| [Background]With the improvement of living standards and the aging of the population, the increasing incidence of diabetes worldwide has brought a great threat to human health and safety. It is predicted that by2025, China, India, America will will have the largest number of people with diabetes, the prevalence rate of diabetes mellitus will reach4.4%, the number of patients will increase from171million in2000to366million in2030. According to a recent survey,50%more than60thousand cases of nontraumatic operation is diabetic patients in the United States each year; patients with nontraumatic amputations due to diabetes is about1/3in Hospital, patients with diabetic foot complicated with infection is70%of all diabetic people in china, the rate of amputation in diabetic patients is40times that of nondiabetic patient.Diabetic wound healing delay had many factors, including peripheral neuropathy, efects in the immune response, periphera1microvascular disease, hemoglobin glycosylation induced transport problems, the changes of erythrocyte, III type and I type skin collagen ratio change, biomechanical properties of diabetic skin changes, fibroblast and keratinocyte migration, proliferation weaken, keratinocytes and endothelial cell apoptosis and so on. In addition, diabetic wound healing delay are closely related to deficiency of growth factor in tissue remodeling, including PDGF, VEGF, NOS, KGF and FGF.Compared with non-diabetic wound, at the cellular level, the ability of proliferating in fibroblasts, keratinocytes and endothelial cell is abate, a decrease of collagen synthesis, angiogenesis and epithelialization delay in diabetic wound, these defects in diabetic wound Combined with infection and osteomyelitis so easily lead to diabetic foot and amputation. Aside from these defective differentiated cells, abnormal stem cells also play a role in stalling wound healing. In a study of rat diabetic wound, it found that epidermal stem cells isolated from diabetic wounds showed decreased clone formation and differentiation ability. Differing from the afore mentioned cells, ADSCs from both diabetic wounds and normal skin secreted identical amounts of growth factors, cytokines, and type I collagen. Diabetic ADSCs even proliferate at the same rate as those from normal wound healing. These characteristics of ADSCs from diabetic wounds render them a potential cell source for wound healing and regeneration. Topical administration of high glucose to wounds of non-diabetic rats resulted in inhibition of the normal angiogenic process. Lack of new blood vessels will limit inflammation cells into tissue, growth factors releasing from inflammatory cells and oxygen supply to the wound will also reduce at the same time. Fergusonf and Delamaire M found that acute inflammatory cells in diabetic wound increased, but neutrophil and macrophage migration ability is insufficient, leukocyte chemotactic ability weakened, which are caused by the metabolic disorder. Therefore, changes in cell level increases the risk of wound infection and insufficient of the vessel. On the molecular mechanism, during wound healing, growth factors influence every phase of wound healing by providing signals for various cellular activities. They are found to be closely related to epidermal cell proliferation, and some of them have been found to play an important role in angiogenesis by modulating endothelial cell migration and proliferation. No matter in animal models or clinical trials with type I and type II diabetic patients, HIF-1and its target genes decreased in diabetic wound, such as VEGF, PDGF, ANGPT, SDF-1and eNOS. Decreased expression of HIF-1and VEGF in lower limb in diabetic patients lead to poor vascular density, poor blood circulation and impaired wound healing, which is responsible for diabetic foot. Yet Maintaining the stable expression of HIF-1by lentivirus transfection or other method can improve the wound microenvironment, promote angiogenesis and accelerate wound healing.Angiogenesis is a key determinant of successful wound closure. Angiogenesis is induced principally by hypoxia. HIF-1holds a key role in mediating and regulating cells response to hypoxia. It has been found to be implicated in the majority of the essential phases of wound angiogenesis, namely endothelial cell activation, migration and proliferation, vascular smooth muscle cell migration and proliferation; bone marrow derived angiogenic cell mobilization, differentiation and proliferation and extracellular matrix (ECM) remodeling, stem cell migration, proliferation and multi-directional differentiation. Recent studies have confirmed that the main mode of angiogenesisis endothelial progenitor cells proliferation from bone marrow. And the VEGF and SDF-1is an important factor of inducing angiogenesisHIF-1is discovered from hypoxia induced liver cancer cell nucleus for the first time by Semenza and Wang in1992. HIF-1is a heterodimer, consisting of two subunits, an O2-liabile a subunit and a constitutively expressed β subunit. HIF-1a protein has a short half-life (t1/2-5min) and is highly regulated by oxygen. In normoxia, the HIF-la proteins are rapidly degraded. During hypoxia, HIF-la becomes stabilized and translocates from the cytoplasm to the nucleus, where it dimerizes with HIF-P, and the HIF complex formed becomes transcriptionally active. The activated HIF complex then associates with HREs in the regulatory regions of target genes and binds the transcriptional coactivators to induce gene expression。Currently, Even though hypoxia is the main regulator of HIF-1, several other factors influence HIF-1expression and function. Some of these factors are involved in diabetes pathogeny, eg:blood glucose concentration, growth factor, cytokines, reactive oxygen species (ROS), advanced glycosylation end products (AGE) and insulin and so on. Changes in gene expression directly or indirectly regulated by HIF-1extend to more than100genes, which are involved in a plethora of adaptation and survival mechanisms, such as angiogenesis, anaerobic glucose metabolism, erythropoiesis, cell growth, differentiation, survival and apoptosis. One of the best-known target genes induced by HIF-1is VEGF, which encodes vascular endothelial growth factor, a potent endothelial cell mitogen that is crucial for the angiogenic process. In addition, HIF-1also induces the expression of the genes for SDF-1and CXCR4, that control adhesion, migration and homing of EPCs, required for the formation of new blood vessels; nitric oxide synthase (NOS), which results in increased levels of NO, which triggers mobilisation of bone marrow endothelial progenitor cells (EPCs) into the circulation.It is found that vascular endothelial growth factor (VEGF) also known as vascular permeability factor (VPF), is the most powerful and specified factor to stimulate endothelial cell proliferation, besides, it also increases vascular permeability, mobilize vascular endothelial cell. In mammals, the VEGF family including VEGF-A (often referred to as VEGF), VEGF-B, VEGF-C, VEGF-D and placenta growth factor (PLGF). So far, it has found3kinds of VEGF receptor (VEGFRs):VEGFR-1, VEGFR-2, VEGFR-3. VEGFR-2is mainly distributed in the vascular endothelial cells, macrophages, and hematopoietic stem cells. Its main role is to mediate endothelial cell proliferation, chemotaxis of endothelial cells and increased vascular permeability function. Therefore, VEGFR-2is the main functional receptor of VEGF.Stromal cell derived factor1(SDF-1, namely CXCL12) is firstly cloned from the bone marrow in rodents, as part of the CXC chemokines. SDF gene can code two proteins in vivo, namely SDF-1α and SDF-1β. The SDF-la gene is expressed in bone marrow stromal cells.CXCR4is widely expressed in leucocytes, CD34+HSC and CD34+progenitor cells. Initially, CXCR4is regarded as a unique SDF-1receptor and the specific combination of SDF-1and CXCR4, is named as SDF-1/CXCR4biology axis.When SDF-1combined with CXCR4, it mediated its many biological behavior through the activation of various signal transduction pathways such as calcium traffic overload, PI3K-AKT-NF-KB axis activation, phosphorylation of MAPK. The G protein dependent signal transduction pathway can activate ephosphatidylinositol-3kinase (PI3K), that can activate multiple signaling molecules, of which PI3K-AKT is the most important. Effect of PI3K-AKT signal pathway in general is anti-apoptosis and promoting the growth and proliferation of cell, including cell motility, chemotaxis and adhesion.A paracrine loop is formed between SDF-1/CXCR4and VEGF, that strengthens mutually their own biological function. SDF-1can promote VEGF formation, at the same time VEGF can promote expression upregulation of vascular endothelial cells, thereby strengthening the response to vascular endothelial cells on SDF-1. Experiment in tumor proved that SDF-1and VEGF can promote the formation of certain synergistic effect of tumor angiogenesis.Therefore, in the process of angiogenesis, SDF-1not only can promote EPCs mobilization home to wound, but also induce EPCs to differentiate into mature endothelial cells in synergy with VEGF, formatting vascular network structure.PI3K/Akt widely exists in cytoplasm triggered by cytokines. It mainly is involved in the signal transduction pathways of cell differentiation and anti-apoptosis. Oxygen activates of nuclear factor kappa B through PI3K/Akt signal channel. Its subunits can combine with HIF-1alpha, that results in increasing HIF-1alpha mRNA expression. The increased signaling of PKC and PI3K/AKT in turn is involved in the protection of HIF-1a from pVHL-independent degradation, and leads to the upregulation of HIF-1a, which was demonstrated via the use of chemical inhibitors for both pathways. In addition, It has been confirmed that PI3K/Akt increased the activity of HIF-1in hepatoma cells, breast cancer cells. Li Lihua found that hypoxic-ischemia in neurons could activate PI3K/Akt signaling pathway, induce phosphorylation of Akt, and then promote the expression of HIF-la protein, It may treat neonatal hypoxic-ischemic encephalopathy by maintaining the HIF-1alpha activity.Above all, wound healing is closely related to expression and activity of the HIF-1and its target gened. But it has not reported that the expression of the HIF-la and target genes in the process of diabetic wound healing and be used for wound healing in china. The experiment aims to elucidate the mechanism of impaired diabetic wound from the aspects of angiogenesis are in diabetes, and provide new ideas for clinical treatment of diabetic chronic wound by observing the wound healing rate in different time, microvessel density, change of Akt, HIF-1a, VEGF, VEGFR2, SDF-1a and CXCR4gene and protein level in wound tissue with in BALB/c normal and diabetic wound as the research object from april,2013to December,2013.[Objective]To investigate wound healing rate, microvascular expression, the expression profile and possible role of Akt、HIF-1a、VEGF、VEGFR2、SDF-1α and CXCR4mRNA and protein during full thickness cutaneous wound healing in BALB/c mice, To clarify mechanism of diabetic wound refractory mechanism.[Methods]Sixty mice (aged4weeks) were divided into2groups randomly,30mice in each group, control group and diabetic model group. The mice in the experimental group was injected intraperitoneally (i.p.) with a low dose of STZ (40mg/kg), while the mice in control group were injected with sodium citrate buffer (pH4.4) only. Mice were treated with five consecutive daily injections. The mice with the non-fasting plasma glucose of≥16.7mmol/l were considered as diabetes and selected for further studies. Four weeks after the STZ injection, A full thickness wound of6mm in size was inflicted on the back of mice. The skin specimen on the edge of wound was collected on0、3、7、10d after injury respectively, calculating wound healing rate and CD31expression by immunohistochemical staining. The endogenous expressiones of Akt、HIF-1a、VEGF、VEGFR2、SDF-1α and CXCR4mRNA and protein were measured by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis in the obtained specimen.[Results]During the process of wound healing, the wound healing of diabetic mice is significantly less than normal mice (P<0.05), wound healing rate of3,7,10days respectively was7.0±5.8,38.7±6.0and6.0±3.0(%). Expression of HIF-1mRNA in diabetic mice increase significantly after trauma, expresse the highest amount on10th, but is lower than that of non-diabetic group. Expression of VEGF and SDF-1increase after trauma,7days express the highest amount, general expression level was lower; Akt, VEGFR2and CXCR4mRNA expression increase after trauma, the highest amount is lower than the control group. The expression of a variety of objective protein after trauma was significantly higher than that of pre trauma (P<0.05), basically it reached the peak on third or seventh day, then decreased to the level before injury; as a whole it found protein expression in diabetic wound was lower than that in normal mice (P<0.05). A variety of purposes protein expression is significantly higher3,7days after trauma of mice than before trauma (P<0.05), which is basically expression of peak on3and7days, then fell to the level before trauma; Expression level of HIF-1a, Akt in diabetic group is lower than the normal mice, with statistically significant (P<0.05); all purpose protein expression level is lower than normal group (except VEGFR2) on the3rd day after trauma; all purpose protein expression level is lower than normal group on the7th day; protein expression level of VEGF, SDF-1and CXCR4is lower than normal group on the10th day, with statistical significance (P<0.05).[Conclusion]The Akt, HIF-la, VEGF, VEGFR2, SDF-la and CXCR4expressions are impaired during diabetic wound healing, resulting in insufficient angiogenesis, which may be a reason for the delay in diabetic wound healing. |
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