Effect Of Platelets On Vascular Endothelial Cells Injured By Oxidative Stress And Wound Healing

Background and Objective:Skin wound healing is a complex and orderly process,with a variety of cells and active factors intertwined.Vascular endothelial cells are the main bearers of vascularization,forming immature new blood vessels on the basis of the original blood vessels in the form of "budding".Reactive oxygen species(ROS)have dual effects on vascularization,which can not only induce and maintain new angiogenesis at low levels,but also inhibit vascular endothelial cell proliferation,migration and neovascularization at high levels,thus hindering wound healing.Recently,platelet concentrates have been shown to regulate the balance of oxidative stress and reduce apoptosis induced by oxidative stress.Considering the potential of mitochondrial transfer in saving injured cells,mitochondria released by platelets may play an important role in regulating oxidative stress in target cells.However,at present,there are no related studies to show this role and mechanism.Therefore,the purpose of this study is to explore the regulatory effect of platelet concentrates on oxidative stress injury of vascular endothelial cells,and the mechanism of platelet-derived mitochondria regulating oxidative stress balance of vascular endothelial cells through intercellular transfer.Methods:1.Platelet concentrates were prepared and activated by thrombin,repeated freezing and thawing,ultrasound and light,respectively.The contents of growth factors EGF,PDGF-BB,FGF-b and TGF-β1 in the supernatant were detected by ELISA.Extract mitochondria and detect their content and oxygen consumption.The effects of several activated platelet concentrates on the proliferation and migration of HUVECs were compared.2.The injury model of HUVECs induced by hydrogen peroxide was established,and the injured HUVECs was treated with sonicate platelet concentrates(SPC).The level of ROS,mitochondrial membrane potential and apoptosis were measured.Transmission electron microscope,flow cytometry and fluorescence microscope were used to observe the transfer of mitochondria from SPC to HUVECs.After inhibiting the respiratory function of mitochondria in SPC,the protective effect of SPC on injured HUVECs was detected.The influence genes of mitochondria derived from SPC on HUVECs were screened by high-throughput sequencing and verified by Western Blot.After blocking the target gene,the protective effect of SPC on injured HUVECs was observed.3.The mouse model of full-thickness skin defect was established and treated with SPC or SPC with inhibition of mitochondrial.The effect of SPC-derived mitochondria on the rate and quality of wound healing was analyzed by wound size analysis,H&E,Masson collagen staining and CD31 immunohistochemistry.Results:1.Among several activated platelet concentrates,SPC released the most EGF,PDGF-BB,FGF-b and respiratory mitochondria,and thrombin platelet concentrates released the most TGF-β1.The effects of several activated platelet concentrates on HUVECs proliferation were similar,but the effect of SPC on HUVECs migration was more obvious.2.SPC can reduce the ROS level of HUVECs injured by hydrogen peroxide,increase the mitochondrial membrane potential and reduce apoptosis.SPC-derived mitochondria can release two kinds of mitochondria,naked or encapsulated by vesicles.These mitochondria entered HUVECs by dynamin-dependent endocytosis,decreased the ROS level of HUVECs damaged by hydrogen peroxide,increased mitochondrial membrane potential and reduced apoptosis.Platelet-derived mitochondria could play a protective role by up-regulating the expression of survivin.3.SPC-derived mitochondria promoted wound healing,reduced early inflammatory infiltration,increased epithelialization and collagen deposition,and improved the quality of wound healing.Conclusion:In this study,we found that platelet concentrates could reduce the injury of vascular endothelial cells induced by hydrogen peroxide,decrease the level of ROS,increase the mitochondrial membrane potential and reduce apoptosis.Its effect was relied on the mitochondria released by platelets,which transferred to vascular endothelial cells,up-regulated the expression of survivin and thus accelerated wound healing.This study preliminarily expounds the regulatory effect of platelet-derived mitochondria on oxidative stress and its effect on wound healing,expands the understanding of platelets,and provides a new direction for wound healing treatment.