| BackgroundAs population aging, the incidence of diabetes increases gradually, and the population of diabetic skin ulcer such as diabetic foot also increases accordingly. There are many difficulties in the treatment for diabetic skin ulcer clinically, because of its complex factors involved, including diabetes vascular lesion with poor blood supply, nerve paralysis, easy infection due to changes of the wound microenvironment, and hypofunction of various cells and cytokines involved in wound healing, ect. Therefore, it has become a hot topic to study the mechanism of diabetic skin lesion and its refractory wound healing for scholars.Integrin-linked kinase(ILK) is the key kinase in the integrin signaling pathway, and it plays an important role in modulating the cell growth, proliferation, apoptosis, survival, differentiation, migration, invasion and angiogenesis. Previous studies have demonstrated that ILK not only plays an essential role in maintaining normal structure and morphogenesis of skin, but also in wound healing by promoting proliferation of fibroblasts, inhibiting cell apoptosis, inducing the differentiation of myofibroblasts to accelerate wound contraction, and promoting the proliferation and migration of keratinocytes and differentiation of hair follicle epidermal stem cells to accelerate the wound reepithelializaiton.The relative researches between ILK and diabetes mainly focused on diabetic nephropathy and retinopathy currently, the expression of ILK in diabetic skin and its role in diabetic skin wound healing have not been reported yet. Therefore we tried to explore the role of ILK in diabetic skin lesion and its wound healing by establishing diabetes animal modesl in rats and investigating the expression of ILK and its downstream effectors such as AKT/GSK 3β in diabetic skin tissues and wound tissues. PARTⅠ Preparation of diabetic rat’s modelObjective To establish a stable and reliable diabetic rat’s model.Method Thirty-six SD rats were fed for one week, then forbidden to eat overnight(more than 12 hours) with free drinking. Next day the rats in diabetes group was injected intraperitoneally using 1% streptozocin with a dose of 60mg/kg, meanwhile the rats in normal group was injected intraperitoneally using the same amount of citrate buffer solution. Seventy two hours afterwards, random blood glucose levels of all rats were checked, and the standard of diabetic mellitus was that blood sugar level>16.7mmol/L. Two weeks later, the rats’ wound models were established with a full-thickness skin excision 2cm×2cm in size.Results After injecting streptozocin, the rats suffered a typical symptom of diabetics including excessive urine, excessive drink, excessive food, weight loss, sparse hair and dimmed skin, and their blood sugar levels increased to 28.91±9.33 mmol/L. However, the blood sugar levels of rats in normal group were 6.538±0.62mmol/L stably, and their weight increased gradually. After establishing the rat’s wound models, the diabetic rats all went though the experimental observation and obtaining tissues, and no rats died halfway. The diabetic rats suffered impaired wound healing. The healing time in diabetic rats was 30.35±2.51 days, which was much longer than that in normal rats(21.47±1.94 days).Conclusion The method of establishing diabetic rat’ wound model using a single intraperitoneal injection of 1% streptozocin with a dose of 60 mg/kg has the advantages of high success rate, high stability and low mortality. PARTⅡ The expression of ILK and its downstream effectors in skin tissues of diabetic ratsObjective To explore the role of ILK and its downstream effectors in diabetic skin lesion by detecting their expressions in skin tissues of diabetic rats.Method The rats were divided into two groups, including normal group(N=18 rats) and diabetic group(D=18 rats). PCR was used to detect the ILK, AKT and GSK-3β m RNA levels of skin tissues. Western blot was applied to detect the protein levels of ILK and its downstream effectors. Sections with HE staining was used to detect the pathological alteration.Results The thickness of skin and epidermis in diabetic rats decreased gradually with time prolonging. PCR results showed that the m RNA levels of ILK and AKT from diabetic rats were significantly lower than those in normal rats(t values were respectively 4.779 and 3.4403, P<0.05), but their GSK-3β m RNA levels had no statistical difference(t=0.363, P>0.05). Western blot results revealed the protein levels of ILK, AKT and p-AKT from diabetic rats were significantly lower than those in normal rats(t values were respectively 2.947,6.209 and 2.63, P<0.05). Although the GSK-3β protein levels of these two groups had no statistical difference(t=0.652,P>0.05), but p-GSK-3β protein levels of diabetic rats were significantly higher than those in normal rats(t=4.131,P<0.05).Conclusion The skin lesion of diabetic rat may be related to the changes of expression of ILK and its downstream effectors. PART Ⅲ The expression of ILK/p AKT in wound tissues of diabetic rats during wound healingObjective To explore the role of ILK/p AKT pathway in wound healing by observing the expressions of ILK/p AKT in wound tissues of diabetic rats.Method The rats were divided into two groups, including normal group(N=18 rats) and diabetic group(D=18 rats). Two weeks after establishing the diabetes rats’ models, the wound models were established with a full-thickness skin excision 2cm×2cm in size. The wound tissues from normal and diabetic rats were harvested at day 3, 7, 10, 14 and 21 after surgery. The wound healing rates were calculated during wound healing with photography in different time points. Western blot was applied to detect the protein levels of ILK and p-AKT in wound tissues.Results The wound healing rates of diabetic rats were much less than those of normal rats. In the process of wound healing, there were no statistical differences in protein levels of ILK between wound tissues and skin tissues among each group, and also no differences among wound tissues in different points of time from each group. The p-AKT protein levels of these two groups both presented an increasing tendency in the early phase, and then falling to almost normal level. The p-AKT protein levels in wound tissues were significantly higher than those in skin tissues in these two groups(t values range from 4.450 to 22.566,P<0.01), except for at day 14 from diabetic rats. Besides, the p-AKT protein levels in wound tissues of normal rats were significantly higher than those in diabetic rats except for day 3(t values range from 4.091 to 20.555, P<0.05). Conclusion The refractory wound healing in diabetic rats may be related to the reduction of p-AKT protein level in wound tissues. |
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