摘要
目的探讨黄芪甲苷对放射性食管上皮细胞(Het-1A)损伤的保护作用及其可能机制。方法采用6 MV-X射线(2、4、6 Gy)照射Het-1A细胞建立放射性食管炎模型,并以不同浓度的黄芪甲苷(0、5、10、20、40、80、160μmol/L)处理模型细胞,采用CCK-8法检测细胞增殖情况。将Het-1A细胞分为正常对照组、射线损伤模型组、黄芪甲苷(40μmol/L)组、磷脂酰肌醇3-激酶(PI3K)抑制剂LY294002(10μmol/L)组及黄芪甲苷(40μmol/L)+LY294002(10μmol/L)组。采用ELISA法检测细胞肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平;Ed U法检测细胞增殖;流式细胞术检测细胞凋亡;免疫荧光法检测PI3K磷酸化(p-PI3K)蛋白表达;Western blotting检测半胱天冬氨酸蛋白酶3(Caspase-3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X基因(Bax)、PI3K、蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)及其剪切或磷酸化蛋白表达。结果选择4 Gy射线剂量构建Het-1A细胞放射性损伤模型。与0μmol/L黄芪甲苷比较,10、20、40、80μmol/L黄芪甲苷处理可显著提高放射性损伤Het-1A细胞的活力(P<0.05)。与正常对照组比较,射线损伤模型组Het-1A细胞TNF-α、IL-6、凋亡率、Cleaved caspase-3/Caspase-3及Bax蛋白表达均升高,而细胞增殖率、Bcl-2、p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR蛋白表达均降低(P<0.05)。与射线损伤模型组比较,黄芪甲苷组Het-1A细胞上述炎症因子水平、凋亡率及相关促凋亡蛋白表达均显著降低,而增殖能力及PI3K/AKT/mTOR通路相关蛋白磷酸化水平均升高(P<0.05)。与黄芪甲苷组比较,黄芪甲苷+LY294002组Het-1A细胞的炎症反应、凋亡水平及促凋亡蛋白表达上升,而增殖能力及PI3K/AKT/mTOR通路活化水平下降(P<0.05),提示LY294002可抑制黄芪甲苷对放射性Het-1A细胞损伤的保护作用。结论黄芪甲苷可减轻6 MV-X射线放射性Het-1A细胞损伤,促进细胞增殖,抑制细胞炎症反应和细胞凋亡,其机制可能与激活PI3K/AKT/mTOR信号通路有关。
Objective To investigate the protective effect of astragaloside IV on radiation-induced esophageal epithelial cell(Het-1A)injury and explore its underlying mechanism.Methods A radiation-induced esophagitis model was established by irradiating Het-1A cells with 6MV-X-rays(2,4,6 Gy).The model cells were treated with different concentrations of astragaloside IV(0,5,10,20,40,80,160μmol/L).Cell proliferation was assessed using the CCK-8 method to determine the optimal intervention concentration.Het-1A cells were divided into the following groups:normal control group,radiation injury model group,astragaloside IV(40μmol/L)group,phosphatidylinositol 3-kinase(PI3K)inhibitor LY294002(10μmol/L)group,and astragaloside IV(40μmol/L)+LY294002(10μmol/L)group.The levels of tumour necrosis factor-α(TNF-α)and interleukin-6(IL-6)were measured by ELISA.The cell proliferation was evaluated using the EdU method.The apoptosis was detected by flow cytometry,phosphorylated PI3K(p-PI3K)protein expression was assessed by immunofluorescence,and the expression of caspase-3,Bcl-2,Bax,PI3K,protein kinase B(AKT),mammalian target of rapamycin(mTOR),and their cleaved or phosphorylated proteins were determined by Western blotting.Results A radiation-induced Het-1A injury cell model was constructed using a 4 Gy radiation dose.Compared with the 0μmol/L astragaloside IV group,the viability of radiation-injured Het-1A cells treated with 10,20,40,and 80μmol/L astragaloside IV was significantly increased(P<0.05).Compared with the normal control group,the radiation injury model group exhibited elevated levels of TNF-α,IL-6,apoptosis rate,cleaved caspase-3/caspase-3,and Bax protein,as well as reduced cell proliferation rate,Bcl-2,p-PI3K/PI3K,p-AKT/AKT,and p-mTOR/mTOR protein levels(P<0.05).Compared with the radiation injury model group,the astragaloside IV group showed significantly reduced levels of the aforementioned inflammatory factors,apoptosis rate,and related pro-apoptotic protein expression,while the proliferation ability and the phosphorylation levels of proteins related to the PI3K/AKT/mTOR pathway were increased(P<0.05).Compared with astragaloside IV group,astragaloside IV+LY294002 group showed significantly increased inflammatory response,apoptosis level,and pro-apoptotic protein expression,while the proliferation ability and activation level of the PI3K/AKT/mTOR pathway were decreased(P<0.05),suggesting that LY294002 could inhibit the protective effect of astragaloside IV on radiation-injured Het-1A cells.Conclusion Astragaloside IV can alleviate 6MV-X-ray-induced Het-1A cell injury,promote cell proliferation,and suppress inflammatory responses and apoptosis.Its mechanism may be related to the activation of the PI3K/AKT/mTOR pathway.
作者
张建华
李晓飞
李桂君
李明利
袁雷
ZHANG Jianhua;LI Xiaofei;LI Guijun;LI Mingli;YUAN Lei(Sixth Department of Oncology,Handan Hospital of Integrated Traditional Chinese and Western Medicine,Handan 056001,China;Third Department of Oncology,Handan Hospital of Integrated Traditional Chinese and Western Medicine,Handan 056001,China;Second Department of Oncology,Handan Hospital of Integrated Traditional Chinese and Western Medicine,Handan 056001,China;First Department of Rehabilitation,Handan Hospital of Integrated Traditional Chinese and Western Medicine,Handan 056001,China)
出处
《临床肿瘤学杂志》
2025年第11期1074-1079,共6页
Chinese Clinical Oncology
基金
2025年度中医药类科学研究课题项目(2025188)。
