■我们先前的研究已经确定,X射线暴露会诱导人脐静脉内皮细胞(HUVEC)的焦亡,Cx43在这个过程中发挥了调节作用。然而,Cx43调节焦亡的确切机制尚不清楚.这项研究的目的是评估钙信号通路在Cx43介导的X射线诱导的HUVECs焦凋亡调节中的参与。
■HUVEC单独或与Cx43过表达或敲低组合暴露于10GyX射线辐射。使用Fluo-4/AM染色钙离子(Ca2+),并通过流式细胞术和共聚焦显微镜分析。通过用FLICA(荧光标记的半胱天冬酶抑制剂)和碘化丙啶(PI)染色,通过流式细胞术评估焦亡。使用BAPTA/AM抑制钙信号,2-APB,或者硝苯地平.通过蛋白质印迹法评估蛋白质表达水平。
■X射线照射以剂量和时间依赖性方式诱导HUVECs细胞内钙水平增加。结果表明,用BAPTA/AM调节钙的释放,2-APB,或硝苯地平可显着降低焦亡。此外,Cx43的过表达显著减弱了细胞内钙的增加。相反,通过siRNA的Cx43敲低显著增加了细胞内钙水平。此外,使用BAPTA/AM干扰钙信号,2-APB,或硝苯地平可降低Cx43敲低诱导的焦亡水平。
■暴露于高剂量X射线照射的单个HUVEC表现出细胞内钙的增加,导致焦亡。此外,上调Cx43表达通过抑制细胞内钙浓度降低了焦亡水平。这项研究引入了新的概念,用于识别辐射引起的损伤的预防和治疗目标。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.