阿米福汀对小鼠放射性肺损伤的防护作用研究

Protective effect of Amifostine against radiation-induced lung injury in mice and its mechanism

摘要:

目的 探讨阿米福汀（Amifostine，AMI）对小鼠放射性肺损伤（RILI）的防护作用机制。方法 将24 只雌性C57BL/6J 小鼠随机分为对照组、单纯照射组、AMI 组。用直线加速器6MV-X 射线单次12Gy 照射小鼠全肺，照射前30 min 腹腔注射AMI，对照组和单纯照射组注射同等剂量的生理盐水。照射14 d 后，留取小鼠肺组织标本，采用苏木精- 伊红染色法（HE）观察病理改变；酶联免疫吸附法（ELISA）检测支气管肺泡灌洗液中白介素6（IL-6）、肿瘤坏死因子-α（TNF-α）、转化生长因子-β1（TGF-β1）表达水平；凝胶电泳迁移实验检测核转录因子-κB（NF-κB）活性；免疫组织化学法观察NLRP3 蛋白的表达和定位；实时荧光定量聚合酶链反应检测肺组织中NLRP 3 和IL-1β mRNA 的表达；Western blot 检测NF-κB p65、NLRP3、IL-1β 蛋白的表达。结果 照射后第14 天，AMI 组与单纯照射组比较，AMI 组肺组织急性炎症反应减轻；AMI 组支气管肺泡灌洗液中IL-6、TNF-α 水平下降，TGF-β1 水平升高（P <0.05）；AMI 组肺组织中NF-κB 活性下降（P <0.05）；AMI 组肺组织中NLRP3 蛋白表达下降（P <0.05）；AMI 组NLRP3 和IL-1β mRNA 表达下降（P <0.05）；AMI 组NF-κB p65、NLRP3、IL-1β 蛋白表达下降（P <0.05）。结论 AMI 可能通过抑制辐射引起的NF-κB 激活，进而抑制NLRP 3 基因的转录和表达，抑制炎症因子的释放，减轻RILI。

Abstract:

Objective To investigate the radioprotective function of Amifostine (AMI) in mice with acute radiation-induced lung injury and its mechanism. Methods Totally 24 female C57BL/6J mice were randomized into 3 groups as AMI group (treated by AMI 200 mg/kg plus radiation), radiation group and solvent control group.The mouse lungs in the radiation group and the AMI group were irradiated with linear accelerator 6MV X-ray at a single dose of 12 Gy, and those in the solvent control group received sham radiation. The mice in the AMI group were intraperitoneally injected with Amifostine 30 minutes before irradiation, while the same volume of solvent was given to the control and radiation groups. The mice were sacrified and the mouse lung tissue was collected 14 days after irradiation. The pathological changes in the lung tissue were observed after HE staining. The levels of IL-6, TNF-α and TGF-β1 in the bronchoalveolar lavage fluid (BALF) were measured by ELISA. The NF-κB activity was detected by EMSA. The expression and positioning of nucleotide-binding domain and leucine-rich repeat containing protein 3 (NLRP3) in the lung tissue were observed by immunohistochemical method. The expressions of NLRP3 and IL-1β mRNAs in the lung tissue were assayed by qRT-PCR. And the expressions of NF-κB p65, NLRP3 and IL-1β proteins in the lung tissue were assayed by Western blot. Results On the 14th day after irradiation, compared with the radiation group, acute inflammatory reaction of the lung tissue was alleviated, the IL-6 and TNF-α levels in BALF were markedly decreased (P < 0.05), the level of TGF-β1 in BALF rose slightly (P < 0.05), and NF-κB activity in the lung tissue was obviously reduced (P < 0.05) in the AMI group. Immunohistochemical results showed that the expressions of NLRP3 and IL-1β mRNAs were obviously reduced (P < 0.05), and the expressions of NF-κB p65, NLRP3 and IL-1β proteins in the lung tissue were obviously reduced (P < 0.05) in the AMI group compared to the radiation group. Conclusions Amifostine can down-regulate the activitity of NF–κB, then inhibit transcription and expression of NLRP3 gene and effectively reduce the release of inflammatory cytokines, therefore alleviate acute radiation-induced lung injury in mice.