Study On The Relationship Of Diabetic Foot Non-ischemic Ulcers And Dysfunction Of Macrophage

BackgroundDiabetic foot ulcer is one of the common clinical chronic diabetic complications, which often leads to difficult wound healing.Diabetic foot ulcer is one of the main reasons leading to amputation.Diabetic foot ulcer not only make the patiens suffer from great mental stress,but also cause heavy economic burden.According to the statistics,the incidence rate of foot ulcer in diabetic patients is 4%—10%.With the rapidly increased incidence rate of diabetes,the number of the patients with diabetic foot ulcer will increase significantly.It is an important topic to study the mechanism of diabetic foot ulcer in depth and it is necessary to find new methods to prevent and treat diabetic foot ulcer nowadays.Skin wound healing is a complex,orderly biological process,and in each period there are many tissues,extracellular matrix,cytokines participated,related to a number of biological processes,such as hemorrhage,vascular reactivity, inflammatory response,granulation tissue formation,repair of epithelial and the remodeling,etc.Macrophage is an important participant in wound healing. Macrophage not only can phagocytize bacteria,remove foreign bodies and necrotic cell,but also can secrete cytokines involved in repair.Test showed that blocking infiltration and activation of macrophage is a serious impediment of wound repair. Shirafuji found that the blocking infiltration of macrophage is an important reason for the impediment of surgical wound healing.The blocking of secretion in macrophage may result in inadequate inflammatory response,thus affecting the healing process.Studies have shown that in the skin tissue macrophage is one of the main cells that secrete VEGFC.In diabetes,the local high glucose circumstance can lead to macrophage dysfunction.When the macrophage can not be activated or the number of macrophage decrease,VEGFC in local tissue may reduce,that may affect the formation of the lymphatic vessels in the period of the inflammation and repair.The activation of lymphatic vessel's formation is controlled by multiple cytokines,involving a series of continuous and orderly steps.These steps are different from the proliferation,budding generation of vascular endothelial cell.The proliferation of lymphatic endothelial cell begins with single cell migration,and then connect into vessel structure.The proliferation of lymphatic endothelial cell is controlled by many cytokines and factors,mainly divided into positive and negative factors.Major positive factors are vascular endothelial growth factor(VEGF) family, hepatocyte growth factor(HGF) family and epidermal growth factor,tumor necrosis factor-α(TNF-α) is a negative factor.VEGF is a member of cystine growth factor superfamily.It is a multifunctional cytokine that specificly operate in vascular endothelial cell.VEGFC is an important member of the VEGF family.VEGFC can make blood vessel and lymphatic vessel proliferate through the combination with VEGFR2 and the VEGFR3.The VEGFC's combination capability with VEGFR3 is much higher than the combination with VEGFR2,so the VEGFC's impact on the lymphatic vessel may be more important than the impact on blood vessel.VEGFC combine with the specific receptor VEGFR3 in lymphatic endothelial cell,thereby stimulate endothelial cell's migration, proliferation and vessel formation.A study found that the lymphatic vessel can transport dendritic cell to inflammatory area during the inflammatory period of wound in the skin.It showed that the lymphatic vessel of the skin was a participant in the inflammatory response, in addition to the local lymphatic drainage and the removal of reaction products. Study also showed that in the skin ulcers of diabetic db/db mice,the activity and number of macrophage that secreted VEGFC declined significantly,which may result in the decrease of the lymphatic vessel's formation,and ultimately caused difficultly healing ulcers.In the study the author also found that the secretion capability of the bone marrow macrophage which was treated with IL-1βwas restored,and can see that the wound healing accelerated significantly dealed with the macrophage of such restored secretion capability,and can see that the formation of lymphatic vessel increase by the detection of laser scanning confocal microscope. We can infer from above that in diabetes the declined number and function of macrophage in the skin tissue may result in decreased generation of local VEGFC, and then can not create enough lymphatic vessels,and ultimately leaded to the lack of inflammation and the delay of wound healing.The pathogeny of diabetic foot ulcers is various,mainly relate to the vascular factor,neurological factors and infection.We found that some wounds with full supply of blood,ABI＞0.7,and no obvious infection,still healed slowly.Does the declined function of macrophage in the diabetic skin tissue result in decreased generation of local VEGFC,and then can not create enough lymphatic vessels,and ultimately leaded to the lack of inflammation and the delay of wound healing? Does the high blood glucose and/or the AGE lead to the declined ability of VEGFC's secretion and phagocytosis in macrophage,and then result in the lack of inflammation and the delay of wound healing.Based on existed research and thinking on above questions,we have adopted a hospital-based case-control study,and selected the skin of diabetic foot non-ischemic ulcer,the skin of non-diabetic foot ulcer,and normal skin for study,and determined the expression level of VEGFC mRNA and protein in the skin of the three groups by RT-PCR and immunohistochemistry techniques,and evaluate the role which VEGFC take in the mechanism that the diabetic foot non-ischemic ulcer is difficult to heal.We also adopted U937 cell for study,and determined the ability of secreting VEGFC and phagocytosis in the U937 cell cultured with different concentrations of glucose or/and AGE,and then provide new experimental data for further prevention and therapy for diabetic foot non-ischemic ulcer.Study was divided into two parts:Part 1 Study on the Expression of VEGFC in Diabetic Foot Non-ischemic UlcersOBJECTIVETo observe the expression characteristics of VEGFC in marginal skin of ulcers in patients with diabetic foot non-ischemic ulcer(DF),which are compared with that in patients with nondiabetic foot ulcer(NDF) and health adults,then evaluate the effectiveness and clinical significance of VEGFC in the mechanism that the diabetic foot non-ischemic ulcer occurs and is difficult to heal.METHODS1.Samples of marginal skin tissue from ten patients with diabetic foot non-ischemic ulcer and ten patients with nondiabetic foot ulcer,with the skin tissue of health adult as control.2.The age,period of ulceration was recorded by patients with ulcer.3.The expression of VEGFC mRNA in the three group were determined with semiquantitative RT-PCR.4.The expression of VEGFC protein in the three group were determined with immunohistochemistry.5.All values are expressed as means±SD.All the data was measurement data. One-way ANOVA was performed using SPSS 13.0 software.Statistical significance was defined as p＜0.05.RESULTS1.There was no signigcant difference in the ages among the three groups(P＞0.05),there was no significant difference in the period of ulceration between the DF and the NDF group(P＞0.05).2.There was significantly less expression of the VEGFC mRNA in the skin tissue of the DF group than that of normal group(P＜0.001).3.There was significantly less expression of the VEGFC protein in the skin tissue of the DF group than that of normal group(P＜0.001).CONCLUSIONSThere was significantly less expression of the VEGFC mRNA and protein in the skin tissue of the DF group.These may correlate to the mechanism of diabetic foot non-ischemic ulcer. Part 2 Study on the Effect of Glucose or/and AGE on U937 CellOBJECTIVETo observe the ability of secreting VEGFC and phagocytosis in the U937 cell after culture with glucose or/and AGE,which are compared with normal control cell. Then evaluate the effect of glucose or/and AGE on U937 cell.METHODS1.U937 cell line were experimental objects.Based on the result of pre-expeirment, the concentration of glucose was 15mM,and the concentration of AGE was 25μg/ml, and the time was 24h.The experiment was divided into four groups:15mM glucose group(GLU group),25μg/ml AGE group(AGE group),co-culture group(CC group),normal control group(NC group).2.Cell activity was determined by trypan blue staining.3.The expression of VEGFC mRNA in the four groups were determined with semiquantitative RT-PCR.4.The expression of VEGFC protein in the four groups were determined with immunofluorescence.5.The ability of phagocytosis was determined by ink phagocytosis test.6.All values are expressed as means±SD.All the data was measurement data. One-way ANOVA was performed using SPSS 13.0 software.Statistical significance was defined as p＜0.05.RESULTS1.Trypan blue staining showed that the cell activity was same in NC group,GLU group,AGE group and CC group(P＞0.05).2.RT-PCR showed that mRNA level of VEGFC in U937 cell in NC group was more than other groups.Compared to NC group independently,mRNA level of VEGFC was significantly decreased in AGE group(P＜0.05),GLU group and CC group (P＜0.001).Compared to CC group,mRNA level of VEGFC was significantly increased in GLU group(P＜0.05) and AGE group(P＜0.001).3.Immunofluorescence showed that protein level of VEGFC in U937 cell in NC group was more than other groups.Compared to NC group independently,protein level of VEGFC was significantly decreased in AGE group,GLU group and CC group(P＜0.001).Compared to CC group,protein level of VEGFC was significantly increased in GLU group and AGE group(P＜0.001).4.Ink phagocytosis test showed that ability of phagocytosis in U937 cell was highest in NC group.Compared to NC group independently,ability of phagocytosis was significantly decreased in AGE group(P＜0.05),GLU group(P＜0.01) and CC group (P＜0.001).Compared to CC group,ability of phagocytosis was significantly increased in GLU group(P＜0.05) and AGE group(P＜0.01).CONCLUSIONS1.MRNA and protein level of VEGFC in U937 cell will be decreased with culture of glucose and AGE.MRNA and protein level of VEGFC in cell will be decreased more with co-culture.2.The ability of phagocytosis in cell will be decreased with culture of glucose and AGE.