activity changes 24 and 48 h after 0,0.5,1.0,1.5,2.0,2.5,3.0,4.0,6.0,8.0μmol/L LK-A.Edu proliferation assay was used to detect the changes in number of Edu positive cells after 0,1,2 and 3μmol/L LK-A,and cell clonal formation assay was used to detect the changes in number of cell colony formation after 0,0.1,0.2 and 0.3μmol/L LK-A.Flow cytometry was used to detect the changes in cell cycle proportion after 0,1,2 and 3μmol/L LK-A.Expression changes of proliferation-related proteins(Ki-67 and c-Myc)and cell cycle-related proteins(Cyclin B1,Cyclin D1,cyclin dependent kinase 1[CDKl])and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway-related proteins were detected by Western blotting after 0,1,2,and 3μmol/L LK-A.Results U87 and SNB19 cell viabilities decreased gradually after being treated with 0.5,1.0,1.5,2.0,2.5,3.0,4.0,6.0,and 8.0μmol/L LK-A for 24 and 48 h compared with those with 0μmol/L LK-A,with significant differences(P<0.05);cell viability was dose-dependent.The number of Edu positive cells in U87 and SNB19 after being treated with 2,and 3μmol/L LK-A was significantly decreased compared with that with 0μmol/L LK-A(P<0.05).The colony formation number of U87 and SNB19 cells after being treated with 0.1,0.2 and 0.3μmol/L LK-A decreased significantly compared with that with 0μmol/L LK-A(P<0.05).The proportion of U87 and SNB19 cells at G2/M phase after being treated with 2 and 3μmol/L LK-A were increased significantly compared with that with 0μmol/L LK-A(P<0.05).The Ki-67,c-Myc,Cyclin B1,CDK1,p-PI3K and p-AKT expressions were significantly decreased,while Cyclin D1 expression was significantly increased in U87 and SNB19 cells after being treated with 2 and 3μmol/L LK-A compared with those with 0μmol/L LK-A(P<0.05).Conclusion LK-A can inhibit the g

关 键 词：长栲利素A 神经胶质瘤 细胞增殖 磷脂酰肌醇3-激酶/蛋白激酶B信号通路 

分 类 号：R739.41[医药卫生—肿瘤]