| 1 BackgroundWound healing is a complex biological process of self-repair, which involved in many factors of body, and tissue regeneration is the substructure. Normal wound healing process includes three phases:inflammation phase; period of cell proliferation and extracellular matrix reconstruction phase.Skin wound often leads to epidermis, dermis and adnexal injury, namely, it exists cell dysfunction or tissue defect. The progress of wound healing includes stopping bleeding in earlier period, protecting wound surface, controling inflammation, histiooytic hyperplasy, forming granulation tissue, creeping of the wound limbus, and so on. Proliferation of endothelial cells, epithelial cells and fibroblasts is the key to the formation of granulation tissue, also the key to wound healing.There are many factors affecting wound healing, such as age, nutritional status, wound size, and kinds of systemic diseases. Some factors can result in disunion of wound for a long time, which belongs to the thorny issue of clinical treatment. For the inhibition of cell proliferation caused by necrotic tissue, microbial infection, metabolism and other factors, chronic and refractory wounds stay in the inflammatory response period, instead of into the next phase, namely period of cell proliferation. Although new technologies and new means come out one after the other, therapeutic efficacy of refractory wounds such as chronic skin ulcer was still not good. Researches reveal that the number of patients requiring amputation for their chronic skin ulcers will increase 30% in 2030 more than it is now in whole world. Therefore, it is necessary to make more in-depth study and practice to develop effective drugs that they could promote wound healing and accommodate clinical requirements and international standards.Ecdysterone (ecdysterone, EDS) also known asβ-Ecdysone in animals. It was an insect metamorphosis hormone, which could stimulate the insect dermal cell division to generate new skin, result in insect metamorphosis, and regulate insect metabolism. But it was found that ecdysterone exists in plants more than in animal kingdom. Currently, ecdysterone was mostly extracted from plants.Ecdysterone in vertebrates is also necessary substance. The study indicate that it also showed a strong pharmacological activity in higher animals, and its side effects were minor. Nowaday, as we know its actions include:1) promote nucleic acid and protein synthesis; 2) adjust glucose metabolism; 3) modulate lipid metabolism; 4) regulate gene expression; 5) improve immune function; 6) posses antioxidan activity; 7) promote angiogenesis and establishment of collateral circulation in ischemic area; 8) promote proliferation of varied cells.The previous investigations of our study group reveal that Ecdysterone can improve the wound healing process, and Ecdysterone paste(patent number index: 200710030920.6) was produced to use in experimental rabbits. New Zealand white rabbits with full-thickness skin defect on the dorsal skin were as models in this experiment. It was used to investigate the effect and possible mechanisms of ecdysterone on wound healing of cutis in experimental rabbits. And it could provide a basis for the clinical application of Ecdysterone paste in trauma. 2 ObjectiveTo investigate the effect and possible mechanisms of ecdysterone on wound healing of cutis in experimental rabbits, and provide a basis for the clinical application of Ecdysterone paste.3 MethodsTaking 40 healthy New Zealand white rabbits, injecting pentobarbital(35mg/kg) into vein of ear for anesthesia, depilating the back fur with 10% sodium sulfide, using iodophor to disinfect the back skin of rabbits, cutting off a circular full-thickness skin with diameter 1.8cm and depth of sarcolemma, causing the trauma models with full-thickness skin defect on the dorsal skin. Each rabbit has three raw surfaces, labeled randomly A, B and C, respectively as blank control group(group A, only give conventional disinfection), Yunnan white powder group(group B, smear Yunnan white powder, each about 0.5g), and ecdysterone paste group(group C, smear ecdysterone paste with concentration of 2.5%, each about 0.5ml). After applying medicine, all the raw surfaces were covered with vaseline gauze, and then bandaged with dry gauze. The wounds were washed with isotonic Na chloride, disinfected with iodophor everyday, and then deal each wound with original modality. Each rabbit was breeded in a cage by itself.10 rabbits were selected randomly, and the curative effect was to be evaluated by the wound healing rate and the visual changes of wound in each group on 4,8,12 days after operation. And the time of wound healing in each group was recorded, the comparison between groups was made. The supernumerary 30 rabbits were sacrificed to obtain the histological specimen on 4,8,12 days after modeling in batch, each 10. By observing the histopathological indexes to assess the therapeutic effect after HE staining and making section preparatio. And epidermal growth factor receptor(EGFR) expression in wound edge in each group was detected by Immunohistochemical SP method. Basic fibroblast growth factor(bFGF) levels of wound surrounding tissue were measured by ELISA method.The measurement data are expressed as Mean±SD. And SPSS 13.0 statistical software was used to analyze the data(using analysis of variance, including the One-Way ANOVA and Repeated Measures, and the comparison of groups were used LSD or Dunnett's T3 test).4 ResultsThe wound healing rate of each group was increased over time in the period of observation(0-12d). It indicate that the model possess a certain natural healing ability. There was no significant differences among groups in the rate of wound healing on 4 days after modeling(P>0.05). The wound healing rate of group C and group B on 8, 12 days after modeling were higher than group A(P<0.01), but there was no significant differences between group C and group B(P>0.05).The statistical results of wound healing time:The wound healing time of group A, B, C were respectively (18.60±1.776) d, (14.80±1.229) d, (13.80±0.789) d. The wound healing time of group C was the shortest, followed by group B. There was no significant differences between group C and group B(P>0.05). However, the wound healing time of group C and group B was less than the group A(P<0.01). It demonstrated that ecdysterone paste and Yunnan white powder can effectively shorten the wound healing time.The visual observations of wound:On 4 days after modeling, the wound of each group was moist, no apparente purulent infiltration, and no conspicuous wound contraction. The wound of group C showed small amounts necrotic tissue, and the tissue under it was dropsical slightly. The wound of group B showed the crust with necrotic tissue ablated from wound surface, and the epidermal tissue began to grow. The wound of group A could be seen necrotic tissue with a large number of inflammatory cells, and the tissue under it had considerable inflammatory cell infiltration. On 8 days after modeling, the wound of group C and group B were moist, no infection, and apparente contraction of wound surface and subcutaneous tissue. The wound of group A could still see exudation, and individual wound could see ulcers. On 12 days after modeling, the wound of group C and group B could see further wound contraction, dry wound surface, thick crust on superficies, and small wound surface not healed. Compared with the wound of group A on 8 days after modeling, the wound contraction was conspicuous, and the edge of wound formed crust, but there was still large area did not heal.The observation of tissue sections under light microscope:On 4 days after modeling, the wound of each group mainly reflected much exudation and congestion, little granulation tissue. And there was no significant differences to histopathological scores of each group (P>0.05). On 8 days after modeling, the wound of group C and group B displayed no exudation and congestion, some granulation tissue, a few inflammatory cells, more collagen bundles, a few proliferation of epithelial cells. The wound of group A could see inflammatory exudate and ulcer, less collagen bundles, less proliferation of epithelial cells. The histopathological scores of group C and group B were higher than group A(P<0.01), but there was no significant differences between group C and group B(P>0.05). On 12 days after modeling, the wound of group C and group B displayed much granulation tissue, a few inflammatory cells in junction of dermis and epiderm, more collagen bundles, a large amount of proliferation of epithelial cells and fibroblasts. The proliferation levels of collagen, epithelial cells and fibroblasts in group A were lower than group C and group B. The histopathological scores of group C and group B were higher than group A(P<0.01), but there was no significant differences between group C and group B(P>0.05). The immunohistochemical expression of EGFR in wound edge:The expression of EGFR in normal skin was weakly positive. The expression of EGFR in trauma organizations mainly distribute in membrane and cytolymph of the epidermal basal cells, prickle cells, follicular epithelium, dermal fibroblasts and vascular endothelial cells, and sweat gland cells. There was no expression of EGFR in the necrotic tissues. The EGFR level of group C and group B on 4,8,12 days after modeling were higher than group A(P<0.01), but there was no significant differences between group C and group B(P>0.05). The level of EGFR on 8 days after modeling were higher than those on 4 and 12 days after modeling in each group(P<0.01). The level of EGFR on 12 days after modeling were higher than those on 4 days after modeling in each group(P<0.05).The expression of bFGF in wound surrounding tissue:The group C expressed the highest level of bFGF on 4,8,12 days after modeling, followed by group B, the group A expressed the lowest level, there was significant differences among groups(P <0.01). The level of bFGF on 8 days after modeling were higher than those on 4 and 12 days after modeling in each group(P<0.01). The level of bFGF on 12 days after modeling were higher than those on 4 days after modeling in each group(P<0.01).5 ConclusionEcdysterone paste can improve the wound healing process in experimental rabbits. Its mechanism may be that ecdysterone can reduce the local inflammatory response, promote the formation of granulation tissue and induce high expression of EGFR and bFGF. It could promote proliferation of epithelial cells, endothelial cells and fibroblasts, speed up crawling of epithelial cells, increase new capillaries and collagen formation, thereby enhance the speed and quality of wound healing. |
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