杜仲绿原酸通过PI3K/AKT/Nrf-2通路增强视网膜母细胞瘤细胞系HXO-Rb44放射敏感性  

Eucommia chlorogenic acid enhances the radiosensitivity of retinoblastoma cell line HXO-Rb44 via PI3K/AKT/Nrf-2 pathway

作　　者：刘铭[1] 王俊 李丽[2] 成仲夏[1] LIU Ming;WANG Jun;LI Li;CHENG Zhong-xia(Department of Ophthalmology,Affiliated Hospital of Chengdu University,Chengdu 610081;Department of Ophthalmology,Sichuan Provincial People's Hospital,Chengdu 610072,China)

机构地区：[1]成都大学附属医院眼科,成都610081 [2]四川省人民医院眼科,成都610072

出　　处：《中国临床解剖学杂志》2019年第6期644-649,655,共7页Chinese Journal of Clinical Anatomy

基　　金：四川省卫计委科研项目（18PJ150

摘　　要：目的探究杜仲绿原酸(CGA)通过调控PI3K/AKT/Nrf-2通路,对视网膜母细胞瘤细胞系HXO-Rb44放射敏感性的增强作用。方法通过不同浓度的CGA处理HXO-Rb44细胞,检测IC10,作为后续实验中CGA浓度。将细胞分为Ctrl、Radio、CGA、Radio+CGA组,Radio组给予4 MV X射线照射24 h,CGA组使用CGA处理24 h,Radio+CGA组在CGA处理24 h后再给予4 MV X射线照射24 h,流式细胞术检测细胞周期与细胞凋亡,蛋白质印迹检测Ki67、依赖还原型辅酶I/II醌氧化还原酶I(NQO1)、活化型半胱天冬酶(cl-caspase-3)、硫氧还蛋白还原酶1(TrxR1)、活化磷脂酰肌醇3-激酶(p-PI3K)、蛋白激酶B(AKT)、p-AKT、核因子E2相关因子2(Nrf2)蛋白表达。进一步通过PI3K激活剂740Y-P处理细胞,检测细胞周期、凋亡、增殖和相关蛋白表达、PI3K/AKT/Nrf2通路蛋白表达。结果随着CGA浓度的增高,细胞的相对存活率降低,药物毒性呈浓度依赖性,IC10为81.59μmol/L。与Ctrl组相比,Radio和CGA组G2/M期细胞比例显著升高,细胞凋亡比率显著升高(P<0.01);与Radio组相比,Radio+CGA组G2/M期细胞比例和细胞凋亡比率进一步显著升高(P<0.01)。同时在蛋白水平上,与Ctrl组相比,Radio和CGA组Ki67、NQO1、TrxR1、p-PI3K、Nrf2蛋白表达及p-AKT/AKT比率显著降低(P<0.01);放射和CGA联合作用进一步降低以上各指标(P<0.01)。此外,PI3K激活剂可逆转放射对细胞周期、增殖、凋亡以及PI3K/AKT/Nrf2通路的作用,CGA可恢复放射引起的上述各指标的变化(P<0.01)。结论CGA可增强HXO-Rb44细胞的放射敏感性,其作用机制与抑制PI3K/AKT/Nrf2通路相关。Objective To investigate the enhancement of eucommia chlorogenic acid(CGA)on the radiosensitivity of retinoblastoma cell line HXO-Rb44 by regulating PI3 K/AKT/Nrf-2 pathway.Methods HXO-Rb44 cells were treated with different concentrations of CGA,then IC10 was detected as CGA concentration in subsequent experiments.Cells were divided into 4 groups:a Ctrl group,a Radio group,a CGA group and a Radio+CGA group.Radio group was disposed with 4 MV X-rays for 24 h,CGA group was processed with CGA for 24 h,and Radio+CGA group was disposed with 4 MV X-rays for 24 h after CGA treatment for 24 h.Flow cytometry was used to detect the cell cycle and cell apoptosis.The protein expressions of Ki67,NAD(P)H quinone oxidoreductase I(NQO1),cleaved-caspase-3(cl-caspase-3),thioredoxin reductase-1(Trx R1),activated phosphatidylinositol 3-kinase(PI3 K),protein kinase B(AKT),pAKT and nuclear factor-erythroid 2-related factor 2(Nrf2)were determined by Western blot.Furthermore,PI3 K activator 740 Y-P was added to detect,the cell cycle,cell apoptosis,cell proliferation and apoptosis associated protein expressions,expressions of proteins in PI3 K/AKT/Nrf2 pathway.Results With the increasing concentrations of CGA,the relative survival rates of cells were decreased,and the drug toxicity was concentration-dependent.The IC10 value was 81.59μmol/L.Compared with Ctrl group,the cell population in G2/M period was significantly increased,and the cell apoptosis rates were increased in Radio group and CGA group(P<0.01).Compared with Radio group,the cell population in G2/M period and cell apoptosis rates were increased further in Radio+CGA group.Meanwhile,compared with Ctrl group,the protein expressions of Ki67,NQO1,Trx R1,p-PI3 K,Nrf2 and p-AKT/AKT ratios in other groups were decreased significantly(P<0.01),and the combined effect of Radiation and CGA further reduced the above indicators in Radio+CGA group.Furthermore,PI3 K activator could reverse the effects of radiation treatment on cell cycle,cell proliferation,cell apoptosis and PI3 K/AKT/Nrf2 pat

关 键 词：杜仲绿原酸 放射敏感性 视网膜母细胞瘤 PI3K/AKT/Nrf2通路 

分 类 号：R739.7[医药卫生—肿瘤]