Department of Burn, The First Affiliated Hospital of Anhui Medical University Department of Burn, The First Affiliated Hospital of Anhui Medical University.
2018 Class of Clinical Medicine (No. 1813010207), The First Clinical College of Anhui Medical University, Hefei, China.
J Burn Care Res. 2021 Mar 4;42(2):258-268. doi: 10.1093/jbcr/iraa139.
The mechanism underlying burn injury-induced enhanced vascular endothelial permeability and consequent body fluid extravasation is unclear. Here, the rat aortic endothelial cells (RAECs) were treated with the serum derived from rats with burn injury to elucidate the mechanism. Sprague-Dawley (SD) rats were grouped as follows (10 rats/group): control, 2, 4, 8, 12, and 24 hours postburn groups. The heart, liver, kidney, lung, jejunum, and ileum of rats injected with 2% Evans blue (EB) through the tail vein were excised to detect the EB level in each organ. The serum levels of hypoxia-inducible factor-1α (HIF-1α) and endothelin-1 (ET-1) were examined using enzyme-linked immunosorbent assay (ELISA). The effect of serum from 12-hour postburn group on the membrane permeability of RAEC monolayer, as well as on the mRNA and protein levels of ET-1, endothelin receptor A (ETA), ETB, and zonula occludens (ZO-1), was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The membrane permeability of GV230/HIF-1α-transfected or shRNA-HIF-1α-transfected RAECs, as well as the expression levels of HIF-1α, ET-1, ETA, ETB, vascular endothelial (VE)-cadherin, and claudin-5, was analyzed using qRT-PCR and western blotting, whereas the localization of VE-cadherin and claudin-5 was examined using immunofluorescence. The serum HIF-1α and ET-1 levels in the burn groups, which peaked at 12 hours postburn, were significantly upregulated (P < .01) when compared with those in the control group. Additionally, the serum HIF-1α levels were positively correlated with vascular permeability. Compared with the shRNA-negative control-transfected RAECs, the shRNA-II/HIF-1α-transfected RAECs exhibited downregulated expression of HIF-1α, ET-1, ETA, and ETB (P < .01), and upregulated expression of ZO-1, claudin-5, and VE-cadherin (P < .05). Compared with the GV230-transfected RAECs, the GV230/HIF-1α-transfected RAECs exhibited upregulated expression of HIF-1α, ET-1, ETA, and ETB (P < .01), and downregulated expression of ZO-1, claudin-5, and VE-cadherin (P < .05). The GV230/HIF-1α-transfected RAECs exhibited degradation and translocation of VE-cadherin and claudin-5. In addition to degradation of VE-cadherin and claudin-5, HIF-1α mediated enhanced endothelial cell permeability through upregulation of ET-1, ETA, and ETB, and downregulation of ZO-1 and VE-cadherin in rats with burn injury.
烧伤诱导的血管内皮通透性增强和随后的体液渗出的机制尚不清楚。在这里，用来自烧伤大鼠的血清处理大鼠主动脉内皮细胞（RAEC），以阐明其机制。将 Sprague-Dawley（SD）大鼠分为以下几组（每组 10 只）：对照组、烧伤后 2、4、8、12 和 24 小时组。通过尾静脉向注射 2% Evans 蓝（EB）的大鼠心脏、肝脏、肾脏、肺、空肠和回肠中注入 EB，以检测各器官中的 EB 水平。使用酶联免疫吸附测定（ELISA）检测低氧诱导因子-1α（HIF-1α）和内皮素-1（ET-1）的血清水平。通过定量实时聚合酶链反应（qRT-PCR）和 Western blot 分析来自烧伤后 12 小时组的血清对 RAEC 单层的膜通透性以及 ET-1、内皮素受体 A（ETA）、ETB 和紧密连接蛋白（ZO-1）的 mRNA 和蛋白水平的影响。通过 qRT-PCR 和 Western blot 分析 GV230/HIF-1α 转染或 shRNA-HIF-1α 转染的 RAEC 以及 HIF-1α、ET-1、ETA、ETB、血管内皮（VE）-钙粘蛋白和 Claudin-5 的表达水平，使用免疫荧光检查 VE-钙粘蛋白和 Claudin-5 的定位。烧伤组的血清 HIF-1α 和 ET-1 水平在烧伤后 12 小时达到峰值，与对照组相比显著升高（P<0.01）。此外，血清 HIF-1α 水平与血管通透性呈正相关。与 shRNA 阴性对照转染的 RAEC 相比，shRNA-II/HIF-1α 转染的 RAEC 下调了 HIF-1α、ET-1、ETA 和 ETB 的表达（P<0.01），上调了 ZO-1、Claudin-5 和 VE-钙粘蛋白的表达（P<0.05）。与 GV230 转染的 RAEC 相比，GV230/HIF-1α 转染的 RAEC 上调了 HIF-1α、ET-1、ETA 和 ETB 的表达（P<0.01），下调了 ZO-1、Claudin-5 和 VE-钙粘蛋白的表达（P<0.05）。GV230/HIF-1α 转染的 RAEC 表现出 VE-钙粘蛋白和 Claudin-5 的降解和易位。除了 VE-钙粘蛋白和 Claudin-5 的降解外，HIF-1α 通过上调 ET-1、ETA 和 ETB 以及下调 ZO-1 和 VE-钙粘蛋白来介导烧伤大鼠内皮细胞通透性的增强。
