Study On The Treatment And Mechanism Of Ferrostatin-1 Accelerating Ulcer Healing In High-glucose By Regulating Ferroptosis Pathway

Objectives: Ferroptosis is a newly discovered cell death pattern with a unique signaling pathway triggered by iron overloaded lipid ROS accumulation.GPX4 and ferrostatin-1 as potent scavengers for lipid ROS have been reported to effectively reverse ferroptosis.Diabetic ulcer wound healing has been a worldwide difficult issue and its underline mechanism of pathology was still unknown.Previous studies demonstrated that glutathione,the precursor of GPX4,could also play an important role in alleviating lipid ROS accumulation during the wound healing process of diabetic ulcer.We therefore deduced that ferroptosis might exist in the progress of diabetic ulcers.This study aimed at illustrating the relationship between ferroptosis and the pathology of diabetic ulcers and exploring whether ferrostatin-1 could accelerate diabetic wound healing as well as its possible underline mechanism.Methods: The mechanism of ferrostatin-1 repairing diabetic ulcers was explored by high-glucose induced cell damage models and animal models with diabetic ulcers.(1)EA.hy926 cells and human fibroblasts(HSF)were used as cell damage models.In vitro therapeutic effect of ferrostin-1 was tested by the detection of GPX4,TFRC and SLC7A11 levels through quantitative real time PCR(q PCR)and western blot assays.Cell viability was assessed by cell counting kit-8(CCK-8)experiments and lipid ROS was examined with MDA detection kits.Total ROS and mitochondrial ROS of cells were examined by DCFH-DA and mitosox-red tracers.Healing effectiveness was evaluated through cell wound scratch assays in HSF cells.Angiogenesis was assayed by protein expressions of e NOS and VEGF in vitro.(2)In vivo studies detected the levels of GPX4,TFRC,SLC7A11,e NOS,VEGF through q PCR and western blot assays.Electron microscopy(SEM)test was also employed to verify the existence of ferroptosis in the wound ulcer of diabetic rats.Lipid ROS was determined by MDA detection kits.Anti-inflammation function was determined by IL-1β,IL-6 and TNF-α by ELISA detection kits.H&E and histological examinations were used to evaluate the therapeutic effect of ferrostatin-1 in vivo.(3)Expressions of mRNA and protein of PI3 K,phosphorylated-PI3K(p-PI3K),AKT,phosphorylated-AKT(p-AKT)were tested by western blot to testify whether ferrostatin-1 accelerates wound healing by activating PI3K/AKT pathway in vivo.Results: In vitro study indicated that compared with high glucose and RSL-3intervention groups,m RNA and protein levels of GPX4,SLC7A11 significantly increased while TFRC significantly reduced in high glucose induced cell damage models.The contents of lipid per-oxidation products malondialdehyde(MDA),total ROS and mitochondrial ROS were significantly reduced in the ferrostatin-1intervention group.Fibroblasts scratch experiment and angiogenesis of EA.hy926 cells proved that ferrostatin-1 accelerated fibroblast and endothelial cell migration and proliferation.In vivo study presented that compared with diabetic rats group,ferrostatin-1 increased re-epithelialization rates.H&E staining and immunohistochemical results showed that in the wound granulation tissues of ferrostatin-1 intervention group,expression levels of inflammatory factors such as IL-1β,IL-6 and TNF-α was down-regulated,and PCNA and VEGF was up-regulated,further verifying ferrostatin-1 could promote diabetes wound ulcer healing.SEM detection displayed that in the diabetic ulcer tissues,the mitochondria volume became smaller,and crest with a cavity change,conforming to the microscopic morphological characteristics of ferroptosis.GPX4,SLC7A11,TFRC also presented corresponding changes in m RNA and protein levels,namely,in the intervention group,GPX4,SLC7A11 increased,TFRC reduced.Granulation tissues of wound ulcer in ferrostin-1 intervention group showed lower MDA content.In addition,compared with the diabetes group and the RSL-3intervention group,the protein expression ratios of p-PI3K/PI3 K and p-AKT/AKT in granulation tissues increased in ferrostin-1 intervention group.Conclusion: This study preliminarily verified the existence of ferroptosis pathway in the pathology of diabetic wound ulcer.Ferrostinin-1 could effectively inhibit ferroptosis,reduce lipid per-oxidation damage,slow down inflammatory activities and promote cell proliferation and migration,which further accelerates the healing process of diabetic refractory ulcers.These findings are supposed to provide a new theoretical basis for the basic research on diabetic wound ulcer healing.