The Role And Mechanism Of CD100/Semaphorin 4D Involved In Diabetic Chronic Wound Healing

BackgroundWith the development of the economy and the improvement of people’s living standards,the number of diabetic patients has increased year by year.One of the chronic complications of patients with common clinical diabetes is spontaneous ulceration and difficult healing after the formation of wounds.Chronic ulcers that occur in the lower extremities usually manifest themselves as diabetic foot（DF）,which is difficult to treat,can cause amputation,and impose a heavy financial burden on patients,families,and society.The reasons for the impaired recovery of diabetic wounds are very complex.They are generally considered to be the result of a combination of internal and external factors.However,the specific molecular mechanisms are not yet clear,and clinically there is also a lack of effective and economical drug treatment methods.Therefore,it is an important issue for clinicians and researchers to deeply study the molecular mechanisms of diabetic wound healing and provide potential therapeutic targets for clinicians.CD100（Semaphorin4D）is a member of the Semaphorin family（brain signaling protein）.It was initially found to have an important role in regulating nerve cell axon guidance and regulating immune responses.Recent studies have shown that CD100 is critical for the normal wound healing after skin damage:CD100 is not only a key molecule in the initial stage of wound healing,but also plays an important role in promoting angiogenesis,whether isolated from cultured vascular endothelial cells or in vivo.In the environment,CD100 can induce potent angiogenesis.However,the relationship between CD100 and chronic wound healing in diabetes has not been studied yet.Our preliminary clinical research has found that the expression of CD100 in ulcer tissue of diabetic foot patients is significantly lower than that of acute wounds.Moreover,in vitro,the function of human vascular endothelial cells can be regulated by the application of exogenous soluble CD100（sCD100）.These findings strongly suggest that CD100 is involved in regulating the chronic wound healing in diabetes.Therefore,we speculate that the angiogenesis disorder caused by CD100 expression or dysfunction is an important cause of chronic wound healing,and supplementation of exogenous CD100may promote wound healing by promoting endothelial cell function and angiogenesis.Purposes1.By studying the expression characteristics of CD100 in clinical acute wounds,chronic wounds,and normal control skin tissues,the role and mechanism of CD100 in wound healing were elucidated.2.By exogenously supplementing soluble CD100（sCD100）molecules in the db/db mouse and CD100^-/-mouse wound models,the effect of CD100 on chronic wound healing in diabetes and its mechanism were elucidated.Methods1.Evaluated the different expression and positioning of CD100 in tissues of normal skin,acute wound and chronic wounds using the immunohistochemical staining.2.Evaluated the effect and its mechanism of exogenous CD100 on db/db mice wound healing including wound healing rate,wound healing quality,collagen remodeling and angiogenesis by HE staining,Masson staining,immunohistochemical staining of CD31,CD34,VEGF and CD68,Western blot of ColⅠ,ColⅢ,TNF alpha and IL–6.3.Evaluated the effect and its mechanism of exogenous CD100 on CD100-/-mice wound healing including wound healing rate,wound healing quality,collagen remodeling and angiogenesis by HE staining,Masson staining,immunohistochemical staining of CD31,CD34 and CD68.Western blot of ColⅠ,ColⅢ,TNF alpha and IL–6 and PCR of PlexinB1,VEGF,TNF alpha and IL–1β.Results1.CD100 is mainly expressed in the epidermis in normal skin tissue,with a high expression in the epidermal cell membrane,a high expression in the epidermal cytoplasm,and no expression in the dermal tissue.The expression level of CD100 in acute wounds was significantly lower than that in normal tissues contain epidermis and epidermis cytoplasm and dermis.In the chronic wound,the CD100 expression on epidermis cell membrane and cytoplasm were.but expression in dermal tissue was significantly increased.2.After exogenous administration of sCD100,compared with PBS control group,db/db mice in the CD100 treatment group had faster wound healing,shorter wound healing time,and better wound healing quality（scores of scores for thickness of epidermis and dermis,and scores of thickness of granulation tissue were increased）,promoted the remodeling of collagen（Masson staining to assess the density and arrangement of collagen in the wound tissue,the maturity of the fiber is better,the expression of Col I and Col III were increased）,and the angiogenesis was increased（the number of gross observation was increased and CD31,CD34 and VEGF were increased expression）,and inhibited inflammatory response（expression of CD68,TNF alpha and IL–6 were decreased）3.After exogenous administration of sCD100,compared with PBS control group and CD100^+/-mice,the wound healing rate of CD100^-/-mice CD100 group was accelerated,wound healing time was shortened,and wound healing quality was improved(epidermal thickness,epidermis,and The scores of fresh dermis score and thickness of granulation tissue increased,collagen remodeling increased（increased collagen density in the wound tissue,regular arrangement,fine fiber maturity,increased expression of Col I and Col III）,and increased angiogenesis（increased expression of CD31 and CD34）decreased inflammation（CD68,TNF-α,and IL-6 levels decreased）;and the expression of PlexinB1and VEGF in the wound tissue of the CD100 treated group was significantly upregulated,TNF-αand IL-1βDecline in expression.Compared with CD100^+/-mice,the wound healing time of CD100^-/-mice PBS control group was prolonged,the healing quality was decreased,collagen remodeling was reduced,angiogenesis was reduced,and the degree of inflammatory reaction was severe.ConclusionThe imbalance in the expression or function of CD100 is an important cause of the impoverishment of diabetic wounds.Exogenous administration of sCD100 promotes the healing of diabetic wounds by promoting the remodeling of collagen,blood vessel formation,and reducing the inflammatory response.This effect may be mediated through its receptor PlexinB1.