Rip 1-dependent endothelial necroptosis participates in ischemia-reperfusion injury of mouse flap.

BACKGROUND

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.

OBJECTIVE

To investigate the regulation and functional link between necroptosis and ischemia-reperfusion injury of mouse flap.

METHODS

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.

RESULTS

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.

CONCLUSION

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.