Pathological Effects Of Neutrophil Extracellular Traps And Intervention Of Hydrogen Sulfide

Background/Aim: When neutrophils are activated by a variety of factors,a mesh-like DNA fibers will be released,and these fibers are usually coated with cytosolic granule proteins,such as neutrophil elastase(NE)and myeloperoxidase(MPO),known as Neutrophil extracellular traps(NETs).The process through which NETs are produced and released from neutrophils is so called NETosis.Traditionally,NETs can be used to capture pathogenic microorganisms and play very important roles in innate immune responses.However,recent studies have shown that the microenvironment of diabetes primed neutrophils to undergo NETosis,and impeded wound healing through the released NETs.In addition to impairment of wound healing,epidemiologic evidence suggests that morbidity or/and mortality of many types of cancer are significantly increased in diabetes.However,it is still unknown whether NETs participates in diabetes-related cancer,up to date? Engaged in hydrogen sulfide(H2S)related research for a long time,we have shown that H2 S plays a significant antioxidant effect by reducing formation of reactive oxygen species(ROS),necessary for neutrophils’ NETs release.We therefore speculate that by reducing ROS levels H2 S may inhibit NETosis and thus promote diabetic wound healing? Based on all the facts,the purpose of the present study should cover: 1.To determine the serum levels of NETs in diabetic patients with delayed wound healing and health volunteers;2.To observe NETs direct effects on biological behavior of tumor cells,using homemade NETs through a NETosis model in vitro.3.To investigate the effects of H2 S on wound healing in db/db diabetic mice and NETosis related mechanisms.Method 1.The peripheral blood samples from 5 diabetic foot ulcer patients and 5 healthy volunteers were collected.After centrifugation,the serum was obtained,and then the content of ds NDA and activities of NE were detected to observe the content of NETs.2.Peripheral blood neutrophils were isolated from healthy volunteers.The neutrophil’s morphology and purity in the isolated cells were preliminarily observed under microscope after Wright-Giemsa stained.The neutrophils were further labeled with CD11 b antibody in conjunction with APC,and then the number and percentage of neutrophils were analyzed with Flow Cytometry.3.The isolated neutrophils were treated with Phorbol-12-myristate-13-acetate(PMA)to establish a NETosis model in vitro.The expression of NETosis related proteins,peptidylarginine deiminase 4(PAD-4)and citrulline histone 3(Cit-H3),was detected by Western Blot to determine the optimal concentration and time of PMA to induce NETosis in neutrophils.4.After the treatment of Neutrophils with 25 n M PMA for 2 h,the NETs were extracted by differential centrifugation.The expression of MPO,Matrix Metalloproteinase 9(MMP-9)and NE in the extracts were detected by Western Blot,and the content of ds DNA was tested by Pico Green.5.The extracted NETs was used to co-culture with 3 common tumor cells(SMMC7721,A549 and Hela cells)for 48 h.The effects of NETs on the proliferation of tumor cells were observed by CCK-8 assay.After the treatments,the cells were digested with trypsin and inoculated on the 96-well plates followed by a 6 h incubation.The numbers of the total cells and adherent cells were measured to calculate the cell adhesion rate.Different concentrations of NETs were placed in the sub-plate chamber of Trans-well plates and co-cultured with the tumor cells in the upper chamber for 48 h.The migrated cells were stained with 0.5% crystal violet.The effect of NETs on the migration ability of tumor cells was observed by counting under a microscope.6.A full-thickness skin wound with a diameter of 6 mm was made with a skin perforator on the back of mice.The expression of NETosis-and NETs-related proteins in the wound tissue was detected by Western Blot assay to investigate the effects of NETosis levels during diabetic wound healing.By intraperitoneal injection of Na2S(a H2 S donor),the effects of H2 S on wound healing in diabetic mice were investigated by detecting the area of skin wound,and on NETosis by measuring the content of ds DNA and the activity of NE in wound tissue.7.Neutrophils were pretreated with 25 μM H2 S for 1 h and then treated with 25 n M PMA for 2 h.The effects of H2 S on NETs release which induced by PMA was observed by nucleic acid staining.In addition,the changes of NETs content,such as ds DNA,NE and MPO activity at the bottom of petri dish,were further detected.Determination of ROS by dihydrorhodamine 123.Finally,the expression of NETosis related proteins PAD-4 and Cit-H3 were detected to reveal whether H2 S could antagonize PMA-induced NETs release.Result 1.The serum ds DNA content of diabetic patients was significantly increased comparing with that of healthy volunteers(P < 0.01),and the diabetic NE activity was also statistically increased comparing with that of healthy volunteers(P < 0.05).2.After Wright-Giemsa staining,it was shown that the isolated cells were mainly polymorphonuclear neutrophils,whose content was over 95% under microscope.The quantitative analysis with Flow Cytometry showed that the cells were mainly lobular nuclei and the positive rate of APC CD11 b was 99.95%.3.After the treatment with different concentrations of PMA,the NETosis related proteins,including PAD-4 and Cit-H3,were upregulated at first and then gradually decreased.At 25 n M,the expression appeared to peak,indicating that PMA optimal concentration to trigger NETosis was 25 n M.In addition,the expression of PAD-4 and Cit-H3 was increased in a time-dependent manner within a 2-h treatment.The analysis of the extracts showed that ds DNA,MPO,MMP-9 and NE were rich in it,and the extracts had MPO and NE catalytic activity.4.After the treatments with different concentrations of NETs,the biological behavior of 3 kinds of common tumor cells were observed.The results of cell viability showed that at low concentration(0.13 μg/ml)NETs could induce a proliferation in SMMC7721,a common hepatocellular carcinoma cells,without altering the viability of lung cancer A549 cells and cervical cancer Hela cells.In addition,through testing the adhesion and migration ability in SMMC7721 cells,it was found that the exposure to NETs significantly enhanced the adhesion and migration function(P < 0.05).However,when the concentration of NETs was too high,the proliferation,alongside adhesion and migration,was significantly inhibited(P < 0.05).5.In db/db diabetic mice,it was shown that the wound areas closed significantly slowly comparing with those of the db/+ control mice from day 6 to day 15(P<0.01).The results of H&E and Weigert staining showed the thinner skin tissue,lower elastic fibers in the diabetic mice than those in the control mice;In addition,the expression of NETosis related proteins,PAD-4 and Cit-H3,was significantly higher in diabetic mice wound tissue than that in control mice.Notably,the NETs components,MPO and NE,were also higher than those in the control mice.Importantly,after the treatment with H2 S,the healing of skin wound in diabetic mice was significantly quicker than that in the model group(P < 0.01).The microscope photographs further showed that the H2 S treatment enhanced the thickness of the skin and the length of the elastic fibers,while reduced the content of ds DNA(P < 0.05)and NE activity(P < 0.05)in wound tissue.6.Last but not least,although PMA could promote the release of NETs,including mesh-like ds DNA fibers,NE and MPO protein(P<0.05),along with ROS(P<0.01),as well as the expression of NETosis related proteins PAD-4 and Cit-H3(P<0.05),all the effects of PMA were attenuated by the pretreatment of neutrophils with H2 S for 1 h,characterized by the decreased activities of NE and MPO(P < 0.05),inhibition the release of ROS(P < 0.01),as well as the downregulated expression of NETosis related proteins(PAD-4 and Cit-H3)(P < 0.05).Conclusion The present study demonstrated that diabetes are prone neutrophils to undergo NETosis and release NETs,promoting the proliferation,adhesion and migration of hepatocellular carcinoma SMMC7721 cells and delaying wound healing.The treatment with H2 S can inhibit NETosis and NETs release,thus improving the diabetic wound healing.Our research provides a clue to the treatment of diabetic complications with H2 S by targeting NETosis.