缝隙连接蛋白 43 调节人脐静脉内皮细胞 X 射线诱导的细胞焦亡。

Connexin43 Modulates X-Ray-Induced Pyroptosis in Human Umbilical Vein Endothelial Cells.

机构信息

China CDC Key Laboratory of Radiological Protection and Nuclear Emergency, National Institute for Radiological Protection, Chinese Center for Disease Control and Prevention, Beijing 100088, China.

Medical and Health Analysis Center, Peking University, Beijing 100191, China.

出版信息

Biomed Environ Sci. 2019 Mar;32(3):177-188. doi: 10.3967/bes2019.025.

DOI:10.3967/bes2019.025

PMID:30987692

Abstract

OBJECTIVE

Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs).

METHODS

HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively.

RESULTS

Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown.

CONCLUSION

Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1.

摘要

目的

细胞焦亡是一种炎症形式的程序性细胞死亡。最近有报道称，这种现象在放射性诱导的正常组织损伤中起着重要作用。连接蛋白 43（Cx43）是一种调节细胞生长和凋亡的缝隙连接蛋白。在本研究中，我们研究了 Cx43 对人脐静脉内皮细胞（HUVECs）中 X 射线诱导的细胞焦亡的影响。

方法

用 10Gy 照射 HUVECs、Cx43 过表达和 Cx43 敲低株。采用 Western blot 分析检测蛋白。通过流式细胞术和共聚焦显微镜用荧光标记的半胱天冬酶抑制剂（FLICA）和碘化丙啶染色评估细胞焦亡。通过扫描电子显微镜和乳酸脱氢酶释放试验分别检测细胞形态和细胞毒性。

结果

10Gy X 射线照射诱导 HUVECs 发生细胞焦亡并降低 Cx43 表达。Cx43 过表达显著减弱 HUVECs 的细胞焦亡，降低活性半胱天冬酶-1 水平。然而，用 siRNA 干扰 Cx43 表达则显著通过增加活性半胱天冬酶-1 水平促进细胞焦亡。缝隙连接蛋白 Pannexin1（Panx1）调节细胞焦亡，其裂解形式用于评估通道开放和活性状态。在 X 射线存在的情况下，HUVECs 和 Cx43 敲低株中的裂解 Panx1 水平增加，但在 Cx43 过表达株中降低。此外，用 siRNA 干扰 Panx1 减轻了 Cx43 敲低引起的细胞焦亡的上调。