Chen Pengxiang, Liu Hui, Xin Huixian, Cheng Bo, Sun Changhua, Liu Yuchen, Liu Tianyu, Wen Zhihua, Cheng Yufeng
Department of Radiation Oncology; Laboratory of Basic Medical Sciences.
Department of Clinical Laboratory, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan, People's Republic of China.
Int J Radiat Oncol Biol Phys. 2023 Feb 1;115(2):476-489. doi: 10.1016/j.ijrobp.2022.03.008. Epub 2022 Apr 19.
Radiation-induced lung fibrosis (RILF) is a serious late complication of thoracic radiation therapy. Inflammation is crucial in fibroblast activation and RILF, and arachidonic acid (AA) is an important inflammatory mediator released by cytosolic phospholipase A2 (cPLA2) and reduced by arachidonyl trifluoromethyl ketone (ATK)-targeting of cPLA2. Here, we aimed to investigate the roles of the cPLA2/AA pathway in RILF and assess the potential of targeting cPLA2 to prevent RILF.
A computed tomography scan was used to obtain the mean lung density, and hematoxylin-eosin, Masson's trichrome, and Sirius Red staining were used to assess the histopathologic conditions in mouse models. AA levels in mouse serum and cell supernatants were tested by enzyme-linked immunosorbent assay. Fibroblast phenotype alterations were examined by a Cell Counting Kit-8, manual cell counting, and a Transwell system. The protein levels were evaluated via Western blotting, immunofluorescence, and immunohistochemistry.
AA protected fibroblasts against radiation-induced growth inhibition and promoted fibroblast activation, which was characterized by enhanced α-smooth muscle actin expression and migration capacity. Radiation could activate fibroblasts by upregulating cPLA2 expression and AA production, which could be reversed by ATK. Moreover, inhibiting cPLA2 with ATK significantly attenuated collagen deposition and radiation-induced pulmonary fibrosis in mouse models. We further identified extracellular-signal regulated protein kinase (ERK) as the downstream target of the radiation-AA regulatory axis. Radiation-induced AA increased phosphorylated-ERK levels, promoting cyclinD1, cyclin-dependent kinase 6, and α-smooth muscle actin expression and contributing to fibroblast activation. Inhibiting P-ERK impaired radiation- and AA-induced fibroblast activation. The related molecular mechanisms were verified using specimens from animal models.
Our findings uncover the role of the cPLA2/AA-ERK regulatory axis in response to radiation in pulmonary fibroblast activation and recognize cPLA2 as the key regulatory molecule during RILF for the first time. Targeting cPLA2 may be a promising protective strategy against RILF.
放射性肺纤维化（RILF）是胸部放射治疗严重的晚期并发症。炎症在成纤维细胞活化和RILF中起关键作用，花生四烯酸（AA）是由胞质磷脂酶A2（cPLA2）释放的重要炎症介质，通过靶向cPLA2的花生四烯酰三氟甲基酮（ATK）可使其减少。在此，我们旨在研究cPLA2/AA途径在RILF中的作用，并评估靶向cPLA2预防RILF的潜力。
使用计算机断层扫描获取平均肺密度，并用苏木精-伊红染色、Masson三色染色和天狼星红染色评估小鼠模型的组织病理学状况。通过酶联免疫吸附测定法检测小鼠血清和细胞上清液中的AA水平。通过细胞计数试剂盒-8、手动细胞计数和Transwell系统检测成纤维细胞表型改变。通过蛋白质印迹法、免疫荧光法和免疫组织化学法评估蛋白质水平。
AA可保护成纤维细胞免受辐射诱导的生长抑制，并促进成纤维细胞活化，其特征为α-平滑肌肌动蛋白表达增强和迁移能力增强。辐射可通过上调cPLA2表达和AA产生来激活成纤维细胞，而ATK可使其逆转。此外，在小鼠模型中，用ATK抑制cPLA2可显著减轻胶原沉积和辐射诱导的肺纤维化。我们进一步确定细胞外信号调节蛋白激酶（ERK）为辐射-AA调节轴的下游靶点。辐射诱导的AA增加磷酸化-ERK水平，促进细胞周期蛋白D1、细胞周期蛋白依赖性激酶6和α-平滑肌肌动蛋白表达，促进成纤维细胞活化。抑制磷酸化-ERK可损害辐射和AA诱导的成纤维细胞活化。使用动物模型标本验证了相关分子机制。
我们的研究结果揭示了cPLA2/AA-ERK调节轴在肺成纤维细胞对辐射反应中的作用，并首次将cPLA2识别为RILF期间的关键调节分子。靶向cPLA2可能是一种有前景的预防RILF的保护策略。
