萝卜硫素对小鼠放射性肺损伤的防护作用机制

Mechanism of protective effect of suiforaphane against radiation-induced lung injury in mice

目的 探讨萝卜硫素（sulforaphane,SF）对小鼠急性放射性肺损伤的防护作用及其机制.方法 40只雌性C57BL/6J小鼠,按随机数字表法分为健康对照组、单纯照射组、照射＋SF3 mg/kg组、照射＋SF 5 mg/kg组、照射＋SF 10 mg/kg组,每组8只.以6MVX射线全肺单次12 Gy照射,各给药组自照射前7d开始至照射后7d,隔天腹腔注射不同浓度的SF,健康对照组和单纯照射组注射同等剂量的二甲基亚砜溶剂（DMSO＋生理盐水）.照后14 d,留取小鼠肺组织标本,HE染色观察病理改变,免疫组织化学法观察NLRP3的定位与表达,ELISA法检测支气管肺泡灌洗液中IL-6、TNF-α、TGF-β1表达水平,实时荧光定量反转录聚合酶链反应（qRT-PCR）检测肺组织中NLRP3、IL-1β mRNA的表达,Western blot检测NF-κB p65、NLRP3、IL-1β蛋白表达,凝胶迁移实验（EMSA）检测NF-κB活性.结果 HE染色结果显示,各照射＋SF组与单纯照射组比较,肺组织急性炎性反应减轻.肺组织中NLRP3表达下降,照射＋SF 10 mg/kg组NLRP3降低显著（F=42.750,P ＜0.05）.支气管肺泡灌洗液中IL-6、TNF-α、TGF-β1水平下降（tIL-6=-62.65 ～-21.00,tTNF-α=-32.18～-16.57,tTGF-β1=-58.22～-46.11,P＜0.05）.肺组织NLRP3和IL-1β mRNA表达显著下降（tNLRP3=-6.56 ～-5.68,tIL-1β=-29.75 ～-21.20,P＜0.05）.NF-κB p65、NLRP3、IL-1β蛋白表达下降（tNF-κB p65=-34.00 ～-1.71,tNLRP3=-25.01～-16.91,tIL-1β=-73.70 ～-55.14,P＜0.05）,其中NF-κB p65、NLRP3相对表达量和SF呈剂量依赖性降低（r=0.945、0.926）.EMSA结果显示,NF-κB活性下降,且和SF呈剂量依赖性降低（tNF-κB=-38.68、-614.82、-2 831.40,P＜0.05）.结论 萝卜硫素可通过降低小鼠肺组织NLRP3表达,有效地降低炎性因子的表达,减轻辐射诱导的炎症反应,对放射性肺损伤具有防护作用.

Objective To investigate the radioprotective function and its mechanism of Sulforaphane （SF） in mice acute radiation-induced lung injury.Methods Totally 40 female C57BL/6J mice were equally divided into 5 groups randomly.Group A,treated by SF 3 mg/kg plus radiation;group B,treated by SF 5 mg/kg plus radiation;group C,treated by SF 10 mg/kg plus radiation;radiation group with a single dose of 12 Gy in 6 MV X-ray by a linear accelerator,and control group with sham radiation.The mice in drug group were administered intraperitoneally with different concentration of SF every other day from 7 d before irradiation to 7 d after irradiation,while the same volume of DMSO plus physiological saline solvent was given in the control and radiation groups.After being sacrificed at 14 d of SF administration,the pathomorphological changes of mice were observed in trauma lung tissue,the positioning and expression of NLRP3 was observed by immunohistochemical staining,the levels of IL-6,TNF-α and TGF-β1 in bronchoalveolar lavage fluid （BALF） were measured by ELISA,the expressions of NLRP3 and IL-1β mRNA in lung tissue were assayed by qRT-PCR,the expressions of NF-κB p65,NLRP3 and IL-1β proteins in lung tissue were assayed by Western blot,the activity of NF-κB was detected by EMSA.Results In comparison with radiation group,there was an obvious amelioration in pathological injury of lung tissue in the treatment groups：the expression of NLRP3 in lung tissue decreased;the concentration of NLRP3 in the drug intervention group （SF 10 mg / kg） markedly decreased （F =42.750,P 〈 0.05）.the IL-6,TNF-a and TGF-β1 levels in BALF decreased （tIL-6 =-62.65-21.00;tTNF-α =-32.18-16.57;tTGF-β1 =-58.22-46.11,P 〈 0.05）;the expressions of NLRP3 and IL-1β mRNA markedly decreased （tNLRP3 =-6.56-5.68;tIL-1β =-29.75--21.20,P 〈 0.05）,and the expressions of NF-κB p65,NLRP3 and IL-1β proteins decreased （tNF-κB p65 =-34.00--1.71,tNLRP3 =-25.01--16.91,tIL-1β =-73.70--55.14,P 〈 0.05）;the relative expressions of NF-κB p65 and NLRP3 were reduced in a dose-dependent manner （r =0.945,0.926）;and the activity of NF-κB were obviously reduced （tNF-κB =-38.68,-614.82,-2 831.40,P 〈 0.05）.Conclusions Sulforaphane effectively alleviates the RILI in lung of mice by downregulating the expressions of inflammatory factor NLRP3.