Preparation Of Compound Insulin Gel And Its Primary Experimental Study On Promoting Healing Of Skin Ulcer In Rats

Background and Objective:Chronic skin ulcer (CSU) is a refractory disease with complicated etiology. Its healing needs to experience three completely distinguished phases, which is still related each other. It is also a complex but continuous biologic reactive process. High incidence and seriousness of CSU have aroused much concern of the people. In European nations, diabetic ulcer and leg vascular ulcer lead a dominant station in the incidence of CSU. However, trauma or trauma with local infection is popular in China. But in recent years, the incidence of diabetic foot (DF) and vascular ulcer have moved up.No matter what reasons cause skin ulcer, such as trauma and diabetes, local treatment is an important clinic therapeusis. But up to present, healing treatments have not delivered satisfactory results. Recently, some scientists inside and outside the country have tried to apply insulin to treat various trauma or skin ulcer (general application or local application). They found the wound could heal up ahead of time and accepted better curative effects.Insulin is a water soluble and acidic protein molecule of 56kD molecular weight. It is composed of two polypeptide chain of A and B. It has three disulfide bonds and the minimum solubility at isoelectric point. Insulin is one of the multifunctional protein hormones. It can not only mediate substance metabolism, but also speed wound repair. Abundant research work has been done to confirm that insulin can increase or sustain proliferation and differentiation of the cells cultured in vitro. And it may act as a growth regulator in vivo.We have locally applied insulin mixed solution in the clinic practice to treat multifarious refractory wounds of skin ulcer and acquired satisfactory clinic curative effects. The detection of blood sugar concentration has shown no signs of hypoglycemia. Insulin usage is safety and economical. But the experimental study on the preparation of insulin agent for external use is absent inside and outside the country. In this study, we tried to prepare compound insulin gel to locally treat the wound of skin ulcer and investigate the possible mechanisms of enhancing wound healing. It may offer the theoretic and experimental evidences for the clinic practice.Methods:1. According to generate orthogonal design, the optimization prescription was filtrated. The compound insulin gelagent was prepared by taking Carbpol as base material. The change of insulin content was determined with UV spectrophotometry and its stability was estimated.2. Wistar rats were taken as the objects of investigation. The acute trauma animal model was repeated based on the references. We designed three groups including the compound insulin gel treated group(A), the Carbpol base material group(B) and the negative control group(C). The repair effects were valued by observing the change of ulcer area size, healing speed of ulcer wound in all the groups at different time points and the tissue pathology.3. Diabetic rats were taken as the objects of investigation. The diabetic skin ulcer model was repeated based on the references. We designed three groups including the compound insulin gel treated group(A), the GeneTime (Recombinant Human Epidermal Growth Factor derivative For External Use, rhEGF) positive control group(B) and the negative control group(C). After treatment, the repair effects were valued by observing the change of wound area size, the percentage of wound healing in all the groups at varied time points. The morphological methods such as HE staining, immunohistochemistry and transmission electron microscopy (TEM) were also used to analyze the repair effects.4. Statistical analysis: SPSS10.0 statistical soft package was used. The results were expressed as Mean±SD (x±s). Repeated Measures and Factorial analysis were used separately to make area and healing rate of wound and PCNA, VEGF. One-way ANOVA and Least-significant Difference (LSD) test were applied to make multiple comparisons between the groups. P≤0.05 was considered to indicate significant difference.Results:1. The results from experiment of generate orthogonal design showed pH affected stability of insulin the most. The effect of the concentration of Carbpol was in the second place and the concentration of insulin, the least. Construction of the optimization prescription was A3B1C1, which meant pH was 6 and the concentration of Carpbol and insulin was 1.5% and 14U/g, respectively.2. We found the compound insulin gel prepared had a good stability when putting it in the refrigeratory (4℃±3) or at room temperature.3. Pharmacodynamics research3.1 Acute trauma animal model experimentsâ‘ The wound area among the groups was distinctly different (F=5.880, P＜0.01). From 7 days on, there was significant difference of the wound area between group A and group B,C (P＜0.01 or P＜0.05). However, there was no significant difference between group B and group C (p＞0.05). â‘¡By observing under microscope, granulation tissue of the wound at 7d after trauma in group A was in a bloom hyperplasia, in which fibroblasts and capillary vessels were affluent. But in group B and C, the proliferations of granulation tissue, fibroblasts and capillary vessels were relatively less than those in group A. At 14d after trauma, the wound in group A was epitheliogenic and the frame of skin was integrated. But epithelialization of the wounds in group B and C were poor and the construction of skin was half-baked.3.2 The diabetic skin ulcer animal model experimentsâ‘ The percentage of wound healing: there was significant difference of the percentage of wound healing among the groups (F=8.302, P＜0.01). the three days later after injury, the percentage of wound healing between each group at various time points was significantly different (P＜0.05 or P＜0.01). In detail, the percentage of wound healing in treated and positive control group was all obviously different from that in negative group at various time points (P＜0.05 or P＜0.01). But there was no distinct difference between treated group and positive control group (P＞0.05).â‘¡Under microscope, we found at 7d after injury, granulation tissue in the wound of treated group and positive control group grew actively and obviously excessive capillary vessel budding and fibroblasts also presented, compared with negative control group. At 14d after injury, the wound of treated and positive control group was basically covered by epidermis. Mass fusiform fibroblasts and organized collagenous fibers were also seen. While in negative control group, the remained wound was still large. Collagenous fibers aggradation was not obvious and orderly. At 21d after injury, the wound of treated and positive group was basically healed, but that of negative control group still remained.â‘¢Expression of PCNA and VEGF antigen in the wound tissues were examined by immunohistochemistry. There was significant difference of expression of PCNA and VEGF among. the groups. At 7d after injury, higher expressions of the two kinds of antigen were found in the treated group and positive control group, compared with the negative control group. There was significantly difference among the groups(P＜0.05). But at 14d after injury, there was no significantly difference among its (P＞0.05). And there was not significantly different about the expression of PCNA and VEGF between treated group and positive control group at various time points.â‘£The observation under TEM suggested at 14d after injury, plentiful and thick collagenous fibers proliferated in intercellular substance of the wound tissue in treated and positive control group and arranged regularly. The cytoplasm and organelle such as rough surfaced endoplasmic reticulum (RER) and mitochondrion were still spare. However, collagenous fibers in negative control group were thin, spare and disorganized. The cytoplasm of fibroblast was full and the nucleus was big. Large quantities of RER and mitochondrion were also found in the cell.Conclusion:1. According to generate orthogonal design, the filtrated optimization prescription was include the neutral insulin injection 140 U, vitamin B₁₂ 8.75 mg, Carbpol 941 0.15g, glycerol 1.0g, pH 6.0～6.5 which is adjusted by using proper triethanolamine. Finally add distilled water to 10g.2. The gelagent is designed reasonably and can be prepared easily. Its property is stable and it is not irritant for skin. Its quality can be measured rapidly and exactly at same time.3. Compound insulin gel can promote the proliferation, division and differentiation of the repairing cells, such as vascular endothelial cell, fibroblast and epithelium, etc. It can speed up the growth of granulation tissue and make it epitheliogenic in advance. It can improve the wound healing of skin ulcer and have repairing effects at three phases in the process of the wound healing.