Efficacy And Mechanism Of Netrin-1 In Diabetic Wound Healing

Background:Diabetes is a common and serious systematic desease with metabolic disturbance.It has become a global disease that endangers human health with the increase people affected.Diabetic ulcer,as the most highly susceptible complication of diabetes,is characterized by difficult wound healing,high recurrence rate,high amputation incidence and high mortality rate,which brings heavy psychological pressure and economic burden to patients,families and society.Current conventional treatments of diabetic wounds include blood glucose regulation,wound debridement,infection control,dressing coverage,revascularization and wound decompression,etc.The overall therapeutic effect of diabetic wounds is still unsatisfactory.Wound repair is a multi-level dynamic process.Coagulation,inflammation,proliferation,and remodeling coordinate with each other and cooperate precisely to achieve rapid wound healing.However,the persistent hyperglycemic environment of diabetes leads to abnormal wound inflammatory response,and granulation tissue formation,angiogenesis and epithelialization are blocked.Diabetic wounds are a complex problem involving multistages and multicells.Further exploring the pathogenesis of diabetic wounds and new therapies to modulate diabetic wound healing from multiple perspectives have important clinical significance for the treatment of diabetic wounds.Netrin-1 was originally discovered as a nerve axon guidance factor.Recent studies have found that Netrin-1 is not only involved in neural development and peripheral nerve regeneration,but also in inflammation regulation,angiogenesis,organ fibrosis,epithelial cell repair and tumor development.The process of skin wound repair involves early inflammatory response;middle-late stage angiogenesis,extracellular matrix deposition,wound shrinkage and wound epithelization.Therefore,it is of much feasibility and innovation to explore the role of Netrin-1 in wound healing and apply it to the treatment of diabetic wounds.Netrin-1 may be a new target molecule for chronic wound repair.Objectives:Part I:To detect the Netrin-1 expressions in the stages of normal wound healing and observe the whole process of wound healing with mouse Netrin-1 antibody injected intravenously.Part II:To compare the expressions of Netrin-1 in normal wound and diabetic wound at each repair stage and observe the improvement of diabetic wound repair with local application of Netrin-1 protein.Part III:To explore the direct function of Netrin-1 on wound repair-related cells under continuous high-glucose environment.Part IV:To clarify the mechanism of Netrin-1 promoting cell function related to wound repair with experiments for further verification.Materials and Methods:Part I:(1)C57BL/6 mice were used for wound modeling without any treatment.The wound samples were harvested on time and Western Blotting(WB)experiment was adopted to evaluate the expressions of Netrin-1 in normal wound phases of inflammation,proliferation and healing.(2)The mice(C57BL/6)were used for wound modeling and randomly divided into two groups.One group was intravenously injected with mouse Netrin-1 antibody to antagonize Netrin-1 protein in wound healing with the other group operated with the relative control antibody.The wound healing trajectory of the two groups was observed,and tissue immunofluorescence(F4/80~+CD206~+),hematoxylin-eosin(HE)staining,immunohistochemistry(CD31~+)and sirius red staining were performed to evaluate wound macrophage polarization,the general structure of the wound,the wound vascularization and extracellular collagen deposition.Part II:(1)Clinically obtain skin samples from healthy people and diabetic patients and use WB to verify the expressions of Netrin-1 in normal and diabetic skins.(2)db/db mice and littermate db/m mice were used for wound modeling;observe wound healing and collect samples on time.Immunohistochemistry and immunofluorescence staining Netrin-1weres appiled to explore the time-series differences of Netrin-1 expression in normal and diabetic wounds at the phases of inflammation,proliferation and healing.(3)The db/db mice were used for wound modeling,and exogenous Netrin-1 was injected into the wounds during the inflammatory and proliferative phases to explore the improvement of diabetic wound repair.Tissue immunofluorescence(F4/80~+CD206~+),enzyme-linked immunosorbent assay(ELISA,inflammatory factors:TNF-α,IL-1β,IL-6,growth factors:TGF-β1,IGF-1,VEGF-A),HE staining,immunohistochemistry(Ki67~+,CD31~+),sirius red staining and WB assay(Collagen I,Collagen III,α-SMA)were used to evaluate overall wound structure,wound microenvironment,cell proliferation,vascularization,extracellular matrix arrangement and composition.Part III:(1)Establish high-glucose cell culture model and use the relevant experiments to evaluate cell functions(macrophages,vascular endothelial cells,fibroblasts)to verify the feasibility of this cell model.(2)Bone marrow-derived macrophages were cultured under high glucose,and TNF-αand IFN-γcombined with Netrin-1 were simultaneously stimulating marophages for 48 hours.Microscope observation,flow cytometry,cellular immunofluorescence and quantitative real-time polymerase chain reaction(q RT-PCR)were conducted to verify the effect of Netrin-1 on macrophage polarization and the cytokine secretion.(3)Umbilical vein endothelial cells were cultured with high-glucose cell culture model;CCK8 assay,scratch and Transwell assays,and tube formation assay were used to detect the improvement of Netrin-1 on proliferation,migration,and angiogenesis of endothelial cells inhibited by high glucose.(4)Human fibroblasts were cultured with high-glucose cell culture model;CCK8 assay,scratch and Transwell experiments,q RT-PCR and WB experiments were conducted to detect the effect of Netrin-1 on fibroblasts’proliferation,motility and collagen and actin synthesis under high-glucose environment.Part IV:(1)Collect the cellular RNA of bone marrow-derived macrophages cultured with Netrin-1 protein under high-glucose and inflammatory stimulations and perform high-throughput transcriptome sequencing(RNA-seq),and the differential genes were analyzed by GO and KEGG to enrich the possible mechanisms of Netrin-1 on macrophage polarization.(2)WB were used to detect the key proteins in the above candidate pathway,and inhibitors antagonizing core protein and Netrin-1 receptor were applied to confirm that Netrin-1 indeed regulated macrophage polarization through certain receptor and certain pathway.(3)Cell immunofluorescence and q RT-PCR were used to re-verify the above Netrin-1 receptor and pathway from the perspective of macrophage markers and secretions.Results:Part I:(1)Netrin-1 was expressed in normal mouse skin,temporally increased during inflammatory and proliferative phases,and then gradually decreased when the wound was completely repaired.(2)Using mouse Netrin-1 antibody to antagonize the function of Netrin-1 protein in wounds significantly delayed wound healing;immunofluorescence staining of macrophages showed that comparing with the control group,Netrin-1 antibody group on day 6 presented lower F4/80~+CD206~+cells;HE staining showed that on day 9,the wound in control group had been nearly epithelialized with abundant granulation tissue,while Netrin-1 antibody group still showed obvious naked wound with thin granulation tissue;CD31~+immunohistochemistry and sirius red staining showed that after antagonizing Netrin-1 protein,wound angiogenesis was attenuated and extracellular matrix deposition was insufficient.Part II:(1)The skin of diabetic patients showed significantly lower expression of Netrin-1 than that of healthy people.(2)Immunohistochemitry and immunofluorescence staining Netrin-1 showed that nerin-1 expression was temporally increased in inflammatory and proliferative phases,while Netrin-1 expression was abnormal in diabetic wounds at the same time,showing a significantly lower expression.(3)Netrin-1 locally injected significantly promoted the repair of diabetic wounds;immunofluorescence showed that Netrin-1 significantly increased the number of F4/80~+CD206~+cells in the treatment group on day 6;ELISA showed that the wounds in Netrin-1 group presented the decreased expressions of inflammatory factors(TNF-α,IL-1βand IL-6)and the increased levels of growth factors(TGF-β1,IGF-1,VEGF);histopathological detection and WB experiment revealed that compared with the control group,the Netrin-1 group had completely achieved epithelization on day 9,with abundant granulation tissue,significantly enhanced wound-related cell proliferation,obvious wound vascularization,and closely arrangement of extracellular matrix and increased synthesis of collagens and actin.Part III:(1)The polarization efficiency of macrophages into M2 type decreased under high-glucose culture;the proliferation,migration,angiogenesis and matrix synthesis of endothelial cells and fibroblasts were also inhibited;these results indicated that high-glucose cell culture model was feasible.(2)The general image under light microscope showed that Netrin-1 could induce macrophage morphology from round M1 type with dendritic bulges to long M2 type;flow cytometry and cellular immunofluorescence presented that Netrin-1 promoted CD206 expression in macrophages under high-glucose and inflammatory environment;q RT-PCR results showed that Netrin-1 could significantly inhibit the M1-type markers(CD86,TNF-α,IL-1βand IL-6)and promote the M2-type markers(CD206,ARG-1,IL-10 and VEGF).(3)Netrin-1 significantly improved the proliferation,migration and tube-forming function of vascular endothelial cells inhibited by high glucose.(4)Netrin-1 promoted the motility of fibroblasts and the synthesis of collagens and actin in high-glucose environment,but had no significant effect on cell proliferation.Part IV:(1)RNA-seq results showed that there were 640 differential genes between the Netrin-1 stimulation group and the control group;further GO analysis enriched the significantly regulated biological processes,cellular components and molecular functions,and KEGG analysis enriched the possible pathways;referring to the relevant background,we speculated that the enriched PPAR signaling pathway may play an important role in regulating macrophage polarization.(2)Netrin-1 promoted macrophage polarization into M2 type,which was reversed by GW9662,the PPARγ-specific inhibitor;Netrin-1significantly enhanced the expression of core proteins(p STAT3,p STAT6 and PPARγ)in the PPAR signaling pathway of macrophages under high-glucose and inflammatory environment,and the efficacy was inhibited by Netrin-1 A2b receptor antagonist MRS.(3)Cell immunofluorescence showed that GW9662 and MRS inhibited the expression of CD206 in macrophages increased by Netrin-1 under high-glucose and inflammatory environment;q RT-PCR indicated that Netrin-1 induced macrophage to express M2-related markers(CD206,ARG-1,IL-10,VEGF)and suppressed M1-related markers(CD86,TNF-α,IL-1β,IL-6);the above effects were dramatically reversed by addition of GW9662or MRS.Conclusions:The study confirmed that Netrin-1 presented a temporal increase in inflammatory and proliferative phases of normal wound healing and antagonizing Netrin-1 significantly delayed wound repair,indicating that Netrin-1 played an important role in wound healing.Comparing with normal wound,Netrin-1 was in abnormally low expression in diabetic wound at different healing stages.Therefore,we speculated that the possible mechanism of delayed diabetic wound repair may be caused by the abnormal time-ordered low expression of Netrin-1.Exogenous Netrin-1 was further injected into diabetic wounds and cultured with wound-related cells and the results proved that Netrin-1 promoted diabetic wound healing mainly by regulating macrophage polarization,promoting angiogenisis and enhancing fibroblast contraction and collagen synthesis.Netrin-1 modulated macrophage polariztation under high-glucose and inflammatory stimulates mainly through A2b R/STAT/PPARγsignaling pathway.