| Wound healing is a complex cytological, biochemical and histological process. Diabetic patients are generally vulnerable to skin damage and injury, and often experience delayed or difficulty of wound healing. There are several studies suggest that the impaired wound healing may be related to glucose metabolism disorders and the metabolites of disordered system which in turn resulting in biological behavior change of tissue repair cells, extracellular matrix, and growth factors. Chronic hyperglycemia may lead to pathological and functional change of many body systems, in varying degrees. For the wound healing process, the greatest impact mainly focuses on three aspects. Firstly, the vascular pathological changes: endothelial cell damage is the basis of vascular disorder, Diabetes, hyperglycemia, high insulin, high oxidative stress, and other factors may cause function and structural change of endothelial cell, which in turn trigger vascular damage. Secondly, the diabetic neuropathy, and thirdly, hyperglycemia caused oxidative stress. Currently, more and more research for chronic complications of diabetes is focused on mechanisms of induction of new blood vessel formation, protection of endothelial cell function, and anti-oxidative stress. Previous studies have shown that patients with diabetes mellitus often had EPC number decrease and dysfunction, indicating that impaired EPC may play an important role in the development of diabetic vascular complications. At present time, it has been proved that low dose radiation (LDR) has significant excitatory effect on the proliferation of hematopoietic progenitor cells, can promote bone marrow hematopoietic stem/progenitor cell proliferation, and can induce the mobilization of peripheral blood. However, limited studies were carried out regarding the effect of LDR on diabetes and diabetic complications. In this study, diabetic rat with skin wound model was constructed to mimic refractory diabetic skin ulcers of human beings. Low-dose radiation, zinc sulfate, basic fibroblast growth factor were given individually or in combination to treat the diabetic wounds, and the dynamic healing process and histological characteristics of refractory diabetic wound were monitored. The result of this study may shed light on the healing mechanism of refractory diabetic skin ulcers and may provide a basis for future clinical treatment.The following methods were used in this study:①Skin wounds of each group were taken photos and specimens of the wounds were taken at different time points. Computer image processing system was used to calculate residual wound area and rate of wound healing;②Flow cytometry technology was used to detect the mobilization capacity of peripheral blood endothelial cell and bone marrow endothelial progenitor cell in diabetic rats with skin wound;③Pathological HE staining and Masson's staining were used to detect changes of epithelial process and the collagen content of skin wounds;④Real-time quantitative PCR was used to detect transcription levels of MMP-2, MMP-9, and TIMP-1 during the skin wound healing process;⑤Immunohistochemical methods were used to analyze the protein levels of TIMP-1, MMP-9, PCNA and CD34 during the skin wound healing process;⑥Gelatin zymography was used to detect the activity change of MMP-2, MMP-9 and TIMP-1 during the skin wound healing process.Results of this study showed that:(1) Rate of healing capabilities: non-diabetic skin defect was completely healed in the first 15 days. For the diabetic rats, after 15-day intervention, DM/R group showed dramatic increase of healing area compared with the saline group. The difference was statistically significant (P<0.05). DM/MF group showed statistically significant increase of healing area comparing with DM/R group (P<0.05). The skin wound healing induction capacity of different factors was also analyzed and the results showed that the multi-factor group ranked the highest, followed by DM/R group, DM/B group and DM/Z group, and DM/SA group. This finding indicates that multi-factor in combination and LDR alone could dramatically induce diabetic wound healing process and the effect began in the 5th day. These factors can induce wound healing and shorten the healing time.(2) Peripheral blood flow cytometry results showed that on the 5th day, the number of CD31 positive cells in the saline group, DM/B group, DM/MF group, DM/R Group,DM/Z Group and non-diabetes group were 20.6%, 38.07%,40.19%,22.63%,19.6%,18.48%respectively. The number was significantly higher in DM/R group comparing with DM/SA group, and higher in DM/B group comparing with DM/SA group. With the extension of the healing time, the difference showed a downward trend. In multi-factor group, the number of CD31 positive cells increased in the first 10 days and decreased in the next 15 days. This result suggests that in the early restoration stage, bFGF can induce the endothelial progenitor cells mobilization to the peripheral circulatory system. In the bone marrow, on the 5th day, CD31 positive cells in the DM/SA group,DM/B group, DM/MF group, DM/R Group, DM/Z Group and non-diabetes group were 29.19%,31.8%,62.27%,58.79%,26.69%,37.66%. respectively. On the 5th day, DM/MF group had significantly higher number of CD31 positive cells comparing with DM/R Group (Pvalue<0.05), and number of the positive cells decreased on the 15th day for both of the two groups, indicating that low-dose irradiation play an important role for bone marrow excitement.(3) Results of wound epithelization and collagen content change using pathological HE staining and staining of Masson's showed: on the 5th day of wound healing, no dramatic difference was found for wound healing between diabetes and non-diabetic group. On the 10th day of wound healing, certain sparse, small new collagen in the wound was detected in the saline group, while in the intervention group, the formation of new collagen was denser. On the 15th day of wound healing, epithelialization process was significant in non-diabetic group, with hair follicle structure, a large number of collagen formation, or a margin of most basic healing, a relatively large basement membrane detected. In the saline group there was sparse, small, polar poor epithelial wound. For the DM/MF and DM/R groups, a small number of inflammatory cell infiltration, fibroblast proliferation, a margin of some epithelial cells, and continuous formation of the basement membrane were found.(4) Capillary technology results showed that on the 10th day of wound healing, for non-diabetic group, a large number of capillary proliferation, highly positive CD34 expression, and dramatic increase of PCNA-positive cell number in the hypertrophic region were found. For the diabetes saline group scattered capillaries hyperplasia was detected. The number of microvessel count was largest in the multi-factor group, followed by DM/B group, DM/R group, and DM/Z group. The number of PCNA positive cells was significantly higher in the intervention group comparing with the DM/SA group (P<0.05). The results suggests that both bFGF and LDR can induce the expression of skin CD34+cells and PCNA in the basement membrane cell of diabetic refractory skin, and can activate wound capillary proliferation and tissue repair.(5) The results of transcription level change of MMP-2, MMP-9, TIMP-1 during the healing process using real-time quantitative PCR showed that for the non-diabetic group, MMP-2 and MMP-2 transcription was increased in the on the 5th day, decreased on the 10th day, and dramatically decreased on the 15th day. The transcription was stable for TIMP-1 on the 5th and 10th day and decreased on the 15th day. In the intervention group, for both DM/B and DM/R groups, MMP-9 transcription decreased on the 5th and 10th day, and the decrease was more dramatically on the 15th day. The level of decrease in the two groups were more than that in the saline group and the difference was statistically significant (P<0.05). On the 5th day, MMP-9 mRNA levels in the intervention group was five times higher than that of saline group, MMP-2 mRNA levels in the intervention group was two times higher than that of saline group. The trend was stable on the 10th day, while on the 15th day, the MMP-9 and MMP-2 mRNA levels were lower in the intervention group comparing with saline group.(6) The results of MMP-2, MMP-9, TIMP-1 protein changes using quantitative immunohistochemical analysis showed that, MMP-2, MMP-9, TIMP-1 was expressed in the cytoplasm of wound tissue. For the non-diabetic group, MMP-2 and MMP-9 expression was significantly increased on the 5th day, and decreased dramatically on the 15th day. MMP-2 and MMP-9 expression in the saline group was significantly lower than that of the non-diabetic group (P<0.05). Positive staining density measurement showed that the expression gray value of MMP-2 and MMP-9 was significantly increased on the 5th day in the multi-factor group, DM/R group and DM/B group. The expression was gradually decreased with the extension of healing time. TIMP-1 transcription and TIMP protein level changes are consistent.(7) The results of MMP-2 and MMP-9 activity changes using gelatin zymography approach revealed that on the 5th day, in the non-diabetic group, DM/MF and DM/R groups had dramatically higher MMP-2 and MMP–9 activity comparing with the DM/SA group (P<0.05). This difference was also showed on the 10th day. While on the 15th day, the non-diabetic group and the multi-factor group had decreased MMP-2 and MMP-9 activity, DM/SA group, DM/Z Group, and DM/R Group had increased MMP-2 and MMP-9 activity.Conclusion: 1. A variety of factors including LDR, Zinc, and bFGF can induce healing of skin wounds of diabetic rats, in which, multi-factor and LDR showed dramatic effect.2. In the early stage of wound healing, LDR and bFGF can strongly excite bone marrow and induce endothelial progenitor cells mobilization from bone marrow to peripheral circulation.3. Multiple factors and LDR can accelerate wound healing, induce wound epithelization, and increase collagen synthesis.4. The effect of multiple factor and LDR in healing process of diabetic skin wound may be related to induction of MMP-2, MMP-9 and TIMP-1 expression in the early healing stage.5. LDR can induce CD34 expression in diabetic skin cells and PCNA expression in the basement membrane, which in turn induce wound capillary proliferation and tissue repair.6. Multiple factor may induce wound healing by activate wound cell proliferation in the granulation tissue, increase collagen synthesis and deposition, and enhance the synthesis of cell metabolism.In conclusion, in this study, by constructing a diabetic rats model with skin wounds and using a variety of interference factors to treat the skin wound, we studied the healing induction effect of LDR, including the induction of endothelial progenitor cell proliferation and mobilization mechanism, and the protective effects of LDR in anti-diabetic rats for oxidative damage. The effects of LDR for the EPC excitement in the bone marrow as well as the induction of the EPC mobilization to peripheral blood circulation were also showed. The mechanism of endothelial progenitor cells inducing skin wound partial neovascularization was also described. The important role of bFGF and Zinc in the diabetic skin wound healing was elaborated. The possible synergetic effect of three factors in the treatment of chronic diabetic skin wounds was discussed. Scientific basis was provided to guide the future clinical treatment of diabetic chronic skin wound.
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