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

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81.59 μmol/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。

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

Objective　To investigate the enhancement of eucommia chlorogenic acid(CGA) on the radiosensitivity of retinoblastoma cell line HXO-Rb44 by regulating PI3K/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 (TrxR1), activated phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), p-AKT and nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined by Western blot. Furthermore, PI3K activator 740Y-P was added to detect, the cell cycle, cell apoptosis, cell proliferation and apoptosis associated protein expressions, expressions of proteins in PI3K/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 81.59 μmol/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, TrxR1, p-PI3K, 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, PI3K activator could reverse the effects of radiation treatment on cell cycle, cell proliferation, cell apoptosis and PI3K/AKT/Nrf2 pathway. CGA could restore the changes of the above indicators caused by radiation (P<0.01). Conclusions CGA can enhance the radiosensitivity of retinoblastoma cell line HXO-Rb44, and the protective effect of CGA may be related to its inhibition of PI3K/AKT/Nrf2 pathway.