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低剂量放射治疗可通过激活NLRP3炎性小体促进放射性肺炎

2021-11-29 姜新 姜新

肺癌是世界范围内最大的健康挑战,在男性和女性中发病率和死亡率均居第二位。

原文出处
Li X, Gong Y, Li D, et al. Low-Dose Radiation Therapy Promotes Radiation Pneumonitis by Activating NLRP3 Inflammasome. Int J Radiat Oncol Biol Phys. 2020 ; 107(4): 804-814.


研究背景


肺癌是世界范围内最大的健康挑战,在男性和女性中发病率和死亡率均居第二位。放射治疗在肺癌的治疗中起着重要的作用。放射性肺炎(RP)是接受肺部放射治疗患者最常见的临床毒副作用之一。根据NCCN指南,常规放疗肺部的正常组织剂量-体积限制为:V20 ≤35%,V5 ≤65%,肺平均受照剂量(MLD)≤20 Gy。在临床实践中,放疗科医生在做治疗计划时要考虑这些因素。临床研究表明,低剂量辐射,如V5和V10,是影响RP发生率和严重程度的重要因素,对肺部接受5 Gy和10 Gy的区域进行剂量优化可减少RP的风险。在小鼠辐射损伤模型中,4 Gy和8 Gy照射均可增加弥漫性肺泡损伤,增大肺泡壁厚度。但其中的分子机制尚不清楚。


RP的特点之一是细胞因子的激活和释放。IL-1是最有效的早期细胞因子之一,可诱导其他细胞因子的产生。Caspase-1介导的IL-1蛋白表达受NLRP3的调控。NLRP3炎性小体在多种炎性疾病的发生和发展中起着重要作用。近年来,NLRP3炎性小体也被报道在哮喘、特发性肺病和辐射引起的肺部炎症中发挥重要作用。本研究的目的是研究NLRP3炎性小体在体内外低剂量照射后发挥的作用。此外,本研究旨在探索NLRP3激活以及NLRP3抑制剂在体内实验治疗效果的可能机制,并确定NLRP3炎性小体的抑制或缺失是否对RP的保护起至关重要的作用。


研究结果


结果一、NLRP3炎性小体在低剂量辐射条件下被激活
研究人员对培养的THP-1细胞(人单核细胞系)和分化的THP-1细胞进行不同剂量的辐照处理,以确定低剂量的辐射是否可以在体外激活NLRP3炎性小体。Western blot结果显示,2 Gy照射增加了NLRP3和裂解型Caspase-1在蛋白水平的表达(图1A)。研究者通过ELISA法检测THP-1细胞上清液,发现2 Gy照射后细胞分泌IL-1和IL-18水平升高。然而,这些细胞因子的表达水平与辐射剂量的增加无关(图1B、1C)。




结果二、THP-1细胞经辐射后产生的ROS可激活NLRP3炎症小体
ROS的产生在辐射引起的肺损伤中发挥重要作用,也是参与NLRP3炎症小体激活的途径之一。研究人员通过流式细胞术检测了2 Gy照射和LPS干预后细胞内ROS的产生情况。将未处理的细胞作为阴性对照,平均荧光团强度(MFI)分析显示,在照射后的1h、4h和6h时,照射组的ROS生成较对照组增加,在照射后1h,照射组和LPS干预组的ROS产生没有差异(图2A)。图2B显示,在照射后4h,照射组和LPS干预组的ROS生成均较对照组有所增加(图2B)。


随后,研究人员使用不同浓度的NAC(一种ROS的抑制剂),来研究低剂量辐照对细胞内NLRP3炎性小体激活的抑制作用。与对照组细胞上清相比,加入ROS抑制剂的THP-1细胞上清中的IL-18和IL-1蛋白含量明显降低。(图2C、2D)。综上所述,这些结果表明,低剂量辐射诱导的炎性小体激活需要ROS的参与。

 


结果三、低剂量和高剂量辐射可引起小鼠肺内NLRP3的激活
为了检测NLRP3炎症小体在体内的激活情况,野生型雄性C57BL/6小鼠接受2 -16 Gy不同剂量的胸部放射治疗。小鼠肺组织免疫荧光染色显示,NLRP3在2 Gy的剂量下被激活,且激活程度与辐射剂量无关(图3C、3D)。此外,NLRP3炎症小体的激活主要出现在气道,而非肺实质(图3A、3B)。
研究人员在不同的时间点收取了受照射的小鼠肺组织,以研究NLRP3在体内的激活是短暂的还是持续的。结果显示,NLRP3炎性小体在照射后1周表达明显,并在8周时仍保持激活状态(图3)。此外,NLRP3的表达水平不随时间发生变化(图3C、3D)。



结果四、NLRP3促进 LPS介导的小鼠肺组织单核细胞浸润
为了检测低剂量辐射对NLRP3基因敲除小鼠的影响,并评估NLRP3的激活是否会增强肺的超敏反应,NLRP3基因敲除小鼠和野生型小鼠分别接受2 Gy剂量的胸部辐射,然后经鼻给予LPS或PBS。未辐照的NLRP3基因敲除小鼠和野生型小鼠分别用LPS或PBS作为对照。图4A显示了各组肺组织H&E染色的代表性图像。炎症细胞聚集到肺部与辐射诱发的肺炎有关。在NLRP3基因敲除小鼠和野生型小鼠中,放疗和LPS均诱导免疫细胞浸润并增加肺泡壁厚度(图4A b-c f-g)。在NLRP3基因敲除小鼠和野生型小鼠中,照射后再给予LPS会加重炎症反应(图4A d、h)。


为了确定相关的免疫细胞亚型,我们测量了NLRP3基因敲除小鼠和野生型小鼠支气管肺泡灌洗液细胞的浸润情况。在照射组中,LPS在野生型或NLRP3基因敲除小鼠中均未增加中性粒细胞、淋巴细胞和白细胞浸润(图4B、4D和4E)。然而,照射后给予LPS处理,野生型小鼠单核细胞数量增加,而NLRP3基因敲除小鼠没有增加(图4C)。这些结果表明,在这一过程中,单核细胞是改变最多的细胞亚型,而在野生型小鼠中,NLRP3炎性小体的激活可能会引起单核细胞浸润。

图4 NLRP3促进 LPS介导的小鼠肺组织单核细胞浸润


结果五、NLRP3抑制剂MCC950可改善胸部辐射损伤小鼠模型的RP情况
MCC950是一种有效的、选择性的NLRP3小分子抑制剂,可阻断典型和非典型NLRP3的激活。MCC950特异性抑制NLRP3,减少IL-1的生成,在小鼠和人类细胞中均具有活性。研究者对2 Gy和16 Gy实验组以及未照射对照组小鼠的肺组织进行H&E染色(图5A-C),炎症评分结果显示,5mg /kg和10mg /kg MCC950处理降低了2 Gy和16 Gy照射组的炎症评分,尽管16 Gy组的降低没有统计学意义(图5D、5E)。


研究人员进一步检测了2 Gy和16 Gy照射的NLRP3基因敲除小鼠和野生型小鼠以及5mg/kg和10mg/kg MCC950处理组小鼠支气管肺泡灌洗液中细胞因子的水平。在2 Gy照射组中,MCC950治疗降低了IL-6的分泌,但没有降低TNF-α、IL-1β或IL-18的分泌。在16 Gy照射组中,MCC950处理降低了IL-6、IL-18和IL-1β的分泌,但TNF-α的分泌没有降低。这些结果表明,无论是低剂量照射还是高剂量照射,NLRP3抑制剂MCC950都能在一定程度上改善细胞因子的产生(图6)。


图6 NLRP3抑制剂MCC950都能在一定程度上改善细胞因子的产生


研究结论
1、NLRP3炎性小体在接受体内及体外低剂量照射后均可被激活,低剂量放射治疗可通过激活NLRP3炎性小体促进放射性肺炎;
2、NLRP3的抑制或缺失可特异性缓解辐射和LPS诱导的小鼠肺部炎症;
3、本研究揭示了低剂量放射治疗引起放射性肺炎的相关机制,并为放射性肺炎患者提供了一种潜在的治疗策略。


原文链接
https/77726476706e69737468656265737421e0e243912234265e7d0a80e296592e7bb7d62ae2c192eb/32334032/

 

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    2021-12-01 yuandd
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    2021-12-01 vera_1207

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