Affiliations: [a] China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing, China
| [b] Medical and Health Analysis Center, Peking University, Beijing, China
| [c] Department of Radiation Oncology, The First Medical Center of People’s Liberation Army General Hospital, Beijing, China
| [d] School of Computer Science and Engineering, Southeast University, Nanjing, China
Correspondence:
[*]
Corresponding authors: Qiao Gou, National Institute for Radiological Protection, China Center for Disease
Control and Prevention, 2 Xinkang Street, Deshengmenwai, Xicheng District, Beijing 100088, China. Tel.: +86 010
62389626; E-mail: gouqiao76586@outlook.com and Zhong-Jian Ju, Department of Radiation Oncology, The
First Medical Center of People’s Liberation Army General Hospital, No. 28 of Fuxing Road, Haidian District,
Beijing, 100853, China. Tel.: +86 15801234725; E-mail: juzhongjian_jzj@126.com.
Abstract: OBJECTIVE:Our prior research has established that X-ray exposure induces pyroptosis in human umbilical vein endothelial cells (HUVECs), with Cx43 playing a regulatory role in this process. However, the precise mechanism by which Cx43 regulates pyroptosis remains unclear. The objective of this study is to assess the involvement of the calcium signaling pathway in Cx43-mediated regulation of X-ray-induced pyroptosis in HUVECs. METHODS:HUVECs were exposed to 10 Gy X-ray radiation either alone or combined with Cx43 overexpression or knockdown. Calcium ions (Ca2+) were stained using Fluo-4/AM and analyzed via flow cytometry and confocal microscopy. Pyroptosis was assessed through flow cytometry by staining with FLICA (fluorescent-labeled inhibitor of caspase) and propidium iodide (PI). Calcium signaling was inhibited using BAPTA/AM, 2-APB, or nifedipine. Protein expression levels were assessed by western blotting. RESULTS:X-ray irradiation induced an increase in intracellular calcium levels in HUVECs in a dose- and time-dependent manner. The results demonstrated that regulating calcium release with BAPTA/AM, 2-APB, or nifedipine significantly reduced pyroptosis. Also, the overexpression of Cx43 significantly attenuated the increase in intracellular calcium. Conversely, Cx43 knockdown via siRNA significantly increased the intracellular calcium levels. Also, interfering with calcium signaling using BAPTA/AM, 2-APB, or nifedipine reduced the raised pyroptosis levels induced by Cx43 knockdown. CONCLUSION:Individual HUVECs exposed to high-dose X-ray irradiation exhibited an increase in intracellular calcium, leading to pyroptosis. Also, upregulating Cx43 expression reduced the pyroptosis levels by inhibiting intracellular calcium concentration. This study introduces new concepts for identifying targets for the prophylaxis and therapy of radiation-induced damage.
