桃叶珊瑚苷通过调节PD-1/PD-L1信号通路介导的免疫逃逸对肺癌细胞放疗敏感性的影响

Effect of Aucubin on the radiosensitivity of lung cancer cells by regulating the immune escape mediated by PD-1/PD-L1 signaling pathway

目的探究桃叶珊瑚苷(Aucubin)通过调节程序性死亡受体-1(PD-1)/程序性死亡受体配体-1(PD-L1)信号通路介导的免疫逃逸对肺癌细胞放疗敏感性的影响及其作用机制。方法0~300μmol/mL的Aucubin处理人肺癌细胞NCI-H1299,MTT检测细胞活力。将细胞分为正常对照组(Control组)、放疗组(6 Gy组)、Aucubin组、Aucubin+放疗组(Aucubin+6 Gy组),克隆形成实验和Edu实验检测细胞增殖水平,流式细胞仪检测细胞凋亡,酶联免疫吸附试验(ELISA)检测细胞培养液上清中肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白细胞介素(IL)-2水平,蛋白质印迹法(Western blot)检测PD-1、PD-L1蛋白表达。建立肺癌小鼠移植瘤模型,分为对照组(Control组)、放疗组(5 Gy组)、Aucubin组和Aucubin+5 Gy组。测量移植瘤体积与质量,ELISA检测TNF-α、IFN-γ、IL-2水平,Western blot检测PD-1、PD-L1蛋白表达。结果与0μmol/mL比较,25~125μmol/mL的Aucubin能降低细胞活力,选择100μmol/mL的Aucubin进行后续实验。与Control组比较,6 Gy组和Aucubin组细胞克隆形成数、Edu阳性率、TNF-α水平及PD-1、PD-L1蛋白表达水平降低(P<0.05),细胞凋亡率及IFN-γ、IL-2水平升高(P<0.05);Aucubin+6 Gy组细胞克隆形成数、Edu阳性率、TNF-α水平及PD-1、PD-L1蛋白表达水平低于6 Gy组和Aucubin组(P<0.05),细胞凋亡率及IFN-γ、IL-2水平高于6 Gy组和Aucubin组(P<0.05)。小鼠移植瘤实验表明,与Control组比较,5 Gy组和Aucubin组小鼠移植瘤体积和质量、TNF-α水平及PD-1、PD-L1蛋白表达水平降低(P<0.05),IFN-γ、IL-2水平升高(P<0.05);Aucubin+5 Gy组小鼠移植瘤体积和质量、TNF-α水平及PD-1、PD-L1蛋白表达水平低于5 Gy组和Aucubin组(P<0.05),IFN-γ、IL-2水平高于5 Gy组和Aucubin组(P<0.05)。结论Aucubin可能通过阻断PD-1/PD-L1信号通路介导的免疫逃逸增强肺癌细胞放疗敏感性。

Objective To explore the Aucubin by regulating programmed death-1(PD-1)/programmed death ligand-1(PD-L1)signaling pathway mediated immune escape the influence of radiation sensitivity on the lung cancer cells and its mechanism of action.Methods Human lung cancer cell line NCI-H1299 was treated with 0-300μmol/mL Aucubin,and cell viability was detected by MTT assay.Cells were divided into normal control group(Control group),radiotherapy group(6 Gy group),Aucubin group,Aucubin+radiotherapy group(Aucubin+6 Gy group).Cell proliferation were detected by colony formation assay and Edu assay.Cell apoptosis was detected by flow cytometry.The levels of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ)and interleukin(IL)-2 in the supernatant of cell culture medium were detected by enzyme-linked immunosorbent assay(ELISA).The protein expressions of PD-1 and PD-L1 were detected by Western blot.A mouse model of lung cancer xenograft was established and divided into Control group,radiotherapy group(5 Gy group),Aucubin group,and Aucubin+5 Gy group.The volume and mass of transplanted tumor were measured.ELISA was used to detect the levels of TNF-α,IFN-γ,and IL-2,and Western blot was used to detect the protein expressions of PD-1 and PD-L1.Results Compared with 0μmol/mL Aucubin,25-125μmol/mL Aucubin could reduce cell viability,and 100μmol/mL aucubin was selected for subsequent experiments.Compared with the Control group,cell clone formation number,Edu positive rate,TNF-αlevel,PD-1 and PD-L1 protein expression levels were significantly decreased(P<0.05),and the apoptosis rate,IFN-γand IL-2 levels were significantly increased(P<0.05)in the 6 Gy group and the Aucubin group.Compared with the 6 Gy group and Aucubin group,the Aucubin+6 Gy group had significantly lower cell colony formation number,Edu positive rate,TNF-αlevel,PD-1 and PD-L1 protein expression levels(P<0.05),and significantly higher apoptosis rate,IFN-γand IL-2 levels(P<0.05).Compared with the Control group,the trasplanted tumor volume and weight,TNF-αlevel and PD-1 and PD-L1 protein expression levels in the 5 Gy group and Aucubin group were decreased(P<0.05),while IFN-γand IL-2 levels were increased(P<0.05).Compared with the 5 Gy group and Aucubin group,the Aucubin+5 Gy group had significantly lower transplanted tumor volume and mass,TNF-αlevel and PD-1 and PD-L1 expression levels(P<0.05),and significantly higher IFN-γand IL-2 levels(P<0.05).Conclusion Aucubin may enhance the radiosensitivity of lung cancer cells by blocking the immune escape mediated by PD-1/PD-L1 signaling pathway.