Study On The Effect And Mechanism Of Ozone In Patients With Diabetic Foot

BackgroundDiabetes is a chronic disease which causes serious damage to the human health and the incidence rate of the disease increase gradually on a global scale. Wild S once anticipated that by the year of2030incidence rate of diabetes would reach4.4%worldwide and the figure of diabetic was expected to reach366million. World health organization and the international diabetes federation predicted that the people living with diabetes would double in the next15years, with a larger incidence happening in developing countries. And the incidence of diabetes in China increased alarmingly. Professor Yang summarized and analyzed the survey data on the Chinese diabetes epidemic between2007and2008, the results showed that the incidence of diabetes among adult reached9.7%, pre-diabetes incidence was15.5%, about a quarter of adults in China was in a state of abnormal blood sugar, the incidence of diabetes increased by4.2%than the period between2000-2001. With increasing of diabetics, diabetes-related chronic vascular complications are expected to increase as well and this has become the main cause of death and disability in diabetes patients.Diabetic foot is one of the serious complications of diabetes. The definition of diabetic foot, which has been in common use internationally, was first put forward by Oakley in1956. In1999the World Health Organization (WHO) gave its own definition:Diabetic foot is an umbrella term for foot problems in patients with diabetes mellitus. Due to arterial abnormalities and diabetic neuropathy, as well as a tendency to delayed wound healing, infection or gangrene of the foot is relatively common. As the incidence of diabetes increased year by year for the past few years, in our country, there are12%to25%of patients with diabetes complicated with diabetic foot which seriously affects patients’quality of life, and even endanger the patients’lives. Epidemiological data indicate that diabetic foot accounted for about4%-10%of patients with diabetes, diabetes is now the one of the leading causes of disability and fatality. According to the survey statistics that50%of patients with amputation surgery every year all over the world are diabetic foot patients. Foot ulcers probability among patients with diabetes during their lifetime is15%, and as patients grow older, course of disease has a tendency to extend.85%of patient with foot ulcers finally need amputations. Case fatality rate among diabetic1year after the amputation is1%-41%,3years is20%-20%,5years is39%-69%. It is thus clear that amputation will severely affect patients’long-term survival rate. Diabetic foot has long disease course and is difficult to cure. The psychological and economic burden of diabetics is higher. In addition, disability and fatality rate of this disease is higher, so it is a huge threat to people with diabetes. Clinically, the wound of diabetic foot is different from that of common chronic diseases. Diabetic foot is a chronic disease and hard to treat, which brings great pains to the patients both physically and mentally and also causes serious burden to the family and society.Therefore, the treatment of the wound of diabetic foot appears particularly important.Wound healing is a complicated process of regeneration and repair of damaged tissue, its main feature is the formation of granulation tissue and scar tissue. New vessels are the necessary condition for wound healing, for they are not only the main component of the granulation tissue but also provide the necessary nutrients for tissue repair. The time needed for wound repair is different and the repair speed depends on the number of repair cells that can enter into the wound and their proliferation rate. While chemokines and growth factors play an important role during the entry and proliferation of repair cells. In addition, growth factors involved in every stage of wound healing. The problem of wound healing has troubled clinician for a long time, but people also have done a lot of research on the mechanism of wound healing, and so far it has confirmed that VEGF PDGF and TGF beta has very close relationship with the mechanism of wound healing. VEGF is a kind of specific cytokines with multi-functions, acting on specific receptors in the vascular endothelial cell, thereby promoting the proliferation and migration of the cell and inducing the formation of new blood vessels. In addition, it also can increase vascular permeability, change the components of extracellular matrix, provide the environment for angiogenesis, also provide the material basis for fibroblasts collagen synthesis, thus it is conducive to the formation of granulation tissue and scar tissue and promote wound healing.In the body, PDGF has chemotaxis on neutrophils, monocytes and fibroblasts, which can make these cells migrate into the wound tissue; PDGF also is mitogen of fibroblasts, which can induce regeneration of fibroblast and increase the amount of fibroblasts in granulation tissues; In the body, PDGF can also activate macrophages, then make them compound VEGF, further promoting angiogenesis. The chemotaxis, activation and induction functions of PDGF towards different cells are conducive to wound tissue repair and to accelerate wound healing. So far, TGF-β are generally accepted as important cytokines of Fibrosis, which can promote the phenotype transformation of fibroblasts. In the body, there are three types of TGF-β, among which the TGF-β1is closely associated with cicatrix. TGF-β1stimulates the growth and differentiation of fibroblast, thus make them proliferate; It also induces other growth factors to gather together in the wound, and then makes the monocytes drawn to the wound, and turned into the macrophages. Through epithelial cells, endothelial cells and lymphocytes, the growth factor also promotes the proliferation of mesenchymal cells. Studies have found that TGF-β1can maintain large amount of collagen deposition in tissue by means of cell generation and keeping the stability of the cell phenotype [8], meanwhile it can stimulates fibroblast cells to produce large amounts of growth factor of connective tissue, and then promote the formation of cicatrix. Further more, TGF-β1has chemotaxis in endothelial cell to a certain extent, which can induce the formation of new vessels, and then promote wound healing [10].In high sugar environment, cells are damaged, the secretion of growth factors decrease, and their coupling effect with the corresponding receptor drops, which is not conducive to the formation of granulation tissue and cicatrix, and then will affects wound healing. Moreover, in high sugar environment, nutrients exchange is difficult, which is unfavorable to the formation of new blood vessels, and then it is hard to give plenty of nutrients in tissue repair process. Therefore, the formation of granulation tissue is less, the density of microvascular is low, and the wound healing of diabetic foot is slower than normal wound. Studies have confirmed that the reduce of growth factors and their receptors in the wound is an important reason for refractory wound healing of diabetic foot, and partly application of activated growth factors can promote wound healing. Some scholars have studied the relationship between the diabetic foot and VEGF, the results showed that VEGF gene expression in wound tissue of diabetic foot is significantly less than that of normal control or non-diabetic, suggesting that angiogenesis in wound of diabetic foot is affected, and then affects the healing of wound. The wounds of Diabetic foot are different from common chronic wounds. They are chronic and refractory and caused by complex factors, therefore, the treatment of diabetic foot wound appears especially important. In the process of exploring solutions to treat diabetic foot, some European countries developed ozone therapy.Ozone was first discovered by Van Marum in1785, which is a kind of strong oxidizer and is made up of three oxygen atoms. The half-life of ozone is about20minutes under normal temperature, easy to decompose, soluble in water. Studies have found that for patients with limb ischemia, after treatment with ozone, their platelet release large amounts of PDGF and TGF-β1. Another study found that in fibroblasts exposed to ozone environment, mRNA which is related to TGF-β1is in a higher level of steady state. And hydrogen peroxide can induce the expression of VEGF in keratin cells and promote the healing of wounds.To sum up, ozone has certain effect in treating wounds and ulcers, and cell growth factors(VEGF TGF-beta PDGF, etc.) play an important role in wound healing. Ozone can promote the wound healing of diabetic foot. Whether VEGF、TGF-β、 PDGF also participate in that process will be discussed in this paper.[Objectives]1, Understand the effects of ozone treatment on wound healing of diabetic foot.2, Discusses the effect of ozone treatment on the expression change of VEGF, TGF-β, PDGF of diabetic foot.[Methods]The research was conducted between March2012and January2013in endocrinology department, Nanfang Hospital, Southern Medical University. First, the research chose50diabetic foot patients that conformed to the diagnostic criteria. Second, through random grouping, the test patients were divided into two groups: conventional group and ozone group, each group of25cases. There should not have obvious difference in patients’age, gender, course of the disease, illness, etc between the two groups. The patients in both groups received comprehensive treatment: regular treatment, control blood sugar, improve circulation and nerve nutrition. The research regarded20days as a period of treatment to observe the clinical curative effect and healing condition of local wound. Other than regular group, ozone group also receive ozone treatment.(1)Take secretion from wound and make use of enzyme-linked immunosorbent method to examine the expression of VEGF, TGF-β, PDGF in local tissue respectively before the treatment and3,7,11days after treatment.(2) Select10patients and divided them into conventional group and ozone group by random grouping, each group of10cases. Take secretion from wound and make use of immunochemistry and enzyme-linked immunosorbent method to examine the expression of VEGF, TGF-β, PDGF in local tissue, use masson staining and HE staining methods to observe the histopathologic changes and collagen expression respectively before treatment and11days after treatment.[Results]1.Curative effect comparison of the two groups after treatment:the ozone group:6cases healed,12cases were markedly effective,5cases were effective,2had no effect, total effective rate92.000%; conventional group:3cases healed,7cases were markedly effective,6cases were effective,9cases were ineffective, the total effective rate was64.000%. Between conventional group and ozone group, treatment effects have significant differences, suggesting that the curative effect of ozone group is better than that of conventional group (P<0.05).2.Conditions of the wound area before and after treatment between the conventional group and the ozone group:first, intra-group comparison, ulcer area of the two groups after treatment is significant narrowing than that before treatment,(the conventional group was10.824±4.642,7.637±4.900; the ozone group was11.742±3.607,4.902±3.844(P<0.05); second, comparison between the two groups, curative effect on ulcer healing of ozone group was better than conventional group (P<0.05).3.The change of VEGF, TGF-β,PDGF in secretion of local wound:the expression of VEGF, TGF-β,PDGF in the two groups has no significantly difference (P>0.05) before treatment; concentration of PDGF, VEGF, TGF-β in the two groups began to rise3days after treatment, the concentration of VEGF reached peak on seventh day, began to drop on11th day, TGF-β,PDGF reached peak on11th day. The concentration of them all were significantly higher than that before treatment (P<0.05), showing that two groups can have an effect on local wound of diabetic foot. There were significant differences in the quantitative values of VEGF, TGF-β, PDGF between the two groups (P<0.05), suggesting the ozone group had greater effect on the local wound of diabetic foot than the conventional group.4.The changes of VEGF, TGF-β, PDGF in local tissue:Before the treatment, the expression of growth factors (VEGF, TGF-β, PDGF) of the two groups had no significant difference (P>0.05); The expression of growth factors (VEGF, TGF-β, PDGF) of local tissue were significantly increased (P<0.05) after the treatment, showing that two groups can have an effect on local wound of diabetic foot. The difference of the expression of growth factors (VEGF, TGF-β, PDGF) between the two groups after the treatment (P<0.05), suggested the ozone group had a better effect on the local wound of diabetic foot than the conventional group.5.The changes of collagen fibers in local tissue (masson staining):By means of masson staining, the increase of blue collagen fibers in ozone lgroup in greater than that in the ozone group after treatment. That was significant difference (P<0.05).6.Pathological changes (HE staining) of local tissue:fibroblasts increased significantly in the two groups after treatment, and skin tissues in the ozone group gradually returned to normal after treatment. [Conclusion]Ozone treatment can promote wound repair of diabetic foot, increase the expression of VEGF, TGF-β, PDGF in local tissue, boost the expression of collagenous fiber, accelerate the healing of ulcer, improve the cure rate of diabetic foot ulcers and reduce the fatality and disability rate. It has achieved the good curative effect. Furthermore, the application of this treatment is simple, with no obvious side effects. Ozone treatment has certain clinical advantage for the treatment of diabetic foot.