Isoflavone-mediated radioprotection involves regulation of early endothelial cell death and inflammatory signaling in Radiation-Induced lung injury

<b>Purpose:</b> Vascular damage and inflammation are limiting toxic effects of lung cancer radiotherapy, which lead to pneumonitis and pulmonary fibrosis. We have demonstrated that soy isoflavones (SIF) mitigate these toxic effects at late time points after radiation. However, the process by which SIF impacts the onset of radiation-induced inflammation remains to be elucidated. We have now investigated early events of radiation-induced inflammation and identified cellular and molecular signaling patterns by endothelial cells that could be modified by SIF to control vascular damage and the initiation of lung inflammation. <b>Materials and methods:</b> Histopathological, cellular and molecular studies were performed on mouse lungs from C57Bl/6 mice treated with 10 Gy of thoracic radiation (XRT) in conjunction with daily oral SIF treatment given prior and after radiation. Parallel studies were performed in-vitro using EA.hy926 endothelial cell line with SIF and radiation. Immunohistochemistry, western blots analysis, and flow cytometry were performed on lung tissue or EA.hy926 cells to analyze endothelial cells, their patterns of cell death or survival, and signaling molecules involved in inflammatory events. <b>Results:</b> Histopathological differences in inflammatory infiltrates and vascular injury in lungs, including vascular endothelial cells, were observed with SIF treatment at early time points post-XRT. XRT-induced expression of proinflammatory adhesion molecule ICAM-1 cells was reduced by SIF in-vitro and in-vivo in endothelial cells. Molecular changes in endothelial cells with SIF treatment in conjunction with XRT included increased DNA damage, reduced cell viability and cyclin B1, and inhibition of nuclear translocation of NF-κB. Analysis of cell death showed that SIF treatment promoted apoptotic endothelial cell death and decreased XRT-induced type III cell death. In-vitro molecular studies indicated that SIF + XRT increased apoptotic caspase-9 activation and production of IFNβ while reducing the release of inflammatory HMGB-1 and IL-1α, the cleavage of pyroptotic gasdermin D, and the release of active IL-1β, which are all events associated with type III cell death. <b>Conclusions:</b> SIF + XRT caused changes in patterns of endothelial cell death and survival, proinflammatory molecule release, and adhesion molecule expression at early time points post-XRT associated with early reduction of immune cell recruitment. These findings suggest that SIF could mediate its radioprotective effects in irradiated lungs by limiting excessive immune cell homing via vascular endothelium into damaged lung tissue and curtailing the overall inflammatory response to radiation.

<b>研究目的：</b> 血管损伤与炎症是肺癌放疗的限制性毒性反应，可引发放射性肺炎与肺纤维化。本课题组既往研究证实，大豆异黄酮（soy isoflavones, SIF）可在放疗后晚期阶段减轻此类毒性反应，但SIF影响辐射诱导炎症发生的具体过程尚未阐明。本研究聚焦辐射诱导炎症的早期发生过程，明确可被SIF调控的内皮细胞相关细胞与分子信号通路特征，以控制血管损伤及肺部炎症起始。
<b>材料与方法：</b> 对接受10 Gy胸部放射治疗（thoracic radiation, XRT）、且于放疗前后每日口服SIF干预的C57BL/6小鼠肺部开展组织病理学、细胞及分子水平研究。同时采用EA.hy926内皮细胞系（EA.hy926 endothelial cell line）联合SIF与放射处理开展体外平行实验。通过免疫组织化学（Immunohistochemistry）、蛋白质免疫印迹分析（western blots analysis）及流式细胞术（flow cytometry）检测小鼠肺组织或EA.hy926细胞，以分析内皮细胞的死亡/存活模式及参与炎症反应的信号分子。
<b>结果：</b> 放疗后早期时间点，经SIF干预的小鼠肺部（包括血管内皮细胞）的炎症浸润与血管损伤存在组织病理学差异。体外与体内实验均显示，SIF可降低内皮细胞中促炎黏附分子细胞间黏附分子1（ICAM-1）的辐射诱导表达。联合SIF与放射处理的内皮细胞的分子改变包括：DNA损伤加重、细胞活力与细胞周期蛋白B1（cyclin B1）水平降低，以及核因子κB（NF-κB）的核转位受到抑制。细胞死亡分析显示，SIF干预可促进内皮细胞凋亡，并减少辐射诱导的III型细胞死亡。体外分子实验表明，联合SIF与放射处理可激活凋亡通路的半胱氨酸天冬氨酸蛋白酶9（caspase-9）、促进干扰素β（IFNβ）生成，同时减少促炎因子高迁移率族蛋白B1（HMGB-1）与白细胞介素1α（IL-1α）的释放、抑制焦亡相关蛋白焦孔素D（gasdermin D）的切割，以及降低活性白细胞介素1β（IL-1β）的释放——上述事件均与III型细胞死亡相关。
<b>结论：</b> 放疗后早期时间点，SIF联合放射处理可改变内皮细胞的死亡与存活模式、调控促炎分子释放及黏附分子表达，进而早期减少免疫细胞招募。本研究结果提示，SIF可通过限制免疫细胞经血管内皮过度趋化至受损肺组织、抑制整体辐射诱导的炎症反应，从而在受照射肺部发挥辐射防护效应。