| In recent years, with the growing abuse of antibiotics, severe infected wounds caused by drug-resistant bacterae are on the rise lack of effective clinical treatment. This poses a significant impact on the health care system on account of its complications, the chronicity of care required and the associated cost. Traditional Chinese medicine“WuGuChong”is the larvae of Lucilia sericata, Chrysomyia megacephala or other relative Calliphoridae insects. According to traditional Chinese medicine principle and the book Compendium of Materia Medica records,“WuGuChong”is salty in taste, cold in nature and non-toxic. It is a medicine for external application to treat superficial purulent diseases such as furuncle or carbuncle. Nowadays, WuGuChong therapy, as a kind of biological therapy, has been widely used to treat various refractory wounds including diabetes foot and venous ulcers. The therapeutic action contains biological debridement, disinfection and enhancement of wound healing.In previous study of our group, we have obtained an antibacterial peptide. However, there are still several controversial and unsettled problems: 1) in western medicine, WuGuChong therapy using living body of larvae is applied as a surgical debridement technique to treat infected wounds. While in traditional Chinese medicine, WuGuChong is extra-administration as a kind of dried drug after heat processing. There is no report on the relationship and comparative study of the above two applications; 2) the process of wound healing is complicated, referring to migration of related cells, angiogenesis and wound nerve regeneration. The effect of active components from WuGuChong on wound healing and related mechanisms is not clear. 3) based on traditional Chinese medicine documentation, WuGuChong is a animal drug, and it is difficult to analyze due to its complicated components. Therefore dried WuGuChong is limited clinically and falls behind from living body application. So for the animal drugs, new theory and methods must be innovated to develop its active components.To solve the above problems, the present study under the guidance of integrated medicine theory is committed to clarify the effect of WuGuChong on wound healing and its related mechanisms ignoring infection. The contents of present study are as follows: 1) comparative study of different application of WuGuChong on wound healing; 2) effect of active components from living WuGuChong on wound healing and related mechanisms; 3) effect of fatty acid extracts from dried WuGuChong on wound healing and related mechanisms.1. Comparative study of different application of WuGuChong on wound healingIn this part of study, we compared the effects of living WuGuChong homogenate products with that of dried WuGuChong on murine cutaneous wound healing in vivo. It was demonstrated that on day 1 and 3 after murine acute excision wounds creation, there was no statistical difference among homogenate group, died group and control group(P>0.05). On day 7, 10and 14, the percent wound contraction of homogenate group and died group was higher than that of control group(P < 0.05). However, the percent wound contraction in both homogenate group and died group was the same. Histological examination showed that comparing with control group, wounds of both homogenate group and dried group displayed a more accumulation of granulation tissue with a high degree of newly-formed micro-vessels and numerous inflammatory cells and fibroblast, the granulation was matured showing capillary vertically oriented, robust fusiform fibrocytes and moderate well-arranged collagen, and regenerative folliculus and sebaceous glands were detected in the fibrous connective tissue. In conclusion, both living WuGuChong homogenate and dried WuGuChong can reduce the rat skin wound healing course and improve wound healing rate and quality of healing.2. Effect of active components from living WuGuChong on wound healing and related mechanisms(1) WuGuChong excretions/secretions induces human microvascular endothelial cell migration through AKT1 pathwayIn this part of study, we investigated if WuGuChong excretion/secretion(ES) induced cell proliferation and migration during wound healing process using microvascular endothelial cells (HMEC-1) as model. Wound healing and transwell migration assays were performed to study the effects of ES on HMEC-1 cell migration. Our data showed that ES significantly induced HMEC-1 cell migration in both wound healing and transwell assays, and time-dependently (P<0.05) activated AKT1, but not ERK1/2. Moreover LY294002 (a PI3K inhibitor) partially attenuated (P<0.05) ES-induced cell migration in wound healing assay while completely inhibited (P<0.05) ES-induced AKT1 activation. These findings demonstrate that ES directly induces HMEC-1 cell migration and this event is partially mediated by the activation of AKT1.(2) WuGuChong homogenate is associated with neural regeneration and murine cutaneuous wound healingIn this part of study, we evaluated effects of living WuGuChong homogenate on acute skin wound healing and wound nerve regeneration. On days 7, 10, and 14 following injury, the rate of wound healing was significantly greater in the homogenate product group compared with the control group (P < 0.05), and homogenate healing was better than that seen in the control group. On days 3, 7, and 10, SP expression in cells and regenerative nerves was significantly greater in the homogenate product group compared with the control group (P < 0.05). On days 7 and 10, protein gene product 9.5 expression was detected in the regenerative nerve, and expression level was significantly greater in the homogenate product group compared with the control group (P < 0.05). It can be concluded that WuGuChong homogenate up-regulates SP and protein gene product 9.5 expressions during wound healing process, thereby promoting neural regeneration and wound healing.3. Effect of fatty acid extracts from dried WuGuChong on wound healing and related mechanisms.In his part of study, we aimed to investigate the effect of fatty acid extracts from dried WuGuChong on murine cutaneuous wound healing as well as angiogenesis. Results demonstrated that on day 7 and 10 after murine acute excision wounds creation, the percent wound contraction of fatty acid extracts group was higher than that of vaseline group(P<0.05). On day 3, 7 and 10 after wounds creation, the wound healing quality of fatty acid extracts group was better than that of vaseline group on terms of granulation formation and collagen organization(P<0.05). On day 3 after wounds creation, the micro vessel density and vascular endothelial growth factor expression of fatty acid extracts group were higher than that of vaseline group(P<0.05). Component analysis of the fatty acid extracts by Gas chromatography-mass spectrometry showed there were 10 kinds of fatty acids in total and the ratio of saturated fatty acid, monounsaturated fatty acid and polyunsaturated fatty acid (PUFA) was: 20.57%:60.32%:19.11%. In conclusion, fatty acid extracts from dried WuGuChong, four fifths of which are unsaturated fatty acids, can promote murine cutaneous wound healing probably resulting from the powerful angiogenic activity of the extracts. |
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