| Research background and Purpose:Ischemia-reperfusion injury is mainly caused by excessive oxidative stress,and is one of the main reasons for skin flap necrosis after transplantation.Recent research indicates that succinate dehydrogenase(SDH)catalyzes the accumulation of succinate during ischemia,which drives reactive oxygen species generation during reperfusion and constitutes a critical mechanism underlying ischemia-reperfusion injury.The current research is only conducted in the heart,brain,and kidneys.This study explored the metabolic pattern of succinate during the process of skin tissue ischemia-reperfusion and the feasibility of targeted succinate therapy for ischemia-reperfusion injury in free skin flaps.Combining advanced biomaterial,a sequential release microneedle(named DCM,short for DMM/RCDs@MN)was developed,which contains dimethyl malonate(DMM,a competitive inhibitor of SDH),and antioxidant stress carbon dots(RCDs).The effect of DCM on the survival of ischemia-reperfusion skin flaps in rats was observed,and an in vitro model of ischemia-reperfusion injury was established to explore the possible mechanisms of the drugs from the perspective of oxidative stress.Methods:This experiment consisted of three parts:(a)Investigation of changes of succinate metabolism during ischemia-reperfusion in skin flaps and the effect of dimethyl malate on its metabolism.Our study selected the rat abdominal ischemia-reperfusion skin flap model,in which a microvascular clamp was applied to occlude the vascular pedicle for a certain period of time before releasing it to simulate the process of ischemia-reperfusion in free flaps.The rats were randomly divided into the simple ischemia-reperfusion group and the dimethyl malate pretreatment group,and skin flap tissues were collected at four time points(3 rats at each time point)to detect the succinate content: before ischemia,8 hours after ischemia,5 minutes after reperfusion,and 30 minutes after reperfusion.(b)In vitro study of the protective effects of dimethyl malate and antioxidant stress carbon dots against oxygen and glucose deprivation/reoxygenation-induced injury.Human umbilical vein endothelial cells(HUVECs)were cultured,and anaerobic bags were used to establish an oxygen and glucose deprivation/reoxygenation(OGD/R)cell model.The following five groups were divided: control group,OGD/R group,OGD/R+DMM group,OGD/R+RCDs group,and OGD/R+DMM+RCDs group.Cell Counting kit-8 assay was used to select the optimal OGD/R time and drug concentration.The malondialdehyde content and superoxide dismutase activity of each group after reperfusion were detected,and ROS fluorescence staining and mitochondrial JC-1 fluorescence staining were performed.(c)Preparation and characterization of dual drug-loaded microneedles(DCM)and their effects on the survival of rat abdominal wall ischemia-reperfusion injury skin flaps.Fifteen SD rats were randomly divided into five groups,with 3 rats in each group.The grouping was as follows: sham group,8h ischemia group,12 h ischemia group,8h ischemia+DCM group,and 12 h ischemia+DCM group.The microneedle treatment group immediately used microneedles to puncture the skin flap from the inner side at the head end of the skin flap after clamping the femoral artery.The sham group lifted the skin flap blood vessel without microvascular clamping.The blood vessel clamp was removed after 8h/12 h,and the survival area of the skin flap was observed and recorded on the 7th day after surgery.After the observation,skin flaps were collected for subsequent histological HE staining.Results:(1)The content of succinate in the flap tissue increased significantly after 8hours of ischemia compared to normal tissue,and after 30 minutes of reperfusion,the content of succinate decreased and was almost the same as before ischemia.Rats given dimethyl malate in advance showed a decrease in tissue succinate content after5 and 30 minutes of reperfusion compared to the control group when the skin flaps were ischemic for 8 hours.(2)Dimethyl malate and antioxidant stress carbon dots can increase the cell survival rate of umbilical vein endothelial cells after oxygen-glucose deprivation/reoxygenation,reduce the content of malondialdehyde and the level of reactive oxygen species in cells,increase the activity of superoxide dismutase,and inhibit cell apoptosis.(3)Microneedles(DCM)loaded with dimethyl malate and antioxidant stress carbon dots can improve the survival area of rat skin flaps after ischemia-reperfusion injury and reduce the infiltration of inflammatory cells.Conclusion:(1)The metabolism of succinate exhibits certain patterns during the process of ischemia-reperfusion in skin tissue:succinate accumulates in ischemic flap tissue and is rapidly metabolized during early reperfusion stage.Treatment with dimethyl malate can inhibit the abnormal metabolism of succinate during ischemia-reperfusion in flap tissue.(2)Microneedles loaded with dimethyl malate and antioxidant stress carbon dots(DMM/RCDs@MN,DCM)can improve the postoperative recovery of ischemia-reperfusion flaps. |
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