Human Amniotic Epithelial Cells Regulate NETs To Promote Diabetic Wound Healing And Its Preliminary Mechanism

BackgroundDiabetic foot refers to the destruction of the skin and deep tissues below the ankle joint in diabetes mellitus（DM）patients,and often accompanied with various degrees of peripheral vascular disease and neuropathy,leading to lower extremity infections,ulcers and/or deep tissue destruction.As the most common and serious chronic complication of DM,diabetic foot brings a serious burden to society and families.It has been reported that overreaction of neutrophils to injury is also one of the reasons for the delayed healing of diabetic foot ulcers.It may cause more serious damage to the tissue damage and cause chronic inflammation.Inflammation is a typical process of wound healing,and neutrophils are recruited to the wound bed early,and their overreaction to injury can activate Neutrophil extracellular traps（NETs）.The hyperglycemia microenvironment further stimulates Neutrophils produce NETs,and persistent or excessive NETs can cause tissue and cytotoxic damage.Therefore,inhibiting the production of NETs can accelerate wound healing.It has been reported that human amniotic epithelial cells can regulate inflammation and promote angiogenesis to accelerate wound healing.They can also promote the biological functions of human fibroblasts and endothelial cells through exosomes secretion.It is rarely reported whether the human amniotic epithelial cells can inhibit the production of NETs when used in wound treatment to accelerate wound healing.Objective1.To detect the level of serum NETs in diabetic foot ulcers（DFU）patients.2.Cell experiments:To extract and identify human amniotic epithelial cells and human neutrophils,and establish an in vitro NETs model.To collect the conditioned medium of human amniotic epithelial cells for 24hours and 48 hours to interfere with NETs and observe their effects on NETs.3.To investigate the effect of human amniotic epithelial cells on wound healing in DM rats,and explore whether its mechanism is related to the inhibition of NETs production.Methods1.Population studies:A total of 53 DM patients,and 53 DFU patients were enrolled.Peripheral venous blood was drawn to separate serum,and the fluorescence value of 520/480 nm was detected by combining Pico Green and ds DNA to obtain the serum ds DNA concentration.Then compare the concentration of the two groups of ds DNA.2.Cell experiments:1)To separate amniotic membrane from placenta during term cesarean section extract human amniotic epithelial cells by two trypsin digestion methods.To detect the expression of Cytokeratin 19（CK19）by immunofluorescence,and to detect the expression of SSEA-4,CD29,CD105,and CD45 to identify the purity of human amniotic epithelial cells by flow cytometry.2)To detect the expression of CD11b from human neutrophils by flow cytometry,and then use 25n M PMA induces NETs in vitro to observe the morphology of NETs by detecting the concentration of ds DNA and Sytox Green fluorescence.3)To collect the conditioned medium of human amniotic epithelial cells for 24 hours and 48hours to interfere with NETs.The control group was used 25n M PMA to stimulate the production of NETs and intervened with high glucose DMEM.The 24h CM-h AECs group was used 25n M PMA to stimulate the production of NETs,and at the same time was intervened with 24h CM-h AECs.The 48h CM-h AECs group was used 25n M PMA to stimulate the production of NETs,and at the same time was intervened with 48h CM-h AECs.The blank control group（Blank）was intervened with high glucose DMEM.To detect the expression of NETs-related proteins neutrophil elastase（NE）and Citrullinated histone 3（Cit-H3）to explore its role of NETs by Sytox Green fluorescence and Western Blot.3.Animal experiments:1)SD rats were injected with streptozotocin（STZ）to induce DM.The criterion for successful model building was that the blood glucose in the tail vein was≥16.7mmol/L for a week.2)Rats were made a 10mm×10mm rectangular mark on the back,then cut off the full-thickness skin along the mark,and human amniotic epithelial cell suspension was injected subcutaneously around the wound skin.To observe the wound healing rate on the fifth and tenth days.To detect the expression of NE and CIt-H₃on the fifth and 1tenth days with Western Blotting.Results1.The serum concentration of ds DNA in DFU patients was significantly higher than that of DM patients[1067.68（924.06,1258.83）vs 966.72（839.19,1083.18）,P=0.0011].Correlation analysis of ds DNA concentration factors in DFU patients found that ds DNA concentration was positively correlated with Wagner classification（r=0.463,P=0）.As the Wagner grade is higher,the ds DNA concentration gradually increases,which suggested that the higher the ds DNA concentration,the more serious the condition of DFU might be.2.Human amniotic epithelial cells were successfully isolated and extracted.Flow cytometry showed that they highly expressed SSEA-4 and CD29,but did not express CD45,CD105,and immunofluorescence showed positive expression of CK19.All of those suggested high purity of human amniotic epithelial cells.3.Human neutrophils were successfully isolated in vitro.The positive expression rate of CD11b was 99.7%by flow cytometry.Neutrophils were stimulated with different concentrations of PMA for 120 minutes.The NETs morphology was displayed by Sytox Green fluorescence.The net and filament structures are NETs,and the NETs model is successfully induced.According to the increase of PMA concentration,the production of NETs gradually increased,but after the concentration of 75n M PMA,the production of NETs decreased slightly and then gradually tended to balance.The production of NETs did not increase significantly with the increase of concentration.4.They were divided into four groups:control group（CTL）,24h CM-h AECs group,48h CM-h AECs group,and blank control group（Blank）.CTL has significantly more NETs generation than Blank by Sytox Green fluorescence（P<0.0001）.Compared with CTL,the 24h CM-h AECs group and 48h CM-h AECs group had a significant decrease in NETs（P<0.0001）.The 48h CM-h AECs group inhibited NETs more significantly than 24h CM-h AECs group（P=0.0225）.The results showed that the concentration of ds DNA in the control group was significantly higher than that of the Blank,and there was a statistical difference（P=0.0001）.Compared with CTL,the 24h CM-h AECs group and 48h CM-h AECs grouphadasignificantlylowerconcentrationof ds DNA（P=0.006,P=0.0002）.But there was no significant difference between these two groups.5.The content of TSG-6 in the 24h CM-h AECs group and 48h CM-h AECs group were higher than that of CTL by ELISA.6.Human amniotic epithelial cells could promote wound healing in diabetic rats.The wound healing rate on the 5th and 10th day was significantly higher than that of the control group.Western blot test showed that the expression of NETs-related proteins Cit-H₃and NE in the wound tissue of the human amniotic epithelial cell treatment group was significantly reduced on the 5th and 10th days,suggesting that human amniotic epithelial cells may promote the healing of diabetic wounds by inhibiting the production of NETs.ConclusionThis study showed that the serum ds DNA of DFU patients is significantly increased.Human amniotic epithelial cells may inhibit the production of NETs,thereby promoting the healing of diabetic wounds.At the same time,it was found that the inhibition of NETs may be related to TSG-6,which also provides new treatment strategies and targets for the treatment of diabetic wounds.