The Influence Of NO Mixture On Wound Healing Of Diabetes Mellitus (DM) Mice

ObjectiveDiabetes Mellitus (DM) is a chronic metabolic disease. Wound healing impairments are the most common complications in DM. To promote wound repair, local administration would be optimal. However, even with thousands of methods, satisfactory effect can be hardly obtained. Our research was based on the NO's important role in wound healing, by monitoring microenvironment, aiming to provide a new local drug therapy: Nitric oxide generator L - arginine, sodium nitrate, shevchenko and oxygen anion inhibitors oleander hemp meat. We establish a STZ induced type I diabetes and trauma refractory model in mice, and administrated the mixture to the wounds , wound healing time, granulation tissue growth and proliferation of epidermal cells were measured . Immunohistochemistry was applied to locate TGF-β1 expression and its relation with angiogenesis. Through the microscope observation at different time points we measure fibroblasts quantity and collagen fiber content. By researching on wound healing NO generating mixture, and discussing its mechanism, we are looking forward to providing more theoretical evidence for clinical treatment for diabetes refractory wounds.Methods1. Establish Model1.1 Establish STZ induction of type I diabetes model miceAfter weighing , and four consecutive days intraperitoneal injection STZ (50mg/ kg), mice banned food but not prohibiting to drink for 10h,was taken blood vein tail, and was tracked detection it's glucose concentrations and variation of body weight eight weeks.1.2 Establish STZ diabetic mice trauma refractory modelWe choose the modle ,which fasting plasma glucose concentrations, and continuously appear above 11.1 mmol/L, food and drink more than urine .Mice with 10% water chloric aldehyde (30mg/kg) after intraperitoneal injection of anesthesia in line spacing both (back) in diameter, above the 1cm f.DaKongQi 1cm with diameter punch, hemostatic, single cage. Tracking the wound healing rate until healed completely.2. Generate NO of mice wound healing mixture of observationExperimental group: Total 5 group----diabetic mice, diabetic mice L - arginine (L - Arg in treatment group (group), diabetic mice oleander hemp meat (APO) treatment group, sodium nitrate diabetic mice doppler (SNPS) treatment group, diabetic mice NO mixture treatment groups, each group has 10 mice.Medication:After the wound is established in back, according to different groups success by physiological saline, L - 150g/L arg solution (compound, deionized water solution (1), the APO 10-4 mol/L, deionized water dispensing), mmol/SNPS solution (0.1 L, deionized water dispensing) and NO mixture (L - arg APO, SNPS solution, compares with preparation, mixing before in mice with corresponding wound sterile syringes subcutaneous injection, the next time the 0.15 ml.Specimen after treatment: 1, 3, 5, 7, 10 days, the application of digital camera wound healing, under the circumstance of record. In the first 3 and 7 and 10 days were taken and margin, 10% formalin fixed, paraffin slice, wash. continuity, dyeing, observe VG wound fibroblasts, angiogenesis and collagen deposition etc, evaluation wound healing effect.2.1 calculate the wound healing rate Use transparent graph paper depicts wounds, using computer image analysis software area,and according to the following formula and wound healing wound healing rate: initial area ratio = (wound after the formation of the wound - wound formed n day area) / the initial area×100%2.2 Fibroblasts density measurement HE observed under microscope 400 times respectively in the organization, dyeing, the deep and shallow central randomly selected on both sides of the rectangular vision (10), 0.0052 was/vision and calculation counts in implementing slice of fibroblasts quantity per unit area.2.3. Collagen fiber surface density messurement 400 times observed under microscope VG dyeing biopsy,in the shallow,deep tissue and on both sides of the department of central randomly selected five vision, using HMIAS - 2000 high-resolution color medical graphic analysis system, calculation of collagen fiber dyed red surface density.3. NO generation of mice wound angiogenesis mixture and growth factors3.1 Vascular density measurement Use the antiangiogenesis factor VIII antibodies to immunohistochemical specimens,operating procedures in accordance with the kit instructions .According to weidne criteria: Presents the brown single endothelial cells or endothelial cell clusters are as a blood count, muscle thicker and lumen area more than 8 red blood vessels are not count in diameter.Under the microscope 400 times in each section of the central and peripheral zone randomly selected 5 vessels, the vision of dense as the average density of blood samples (MVD) average.3.2 Growth factor measurement With TGF - beta 1 antibodies to immunohisto- chemical specimens,operating procedures in accordance with the kit instructions .Observing indexes with optical microscope (10 by 40 times) TGF - beta 1 observe fiber distribution with positive immune Mplas-500 multi-media color pathological graphic analysis system, through microscopic imaging system amplification 400 times,each section randomly selected in this field, 5 in the field TGF selected beta 1 (+) - standard, image analysis system of automatic measurement results, a unit of mu m2.Using SPSS statistical analysis software, 10.0 all measurement data () says, t-test, p < 0.05 expressed statistically significant.Results1. Eight weeks tracking detection mouse glucose concentrations and variation of body weight;Test results show that mice glucose concentrations were significantly higher (p < 0.05), and weight decreased obviously (p < 0.05), and the signs for 8 weeks, and reports in the literature of the above model STZ diabetic mice, STZ that success in mice induced type I diabetes.2. The whole STZ diabetic mice wound healing process is slower than the control group (p < 0.05), and explain the operation made STZ diabetic mice appeared trauma and diabetes phenomenon of refractory severely damaged skin tissue in the wound healing abilities.3. Controls in 16-18 days after trauma can heal, NO mixture of granulation tissue growth in treatment group than controls, healing time of 3-4 days in advance. Other single-agent intervention group, a group healed ahead 1-2 days, three days after the formation of wounds, each drug treatment group than controls wound healing rate in mice with wounds, after forming most obvious changes within the 7d.4. Each group, three days after traumatic visible cell hyperplasia, collagen formation, 7-10 tiancheng fiber cell hyperplasia of collagen fibers, aligned, In march, after traumatic group, 10 tiancheng fiber cell density (making) were higher than those in the control group (P < 0.05), and in 14 days after trauma were lower than that of the control group (P < 0.05).But NO treatment group than other mixture single-agent therapy is more obvious (P < 0.01). Each collagen fiber surface density (AA) are continuous increasing trend, at all time points AA are higher than the control group (P < 0.01), single-agent intervention group, with no obvious difference in the APO slightly higher than the other two treatment groups.5. TGF - beta cell 1 positive rate:NO mixture treatment group after 3 days, positive dyeing cells,3-7 days of TGF - beta cells in the positive and express enhancement cell peak, traumatic 7-14 days of cells express weakened gradually to the positive relatively stable level;On July 3, group, 10 days of positive point above expression in control group (P < 0.01), the positive control cells express clearly, and express peak lag is less than other groups,as with any other single agent intervention group no obvious difference (P > 0.05).6. NO mixture in treatment group 3,5,7 days after traumatic endothelial cells, hyperplasia of active control more capillary formation (P < 0.01), and other single-agent intervention group, the number of angiogenesis is also has difference (P < 0.05). Control and angiogenesis significantly less serious than any other group of the inflammatory response. NO one in treatment group and control group of mixture is the most significant difference (P < 0.01), L - Arg APO, between treatment group and SNPS difference.ConclusionsIn diabetic mice wound healing process ,single use L - arginine, or Apocynin, or sodium nitroferricyanide can promote wound healing, while promoting NO generation of wound healing mixture with better effect,can significantly reduce diabetic mice, the wound healing time L - Arg APO SNPS, alone, especially.