Department of Plastic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Department of Plastic Surgery, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
J Dermatol Sci. 2020 Jan;97(1):30-40. doi: 10.1016/j.jdermsci.2019.11.009. Epub 2019 Nov 28.
Ischemia reperfusion injury plays an important role in free flap necrosis. However, the detailed mechanism is not clear, and effective methods for improving the survival rate of skin flap are still lacking.
To investigate the regulation and functional link between necroptosis and ischemia-reperfusion injury of mouse flap.
We established a mouse ischemia-reperfusion injury flap model and a cell Oxygen Glucose Deprivation (OGD) model intervened with Necrostatin-1. The mouse flap tissues were harvested in vivo for histological immunofluorescence analysis and western blotting analyses. The HUVECs cells with various treatments in vitro were assessed by using Transwell assay, tube formation assay, cell counting kit-8 analysis and flow cytometry. A Rip3-knockout cell line and a TNFR1-knockout cell line were generated from HUVEC cells using the CRISPR-Cas9 technology and were subsequently used to explore the related mechanisms.
The expression of p-Rip3 is positive in both mouse and cell culture models. When necroptosis is completely or partially inhibited in vivo, damaged tissues are repaired with better efficiency. The cells treated with Necrostatin-1 in vitro exhibit faster migration, proliferation and better tube formation. Deficiency of TNFR1 can block the necroptosis pathway by blocking the phosphorylation of Rip3 in HUVEC OGD/ROG model. Meanwhile, the levels of APJ, HIF-1α, and VEGF are reduced when necroptosis is inhibited by Necrostatin-1.
TNFR1 mediates Rip1/Rip3 in ischemia-reperfusion injury. Inhibition of necroptosis attenuates the ischemia-reperfusion injury of flap and may enhance hypoxic tolerance of HUVECs and vascular homeostasis through regulation of the HIF-1α signaling pathways.
缺血再灌注损伤在游离皮瓣坏死中起重要作用。然而，其详细机制尚不清楚，提高皮瓣存活率的有效方法仍然缺乏。
研究小鼠皮瓣坏死与缺血再灌注损伤之间的调控和功能联系。
我们建立了小鼠缺血再灌注损伤皮瓣模型和细胞氧葡萄糖剥夺（OGD）模型，并使用 Necrostatin-1 进行干预。在体内采集小鼠皮瓣组织进行组织学免疫荧光分析和 Western blot 分析。在体外使用 Transwell 测定、管形成测定、细胞计数试剂盒-8 分析和流式细胞术评估具有各种处理的 HUVECs 细胞。使用 CRISPR-Cas9 技术从 HUVEC 细胞中生成 Rip3 敲除细胞系和 TNFR1 敲除细胞系，并随后用于探索相关机制。
在小鼠和细胞培养模型中，p-Rip3 的表达均为阳性。当体内完全或部分抑制坏死时，受损组织的修复效率更高。体外用 Necrostatin-1 处理的细胞迁移、增殖更快，管形成更好。在 HUVEC OGD/ROG 模型中，TNFR1 缺乏可通过阻断 Rip3 的磷酸化来阻断坏死通路。同时，当用 Necrostatin-1 抑制坏死时，APJ、HIF-1α 和 VEGF 的水平降低。
TNFR1 介导缺血再灌注损伤中的 Rip1/Rip3。抑制坏死可减轻皮瓣的缺血再灌注损伤，并可能通过调节 HIF-1α 信号通路增强 HUVEC 的缺氧耐受和血管稳态。
