| BackgroundWound healing is a complex and dynamic process for restoration of injured cellular structures and tissues,traditionally explained in terms of three overlapping phases: inflammation,proliferation and maturation.There are a number of factors(vascular disease,neurophathy,nutrition,infection,drugs,and immune dysfunction)that can affect this process,thus causing impaired wound healing.Diabetic chronic wound,one of the most common chronic wounds in clinical work,results from multifactor,the pathogenic mechanism of which is still unclear.Advanced glycation end products(AGEs)accumulated in skin can impact negatively on wounds healing and lead to chronic wounds via suppressing the functions of regulatory cells,repaired cells and extracellular matrix under diabetic state.Despite considerable research focused on understanding and treating of diabetic wound,the molecular mechanisms underlying impaired diabetic wounds are poorly understood.Autophagy,the process by which cells recycle cytoplasm and dispose of excess or defective organelles,plays an essential role for survival,differentiation,and homeostasis.It is noteworthy that an immunosuppressive drug of rapamycin for anti-rejection of grafting,as an inducer of autophagy,has been implicated in impeded normal healing in patient studies,suggesting autophagy is associated with wound healing.This study investigated that the effect of autophagy on diabetic chronic wounds by making full-thickness defects on dorsal skin of mice,furthermore,we clarified the mechanism by which autophagy delayed wound repair in vitro.ObjectiveThe aim of this study was to explore the effect of autophagy on wound healing and the underlying molecular mechanism.MethodsPart one: Full-thickness defects were made on C57BL/6 mice and wound healing was observed.The tissue samples were harvested from the skin wounds at days 0,1,3,7,10,14,and 21 post-wounding,and then LC3 and Beclin-1 were detected using Immunohistochemical staining(IHC)and/or immunofluorescence.Electron microscopy was used to monitor the number of autophagic lysosomes.For identify the effect of autophagy on wound healing,rapamycin or 3-MA was applied for inducing or inhibiting autophagy by intraperitoneal injection per day.To determine whether increased autophagy affects the diabetic wound healing process,full-thickness defects were made on db/db mice and db/m control mice,and db/db mice were treated with 3-MA,wound healing was observed and LC3 was detected using IHC.At the same time,we examined the autophagic level in patient tissue,including chronic cutaneous wounds with or without diabetes and unwounded skin,by using LC3 immunostaining assay.Part two: Full-thickness defects were made on C57BL/6 mice,AGEs with 100mg/kg/d were injected into margin tissues of wound,wound healing was observed.LC3 was detected using IHC on days 0,3,7,and14 post-wounding.Furthermore,AGEs treated mice were treated with 3-MA,wound healing was observed and LC3 and F4/80 were detected by immunofluorescence.For identify the effect of autophagy on macrophage polarization,RAW264.7 cells were treated with rapamycin or 3-MA before LC3 was detected using western blotting.At the same time,the gene expressions of M1 related markers(tnf-a,inos,il-1β,and il-6)and M2 related markers(arg-1,mrc-1,il-10,and mgl1)were identified using real-time PCR,on the other hand,CD11c(M1 marker)and CD206(M2 marker)were detected using flow cytometry.Furthermore,the cells were treated with AGEs or AGEs plus 3-MA,LC3 was detected using western blotting and immunofluorescence,autophagolysosome was examined under electronic microscopy.M1 polarization was determined using real-time PCR and flow cytometry.Part three: ShIRF8 RAW264.7 cells were created and treated with AGEs.LC3 was detected using western blotting and immunofluorescence.M1 polarization was determined using real-time PCR.ResultsPart one: IHC staining showed that the levels of LC3 and beclin-1 in the wounded mice skin began to increase on day 1,and reach to the highest level on days 7,and then decreased gradually to the basal level on days 21 post-wounding.A significantly increased the numbers of autolysosome was defined in wounded mice skin on day 7.Furthermore,LC3 levels in the rapamycin treated mice skin were much higher than that in the control or 3-MA treated mice on days 7 or 14 post-wounding,respectively.The wounds from diabetic db/db mice(25.8?0.59 days)were delayed than that from db/m(control)mice(15.9?0.82 days).Significantly rise of autophagic level in db/db mice were especially evident on days 7,14,and 21 post-wounding.However,3-MA treatment did significantly restore delayed wound repair in db/db mice.Furthermore,an increase of autophagy in the patient wounds with diabetes(p < 0.05)was dramatic,compared to that in the patient wounds without diabetes.Part two: IHC staining of AGEs found that AGEs positive staining was observed in db/db mice skin,however,non-staining was found in the skin from db/m mice.Treatment with AGEs apparently delayed wounds closure(18.0?0.67 vs 14.8?0.79 days)in mice skin.Furthermore,administration of 3-MA completely rescued the AGEs delayed wound healing(15.0?0.82 days)in C57BL/6 mice.In addition,LC3 positive cells in the AGEs treated mice skin significantly increased on days 7 and 14 post-wounding,compared to that in the control group.Furthermore,The immunofluorescence staining results of wounded skin from both control and AGEs treated mice revealed that the most of autophagic cells in the dermis were F4/80 positive cells.In vitro,AGEs treated cells contained more autophagolysosome compared to that in cells treated by vehicle under electronic microscopy.In addition,LC3-II isoform also increased by AGEs treatment.Coapplication of AGEs and 3-MA significantly inhibited proteins of LC3 in both western blotting and immunofluorescence results.Rapamycin treated RAW264.7 cells shown consistent results with AGEs treated cells.On the other hand,our results exhibited that gene expressions of M1 related proinflammatory mediators including tnf-a,inos,il-1β,il-6,obviously increased after treatment with rapamycin or AGEs.However,pre-treatment with 3-MA inhibited the gene expressions of M1 related markers.Unexpected,the gene expressions of M2 markers,including arg-1,mrc-1,il-10,mgl1,had no accordant changes to autophagic activity.Flow cytometry results demonstrated that The positive cells of CD11 c in rapamycin treated group(27.86±1.37%)were much higher than that in control(13.61±10.97%).By pre-treatment of 3-MA,the numbers of positive cells were diminished(2.81±0.30%).Moreover,AGEs also increased the expressions of M1 related factors and the numbers of CD11 c positive cells(28.11±0.87%).Part three: By blocking IRF8 with the shIRF8,baseline of LC3 and enhanced LC3 by AGEs were suppressed.Furthermore,gene expressions of M1 markers(tnf-a,inos,il-1β,il-6)in RAW 264.7 cells infected with shIRF8 were significantly reduced,compared to that in the cells infected with control shRNA.Conclusion1.Enhanced autophagy in skin wounds from diabetic mice and patients is related to impaired healing;2.AGEs induced autophagy impacts negtively on wound healing by stimulating macrophage polarization to M1;3.AGEs induces autophagy and promotes M1 polarization by upregulating IRF8. |
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