Study On The Effect And Mechanism Of Natural Antioxidant Fisetin On Wound Healing In Diabetic Mice

Objective: Screening of FDA-approved antioxidant drugs to identify drugs with antioxidant activity on mouse fibroblasts,and verifying their effects and specific mechanisms in promoting diabetic wound healing through in vivo cellular experiments and in vitro animal experiments,to provide strategies for diabetic wound healing.Methods:(1)Mouse skin fibroblasts were cultured and stimulated with hydrogen peroxide at concentrations of 200 μmol/L,400 μmol/L,and 800 μmol/L,respectively,to find out the appropriate concentration of oxidative damage,and further screened for drugs and divided into normal control group and hydrogen peroxide + drug treatment group(Fisetin,Neohesperidin,Hesperidin,and Roflumilast),the drug concentration of drug treatment group were selected 10umol/L,and their reactive oxygen content was measured by reactive oxygen kit and verified by Western Blot experiment for VEGFA,SDF-1 protein expression.(2)Mouse fibroblasts were cultured,and the screened Fisetin was made into a concentration gradient and divided into 6 groups,normal control group,200μmol/L hydrogen peroxide model group,50nmol/L Fisetin+200μmol/L hydrogen peroxide group,500nmol/L Fisetin+200μmol/L hydrogen peroxide model group,10μmol/L Fisetin+200μmol/L hydrogen peroxide model group,and 50μmol/L Fisetin+200μmol/L hydrogen peroxide model group,respectively,for reactive oxygen species assay,and screened the optimal concentration for Western Blot to detect PCNA,Cyclin D1,VEGFA,and SDF-1 protein expression(3)First,40C57 mice were established as diabetic trauma models,and then grouped into 5 groups,namely normal control group,diabetic trauma model group,diabetic trauma positive treatment group(100mg/Kg resveratrol topical),diabetic trauma treatment group(2mg/Kg Fisetin topical),diabetic trauma treatment group(20mg/Kg Fisetin topical),on 7 and 14 days respectively The wounds were taken and the wound healing rate was calculated respectively.The expression of CD31 protein was detected by immunofluorescence,and the expression of PCNA,Cyclin D1,VEGFA and HIF-1α protein were detected by Western Blot.Results: 1,Screening from the antioxidant library(Fisetin,Neohesperidin,Hesperidin,Roflumilast)showed antioxidant activity and promoted VEGFA and SDF-1 protein expression in mouse fibroblasts(n=3,P<0.05),with Fisetin having the strongest antioxidant activity and promoting VEGFA and SDF-1 protein expression at a concentration of 10umol/L(n=3,P<0.05).Fisetin had the strongest antioxidant activity and promoted the expression of VEGFA and SDF-1 protein at 10umol/L(n=3,P<0.05).(2)Fisetin was selected for the next experiment,and the strongest antioxidant activity was found at 50 nmol/L in mouse fibroblasts by reactive oxygen species assay,while the expression of PCNA,Cyclin D1,VEGFA,SDF-1 and HIF-1α proteins was significantly increased at this concentration as determined by protein immunoblotting assay(n=3,P<0.05)(3)In vitro animal experiments to calculate the wound healing rate revealed that Fisetin promoted wound healing,HE staining revealed a significant thickening of epithelial tissue,Masson staining revealed an increase in collagen deposition,and protein immunoblotting experiments promoted the expression of PCNA,Cyclin D1,VEGFA,and HIF-1α in diabetic wounds in mice(n=3,P<0.05).Conclusion: The natural antioxidant Fisetin ameliorates poor diabetic wound healing in mice by contributing to fibroblast proliferation and HIF-1α/VEGFA-mediated angiogenesis.