Inhibition of PLA2G4E/cPLA2 promotes survival of random skin flaps by alleviating Lysosomal membrane permeabilization-Induced necroptosis.

ABSTRACT

Necrosis that appears at the ischemic distal end of random-pattern skin flaps increases the pain and economic burden of patients. Necroptosis is thought to contribute to flap necrosis. Lysosomal membrane permeabilization (LMP) plays an indispensable role in the regulation of necroptosis. Nonetheless, the mechanisms by which lysosomal membranes become leaky and the relationship between necroptosis and lysosomes are still unclear in ischemic flaps. Based on Western blotting, immunofluorescence, enzyme-linked immunosorbent assay, and liquid chromatography-mass spectrometry (LC-MS) analysis results, we found that LMP was presented in the ischemic distal portion of random-pattern skin flaps, which leads to disruption of lysosomal function and macroautophagic/autophagic flux, increased necroptosis, and aggravated necrosis of the ischemic flaps. Moreover, bioinformatics analysis of the LC-MS results enabled us to focus on the role of PLA2G4E/cPLA2 (phospholipase A2, group IVE) in LMP of the ischemic flaps. In vivo inhibition of PLA2G4E with an adeno-associated virus vector attenuated LMP and necroptosis, and promoted flap survival. In addition, microRNA-seq helped us determine that Mir504-5p was differentially expressed in ischemic flaps. A string of in vitro and in vivo tests was employed to verify the inhibitory effect of Mir504-5p on PLA2G4E, LMP and necroptosis. Finally, we concluded that the inhibition of PLA2G4E by Mir504-5p reduced LMP-induced necroptosis, thereby promoting the survival of random-pattern skin flaps.Abbreviations AAV adeno-associated virus; ACTA2/α;-SMA actin alpha 2, smooth muscle, aorta; ALOX15/12/15-LOX arachidonate 15- lipoxygenase; c-CASP8 cleaved caspase; c-CASP3 cleaved caspase 3; CTSD cathepsin D; CTSB cathepsin B; CTSL cathepsin L; DMECs primary mouse dermal microvascular endothelial cells; ELISA enzyme-linked immunosorbent assay; F-CHP 5-FAM-conjugated collagen hybridizing peptide; FISH fluorescence in situ hybridization; HUVECs human umbilical vein endothelial cells; LAMP1 lysosomal-associated membrane protein 1; LAMP2 lysosomal-associated membrane protein 2; LC-MS liquid chromatography-mass spectrometry; LDBF laser doppler blood flow; LMP lysosomal membrane permeabilization; LPE lysophosphatidylethanolamine; LPC lysophosphatidylcholine; MAP1LC3/LC3 microtubule-associated protein 1 light chain 3; MLKL mixed lineage kinase domain-like; NDI N-dodecylimidazole; PECAM1/CD31 platelet/endothelial cell adhesion molecule 1; PLA2G4A/cPLA2 phospholipase A2, group IVA (cytosolic, calcium-dependent); PLA2G4E/cPLA2 phospholipase A2, group IVE; qPCR quantitative real-time polymerase chain reaction; RIPK1 receptor (TNFRSF)-interacting serine-threonine kinase 1; RIPK3 receptor-interacting serine-threonine kinase 3; RISC RNA-induced silencing complex; ROS reactive oxygen species; shRNA short hairpin RNA; SQSTM1 sequestosome 1; TBHP tert-butyl hydroperoxide; TUNEL terminal deoxynucleotidyl transferase dUTP nick end labelling.