Connexin43 Modulates X-Ray-Induced Pyroptosis in Human Umbilical Vein Endothelial Cells
Connexin43 Modulates X-Ray-Induced Pyroptosis in Human Umbilical Vein Endothelial Cells
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DOI：
10.3967/bes2019.025
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发表时间：
2019-03-01
影响因子：
3.5
通讯作者：
Su Xu
中科院分区：
文献类型：
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作者：
Li Chen;Tian Mei;Su Xu
目的细胞凋亡是细胞程序性死亡的一种炎症形式。这种现象最近被报道在辐射诱导的正常组织损伤中起重要作用。缝隙连接蛋白43（Cx43）是一种调节细胞生长和凋亡的缝隙连接蛋白。本研究探讨了Cx43对X射线诱导的人脐静脉内皮细胞（HUVECs）细胞凋亡的影响。方法用10戈伊照射HUVECs、Cx43过表达株和Cx43敲除株。使用蛋白质印迹分析检测蛋白质。采用荧光标记半胱天冬酶抑制剂法（FLICA）和碘化丙啶染色法，通过流式细胞术和共聚焦显微镜观察细胞凋亡。结果10戈伊X射线照射可诱导HUVECs细胞凋亡，Cx43表达减少。过度表达Cx43可显著减弱HUVECs中的细胞凋亡，因为Cx43降低了活性caspase-1的水平。然而，干扰Cx43表达的siRNA显着促进通过增加活性caspase-1水平的细胞凋亡。Pannexin 1（Panx 1）是一种调节细胞凋亡的间隙连接蛋白，其裂解形式可用于评估通道开放和活性状态。在X射线的存在下，HUVECs和Cx43敲低菌株中切割的Panx 1水平增加，但在Cx43过表达菌株中降低。结论单次大剂量X射线照射可诱导HUVECs发生细胞凋亡。此外，Cx43通过激活caspase-1直接或通过裂解Panx 1间接调节细胞凋亡。
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.