The Effects And Mechanism Of 1,25（OH）₂D₃ On Wound Healing Of Diabetic Ulcer

Background:Diabetic foot ulcer,a serious complication of diabetes,is the main cause of non-traumatic amputation and death among diabetic patients.At present,the effect of current clinical conventional treatment is limited.Vitamin D is an essential nutrient for the human body.In recent years,its non-classical effects besides the regulation of calcium and phosphorus metabolism attracted wide attention.It has been reported that vitamin D exerts a regulatory effect on immune disorders,inflammation,and infection,which play a role in diabetic foot ulcer.Therefore,it is of great scientific significance to explore the effects and underlying mechanism of vitamin D on diabetic ulcer wound healing,which can provide theoretical basis for the prevention and treatment of diabetic foot ulcer.Objective:This study aimed to investigate the effects of 1,25(OH)2D3 supplementation on wound healing of diabetic ulcer by vivo and vitro experiments and further explore the possible role of angiogenesis,so as to provide evidence for the treatment of diabetic foot ulcer with vitamin D.Methods:This study constituted of the following three parts:(1)1,25(OH)2D3 intervention improved impaired wound healing in diabetic ulcer model mice.Male C57BL/6J mice aged 3-5 weeks were randomly divided into control group(CON group)and diabetic modeling group.A mouse model of diabetes was established by 8-week high-fat diet combined with intraperitoneal injection of streptozotocin(STZ).At the 9th week,the diabetic model mice were divided into diabetic ulcer control group(DM group),low-dose 1,25(OH)2D3 group[LVD group,1,25(OH)2D3 dose:500 ng/kg]and high-dose 1,25(OH)2D3 group[HVD group,1,25(OH)2D3 dose:1000 ng/kg].The diabetic ulcer model was established at the 13th week.Then,mice in the LVD group and the HVD group were intraperitoneally injected with corresponding doses of 1,25(OH)2D3 every day.The wounds were photographed daily to measure the wound area.Mice were sacrificed on the 4th,7th,and 14th day of wound healing to collect the skin wound tissue samples.The following indicators were measured:1)Random blood glucose(RBG),fasting blood glucose(FBG),body weight and 24-hour water intake were monitored regularly.2)The wound closure was evaluated by measuring wound healing area and calculating daily wound healing rate.3)H&E staining and Masson staining were used to examine histological changes,collagen fibers,and matrix deposition to evaluate wound healing condition.4)The expression levels of vitamin D receptor(VDR)and receptor for advanced glycation end products(RAGE)in skin wound tissue samples were detected by Western blot and RT-qPCR.(2)The effects of 1,25(OH)2D3 intervention on different stages of wound healing in diabetic ulcer model mice.To further explore the effects of 1,25(OH)2D3 intervention on inflammation,vascular endothelial function,angiogenesis,and vascular maturation in the process of wound healing in diabetic ulcer model mice,the cutaneous wound samples were collected and the following indicators were detected:1)Inflammation:The samples on the 4th day of wound healing were used for the following experiments.First,the infiltration of inflammatory cells was assessed by H&E staining.Subsequently,the protein expression levels of CD68 and CD206 were evaluated by immunohistochemical method.Finally,RT-qPCR was performed to determine the expression levels of M1 and M2 macrophages-related cytokines in skin wound tissue samples.M1 macrophages-related cytokines include inducible nitric oxide synthase(iNOS),interleukin 1β(IL-1β)and tumor necrosis factor-α(TNF-α),and M2 macrophages-related cytokines include arginase 1(Arg1)and interleukin 10(IL-10).2)Vascular endothelial function:Immunohistochemistry,Western blot,or RTqPCR were conducted to detect the expression levels of endothelial nitric oxide synthase(eNOS),endothelin-1(ET-1)and vascular cell adhesion molecule-1(VCAM-1)in skin wound tissue samples on the 7th day.3)Angiogenesis:The expression level of angiogenesis marker CD31 in skin wound tissue samples on the 7th day was examined by immunohistochemistry.Then,Western blot or RT-qPCR was used to detect the expression levels of vascular endothelial growth factor(VEGF),vascular endothelial growth factor receptor 2(VEGFR2),platelet derived growth factor(PDGF),platelet derived growth factor receptor β(PDGFRβ),basic fibroblast growth factor(bFGF),epidermal growth factor receptor(EGFR),and hypoxia-inducible factor 1α(HIF-1α)in skin wound tissue of the 7th and 14th days of wound healing.4)Vascular maturation:The expression level of vascular maturation marker αsmooth muscle actin(α-SMA)in skin wound tissue on the 14th day of wound healing was measured by immunohistochemistry.RT-qPCR was used to analyze the expression levels of angiopoietin 1(Ang1)and angiopoietin receptor tyrosine protein kinase receptor 2(Tie2)in skin wound tissue on the 14th day of wound healing.(3)The effects of 1,25(OH)2D3 on angiogenesis in high glucose-induced HUVECs injury model.Human umbilical vein endothelial cells(HUVECs)were treated with high glucose(glucose concentration:33.3 mM)for different time(24 h,48 h,72 h).The cell morphology was observed by the microscope and the cell proliferation ability was detected by CCK-8 method to determine the effect of high glucose treatment.High glucose-induced HUVECs injury models were treated with 1,25(OH)2D3 for 48 hours,and then Western blot was used to examine the expression level of VDR.To explore the effects of 1,25(OH)2D3 on the angiogenesis of high glucose-induced HUVECs injury models and the underlying mechanism,the following experiments were conducted:1)Cell proliferation,migration and apoptosis were detected by CCK-8 assay,wound healing assay,Transwell assay,and Hoechst apoptosis fluorescence staining respectively.2)Tube formation assay was used to determine the changes in the tube formation ability of HUVECs.Western Blot or RT-qPCR was used to detect the expression levels of angiogenic factors,including VEGF/VEGFR2,PDGF/PDGFRβ,bFGF and EGFR.3)The Protein expression of Phosphatidylinositol-3-kinases(PI3K)/Proteinserine-threonine kinase(AKT)/HIF-1α pathway related markers were examined by Western Blot to evaluate the phosphorylation level of AKT.Results:(1)1,25(OH)2D3 intervention improved impaired wound healing in diabetic ulcer model mice.After high-fat diet combined with STZ treatment,compared with the CON group,the body weight of the diabetic model group showed a trend of decline,while 24-hour water intake,RBG and FBG were significantly increased.The RBG reached the standard of diabetic modeling and maintained at a high level at the following experimental period.The results showed that 1,25(OH)2D3 intervention could improve the impaired wound healing of diabetic ulcer model mice and increase the wound healing rate.The histomorphology results indicated that 1,25(OH)2D3 alleviated the excessive infiltration of inflammatory cells in diabetic wounds,promoted the proliferation of granulation tissue,and accelerated the formation of subcutaneous appendage and collagen fiber deposition in wounds.In addition,1,25(OH)2D3 intervention significantly up-regulated the expression level of VDR and inhibited the expression level of RAGE in skin wound samples on the 4th,7th and 14th days of wound healing(P<0.05).These results suggested that 1,25(OH)2D3 intervention could improve wound healing in diabetic ulcer mice,which might be related to the up-regulation of VDR expression and the inhibition of RAGE expression.(2)The effects of 1,25(OH)2D3 intervention on different stages of wound healing in diabetic ulcer model mice.For the inflammatory phase,the skin samples on 4th day of wound healing were selected for analysis.The results showed that 1,25(OH)2D3 intervention could alleviate the excessive infiltration of inflammatory cells in diabetic wounds and significantly reduce the number of CD68-positive cells in diabetic ulcer model mice(P<0.05).In addition,compared with the DM group,the expression levels of Inos,Il-1β,and Tnf-αin the LVD and the HVD groups were significantly decreased(P<0.05).Arg1 and Il-10 expression levels were in an upward trend,while there was no significant difference between the LVD and the HVD groups with the CON group.For vascular endothelial function,the results indicated that on the 7th day of wound healing,compared with the CON group,the protein expression of eNOS was significantly inhibited and the mRNA expression of Vcam-1 was up-regulated in the DM group,while 1,25(OH)2D3 could significantly increase the protein expression of eNOS and suppress the mRNA expression of Vcam-1(P<0.05).As for the angiogenesis,1,25(OH)2D3 intervention could significantly increase the expression level of angiogenesis marker CD31 and angiogenic related factors(VEGF,VEGFR2,PDGF,and PDGFRβ)on the 7th day of wound healing(P<0.05).On the 14th day of wound healing,the protein expression level of PDGF and the mRNA expression levels of Bfgf and Egfr in the LVD and the HVD groups were still significantly higher than those in the DM group(P<0.05),but VEGF expression level in the DM group increased significantly and was higher than that in the CON group(P<0.05).The differences in the expression levels of angiogenic related factors among the groups were gradually reduced.In addition,on the 7th and 14th day of wound healing,the expression of HIF-1α in the DM group was significantly lower than that in the CON group,while 1,25(OH)2D3 intervention significantly up-regulated the expression levels of HIF1α(P<0.05).Regarding the vascular maturation,the results showed that there were no significant differences in the protein expression of vascular maturation marker a-SMA and the mRNA expression levels of Ang1 and Tie2 among the groups on the 14th day of wound healing.These results indicated that 1,25(OH)2D3 intervention may accelerate the wound healing of diabetic ulcer by alleviating excessive inflammation,restoring vascular endothelial dysfunction,and promoting angiogenesis.HIF-1α may play an important role in the process of promoting angiogenesis.(3)The effects of 1,25(OH)2D3 on angiogenesis in high glucose-induced HUVECs injury model.HUVECs were treated with 33.3 mM glucose to establish high glucose-induced injury model to simulate the damage of vascular endothelial cells under the condition of diabetes.Under the microscope,the morphology of HUVECs was deformed under the high glucose background,and the cell proliferation ability was significantly decreased after treatment with 33.3 mM glucose for 48 h(P<0.05).Therefore,the high glucose treatment time was set at 48 h in this study.The protein expression level of VDR in high glucose-induced HUVECs injury model was significantly up-regulated after treatment with 100 nM 1,25(OH)2D3 for 48 h(P<0.05).The results showed that 1,25(OH)2D3 significantly promoted the proliferation and migration of HUVECs under high glucose condition(P<0.05),but had no significant effect on cell apoptosis.Tube formation assay showed that 1,25(OH)2D3 improved the tube formation ability of HUVECs treated with high glucose by increasing the total length of tubule formation,the number of branch points,and the number of tube formation(P<0.05).Moreover,the protein expression levels of VEGFR2 and PDGFRβand the mRNA expression levels of VEGFR2,PDGF and bFGF were significantly increased after 1,25(OH)2D3 intervention(P<0.05).For PI3K/AKT/HIF-1α pathway,the results demonstrated that the phosphorylation level of AKT was significantly decreased under high glucose condition.After the intervention of 1,25(OH)2D3,the phosphorylation level of AKT,the protein and mRNA expression levels of HIF-1α were significantly increased(P<0.05).These results suggested that 1,25(OH)2D3 intervention restored the impaired proliferation,migration,tube formation and angiogenesis of high glucose-induced HUVECs injury model,and this may be related to the phosphorylation activation of PI3K/AKT/HIF-1α pathway.Conclusions:(1)1,25(OH)2D3 could improve wound healing in diabetic ulcer mice by alleviating excessive inflammation,restoring vascular endothelial dysfunction,and promoting angiogenesis.(2)1,25(OH)2D3 might promote the impaired proliferation,migration,tube formation,and angiogenesis of high glucose-induced HUVECs injury model through activating the phosphorylation of PI3K/AKT/HIF-1α pathway.