Adipose Derived Stem Cells And Its Conditioned Medium Protect Vascular Endothelial Cells From Oxidative Stress And Protect Skin Flaps From Ischemia-reperfusion

BackgroundFor pedicle flap or free flap transplantation,one of the main reasons affecting the survival of flap is ischemia-reperfusion injury(I/R).During I/R,the flaps suffer from several pathological injuries,including oxidative stress,inflammatory injury and cell apoptosis.To prevent flaps from I/R,scholars have tried a variety of methods.Among them,adipose derived stem cells(ADSCs)have multidirectional differentiation potential and paracrine function,which have been proved to be effective.The current studies mainly focused on the protective role of single-dose ADSCs in flaps with short-time I/R.However,the most important factor affecting I/R is the ischemia time.Few studies have found the effects of ischemia time and ADSCs dose on the treatment of I/R.During flap I/R,oxidative stress damaged vascular endothelial cells,led to microcirculation disorder and flap injury aggravation.There is increasing evidence that ADSCs play important protective roles in the cellular defense against oxidative stress in several ischemia-reperfusion organs or tissues.Whether ADSCs can protect vascular endothelial cells from oxidative stress has not been studied.The increase of ischemia time will aggravate I/R in flaps.The most effective way to minimize the ischemic time is to find the blood circulation disorder and perform salvage operation as soon as possible.Various methods have been reported for monitoring transplanted flaps,among which flap local glucose measurement is considered a simple and practical method with low cost and easy access.However,there is no research on whether the method can be applied to diabetic models with impaired glucose metabolism.Objective1.To explore whether ADSCs conditioned medium(ADSC-CM)can prevent human umbilical vein endothelial cells(HUVEC)form oxidative stress injury in vitro,and further explore the possible molecular mechanism.2.To explore the effect of ADSCs on preventing skin flaps from I/R under the conditions of different ischemia time and different therapeutic dose.3.To verify the application of continuous interstitial glucose monitoring for flap venous occlusion in a diabetic model.Methods1.Effect of conditioned medium from adipose derived stem cells on protecting vascular endothelial cells from oxidative stress(1)ADSCs were obtained from human adipose tissue.Adipogenic,osteogenic and chondrogenic differentiation of ADSCs were induced.The cell surface markers of ADSCs were detected.(2)The H2O2 induced oxidative stress injury model of HUVEC was established,and median lethal dose was determined.(3)HUVEC were pre-cultured with 50%ADSC-CM and 100%ADSC-CM for 12h.ECM basic medium was used as control.After 600 μM H2O2 treatment for 4h,the cell survival rate was detected by CCK-8.(4)The intracellular reactive oxygen species were detected by DCFH-DA probe and flow cytometry.The oxidative stress products(MDA)and antioxidant enzymes(CAT,SOD,GSH-Px)were detected by test kits.Apoptotic cells were detected by Annexin V method.(5)The mRNA changes in HUVEC were detected by RT-qPCR,including oxidative stress related genes(Keap1,Nrf2,CAT,SOD,GSH-Px,NQO1),apoptosis related genes(Bax,Bcl-2,caspase-3)and inflammation related genes(IL-6,IL-8).2.Effect of adipose derived stem cells on protecting skin flap from ischemiareperfusion injury(1)ADSCs were extracted from groin fat pad of rats.ADSCs of P3 and below were used for flap injection.(2)All rats underwent abdominal ischemia-reperfusion flap surgeries.67 rats were randomly divided into sham group,3h ischemic group,6h ischemic group and 9h ischemic group according to vessel clamping time.Each ischemic group was subdivided into lowdose ADSCs group(5 × 106 cells)and high-dose ADSCs group(1 × 107 cells),according to the number of injected cells.(3)At day 5,flap blood perfusion was measured by laser Doppler;HE staining was performed and scored with a histologic scoring system;Vascular density was calculated by CD31 immunohistochemical staining;Cell apoptosis was detected by TUNEL staining;Ultrastructural changes of the flaps were observed by transmission electron microscope.3.Continuous interstitial glucose measurement for flap venous occlusion monitoring in a diabetic model(1)41 rats were randomly divided into control group,diabetic group and insulin-treated diabetic group.Diabetic rats were induced by intraperitoneal injection of streptozotocin(STZ,50mg/kg).After the diabetic rat model was established,2-4 U/kg insulin was injected cubcutaneously twice one day to control blood glucose to a normal range.(2)All rats underwent bilateral rectus abdominis myocutaneous flap surgeries,with the superior epigastric vessels as the pedicle.Two continuous interstitial glucose monitoring sensors were installed on bilateral myocutaneous flaps.Ligation of one side of the superior epigastric vein was performed to establish a venous congestion flap.The contralateral flap was not treated.(3)From the beginning of venous ligation,the interstitial glucose values of bilateral flaps were measured every 5min until the value fell to the lowest level or stopped falling.The blood glucose in tail vein was measured every 15min.Results1.Effect of conditioned medium from adipose derived stem cells on protecting vascular endothelial cells from oxidative stress(1)ADSCs in this study can differentiate into adipogenesis,osteogenesis and chondrogenesis.The surface markers(CD73,CD90 and CD 105)was positively expressed,and the expression of CD 19,CD34,CD45 and CD11b is negative,which were consistent with the characteristics of mesenchymal stem cells.(2)After pre-culture of 50%ADSC-CM and 100%ADSC-CM,the survival rate of HUVEC was significantly improved after exposure to H2O2.The higher the concentration of ADSC-CM,the higher the survival rate.(3)In ADSC-CM group,the production of reactive oxygen species in HUVEC decreased,the activities of antioxidant enzymes(SOD,CAT,GSH-PX)increased,and the level of oxidative stress product(MDA)decreased.At the same time,the apoptotic cell rate decreased significantly.(4)RT-qPCR results showed that in ADSC-CM group,genes of antioxidant regulatory pathways(Keap1,Nrf2)were up-regulated and genes of antioxidant enzymes(CAT,SOD2,GSH-Px)were up-regulated.Meanwhile,pro-apoptotic genes(Bax,caspase-3)were downregulated,anti-apoptotic gene(Bcl-2)were upregulated and inflammatory genes(IL6,IL-8)were downregulated.2.Effect of adipose derived stem cells on protecting skin flap from ischemiareperfusion injury(1)Flap survival decreased with longer ischemic time.The flap survival in the 3h,6h,and 9h I/R groups was 68.1±9.7%,43.6±6.2%and 24.6 ± 6.8%,respectively.In 6h and 9h ischemic groups,both low and high doses of ADSCs increased flap survival.(2)Laser Doppler results showed that application of ADSCs in all three ischemic groups improved flap blood perfusion.CD31 immunohistochemistry showed that ADSCs enhanced the vessel density in 6h and 9h ischemic groups.(3)HE staining showed that ADSCs improved the pathologic scores and ameliorated tissue damage in 6h and 9h ischemic groups.ADSCs also decreased the cell apoptosis in 9h ischemic group.(4)Under the transmission electron microscope,ADSCs attenuated endothelial cell injury,maintained vascular integrity,and reduced intercellular edema.3.Continuous interstitial glucose measurement for flap venous occlusion monitoring in a diabetic model(1)After venous ligation,the interstitial glucose in all flaps decreased to the lowest level.The time from venous ligation to the lowest interstitial glucose level was significantly longer in the diabetic group than the control group(120.8±5.7min vs 56.5±6.7min,),but was similar between the insulin-treated diabetic and control groups(56.5 ± 6.7min vs 56.0±6.6min).(2)The interstitial glucose in the control group decreased at a steady rate,lasting for 56.5±6.7min.The decline of the interstitial glucose in the diabetic group began with a plateau period lasting 49.0 ± 6.4min,and then slowly declined to the lowest level for 49.0 ± 6.4min.Changes of interstitial glucose in the insulin group all started with a slow descending period of 24.4 ± 4.3min,followed by a rapid decline to the lowest level within 34.3 ± 5.7min.Conclusion1.In vitro,ADSC-CM played a protective role in preventing HUVEC from H2O2 induced oxidative stress injury,through enhancing antioxidant enzyme activities and inhibiting cell apoptosis.The mechanism may be related to activation of the Keap1-Nrf2-ARE signaling pathway or inhibition of inflammatory response.2.ADSCs can protect skin flaps from ischemia-reperfusion injury by promoting revascularization,attenuating inflammatory response,and inhibiting apoptosis.For severe flap injury caused by medium or long-time ischemia,ADSCs exerted protective effects in a dose-dependent manner.3.It is feasible to detect venous congestion in diabetic flaps by monitoring the flap interstitial glucose.However,the impaired glucose metabolism in diabetic models significantly prolongs the monitoring time and dulls the reaction time.Insulin can restore the prolonged monitoring time,but the initial response is slow,which requires attention.