The Clinical And Biomolecular Mechanism Research Of Antibacterial Function Of Chinese Medicine WuGuChongon Infective Wound

The number of patients suffering from chronic infective wounds is on the rise. Those wounds are attributable to diseases such as pressure ulcer after spinal cord injury (SCI), chronic osteomyelitis and infective wounds caused by diabetes mellitus and peripheral vascular disease. Those diseases pose a significant impact on the health care system, because of chronicity of care required, long term treatment and bad healing effects.Those infective wounds are difficult to be treated with common debridement and operation.How to control the infection caused by bacterium is the key point to cure infective wound. Antibacterial drugs posed a significant impact on such kinds of diseases in the past time. Recently, the problem of the rising incidence of antibiotic resistance became serious. Antibiotic resistant bacteria could damage the wound quickly. As a result of the prevalence of antibiotic resistant bacteria, antibacterial function of common antibiotics on the infective wound was declined dominantly. At present, it is a problem to control the bacterial infection and hasten the process of infective wound healing in the field of modern surgery.Chinese traditional medicine"WuGuChong"is the larvae of Lucilia sericata, Chrysomyia megacephala or other relative Calliphoridae insects."Ben Cao Qiu Yuan"and"Ben Cao Bian Du"which are Chinese traditional books record that the pharmic character of the larvae is cool and innoxious, it is a medicine for external application to cure surgical infection diseases such as carbuncle and sore. The application of WuGuChong could offer a new idea to treat infective wound effectively, but antibacterial mechanism of WuGuChong could not be achieved from Chinese traditional medicine. Recently, doctors of western country such as Sherman R.A. began to do clinical studies of WuGuChong for infective wound treatment and found that WuGuChong could be used for cleaning and disinfection of chronic wounds that are sloughy, necrotic, and infected. Nowadays, the researches are mainly focused on case reports and treatment skills. The biomolecular and clinical study about antibacterial function is lacking.Under the guidance of the theory of integrated traditional and western medicine, we would do the clinical and biomolecular research of antibacterial function of Chinese medicine WuGuChong on infective wound. The destinations of the project are as follows: 1. Achieve the systemic rearing and disinfection skills of WuGuChong, offer the materials for clinical and basic research; 2. Achieve the purified antibacterial substance of WuGuChong, get the character of antibacterial activity, confirm the effective antibacterial component on infective wound, realize the molecular structural and biological information, the project will lay the theoretical foundation for farther pharmacological research and gene engineering preparation of WuGuChong; 3. Discuss the antibacterial and healing mechanism of WuGuChong on infective wound, get the scientific application skills for clinical treatment.The idiographic study contents as follows: Part 1.The preparation study of medical WuGuChong and the antibacterial activity study of primary purification; Part 2. Analysis and identification of antibacterial activity of WuGuChong, separation and purification of effective antibacterial substance and molecular structure study.Part 3.The study of antibacterial and promoting healing function about WuGuChong on infective wound.Part1. The preparation study of Medical WuGuChong and the antibacterial activity study of primary purification1. The preparation study of Medical WuGuChongWe investigated the skills of rearing and disinfection for the larvae of Lucilia sericata which was the mostly species of WuGuChong. WuGuChong was propagated and reared with artificial asepsis diets in laboratory, eggs and larvaes were disinfected with"double steps disinfection". The results showed that Lucilia sericata could be reared in doors and the generation was stable. The eggs and larvaes were disinfected with"double steps disinfection", all eggs and larvae were alive, none larvae were dead because of disinfection. No bacteria and virus were found from the the homogenization of the larvae. The experiment approved that the larvae of lucilia sericata could be reared with artificial skill under the asepsis environment indoors. The larvae of lucilia sericata could be used as the materials for the clinical and experimental research.2. Antibacterial activity study of primary purification of WuGuChong on standard bacteriumWe studied the antibacterial activity and biological characters of secretion and hemolymph from the primary purification of WuGuChong. Primary purification of hemolymph and secretion of the larvae were achieved with centrifugation, the antibacterial activity was tested with disc method, took use of standard Escherichia coli and Staphylococcus aureus as the indicator organisms. SDS-PAGE analysis was admitted for the primary purification of hemolymph and secretion. The results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). The experiment approved that not only the primary purification of hemolymph but also the primary purification of secretion had the same broad antibacterial and sterilizing activity to both E. coli and S. aureu. Main antibacterial substance of WuGuChong was some kind of organic component (smaller molecular weight).3. The antibacterial function of WuGuChong against harmful bacterium from infective woundIn this study, we used S. aureus as the indicator organisms which from the infective wounds of clinical DM cases, the primary purification of WuGuChong was collected, the antibacterial activity was tested with disc method, and then recorded the antibacterial effect, cefoperazone sodium and Saline as the contrast groups. Results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). It was found that WuGuChong had the antibacterial activity to common pathopoiesis bacterium of infective wound.Using Pseudemonas aeruginosa as the indicator organisms which from the infective wound of clinical DM cases, the primary purification of WuGuChong was collected , the antibacterial activity was tested with disc method , and then recorded the antibacterial effect , cefoperazone sodium and Saline as the contrast groups. Results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01). It was found that WuGuChong had the antibacterial activity to special pathopoiesis bacterium of infective wound.In the study, the hemolymph of the larvae which was induced by the bacterium with pricking was collected, the hemolymph of the asepsis larvae which was not induced by the bacteria and Cefoperazone Sodium as the contrast groups, Staphylococcus aureus as the indicator organisms which from the infective wound of pressure ulcer after spinal cord injury. The antibacterial activity was tested with disc method, the antibacterial effect was observed. The results showed that the diameters of antibacterial cycle in experimental groups were larger than those in the contrast groups (P<0.01).The experiment approved that the antibacterial substance of WuGuChong was some native component which was not induced by external stimulation. External stimulation could enhance the antibacterial activity.Part2. Analysis and identification of antibacterial activity of WuGuChong,separation and purification of effective antibacterial substance and molecular structure study.1. The study of Turbidometric (TB) assay for antibacterial activity of WuGuChong secretionIn the experiment, we used standard Staphylococcus aureus as the indicator organisms. The secretion of WuGuChong and the indicator organisms were cultured together, measurement of Turbidometric (TB) assay was taken: collected the culture fluid (sample fluid and contrast fluid), measured the OD value on different time with ELIASA.OD-time curve was protracted. Checked and adjusted the protein content of the sample fluid with Commassie Blue, measured the antibacterial activity of sample fluid with different protein concentration. Total culture time was 21 hours, the OD value of sample fluid was declined with the time going on, the OD value of contrast fluid was increased with the time going on, evident antibacterial function was found in the sample fluid. Dominant antibacterial activity was found on the protein concentration of 0.8mg/ml and 0.4mg/ml, no dominant antibacterial activity was found on the protein concentration of 0.2mg/ml and 0.1mg/ml.The results approved that antibacterial activity in the secretion of WuGuChong was changed with the changing of protein concentration.2. Analysis and identification of antibacterial substance of WuGuChongIn this experiment, we collected the secretion of WuGuChong, purified the secretion with DEAE-Sepharose ion exchange, and then collected the gradient elution and the rinse fluid. Not only elution but also rinse fluid was cultured with S. aureus at 37℃for 21 hours. Antibacterial assay was tested and confirmed for measurement of Turbidometric (TB) assay. The sample of secretion, elution and rinse fluid were checked by SDS-PAGE. The results showed that evident antibacterial activity was found in the elution fluid. Similar protein bandages were shown in both secretion and elution, no protein bandage was found in the sample of rinse fluid. The proteins of the secretion of larvae were eluted completely. The experiment approved that the antibacterial material of the secretion of WuGuChong was some kind of protein (PI<8), DEAE-sepharose ion exchange chromatography was elected as primary method and its gradient elution condition was optimized.3. Purification research of antibacterial substance of WuGuChongAnd then, we investigated the purifying effects of DEAE-Sepharose ion exchange chromatography and gel filtration chromatography for antibacterial substance of the secretion from WuGuChong. Antibacterial assay was tested with measurement of Turbidometric (TB) assay. The gradient elution was collected into serial number tubes in the course of chromatography. The gradient elution into serial number tubes was checked by SDS-PAGE and silver stain, searched the objective antibacterial protein, achieved the objective protein bandage. The results showed that protein contents of tube 33 had dominant antibacterial after ion exchange chromatography. The elution of tube 33 was purified with Superdex-75 gel filtration, antibacterial activity was found in 13, 14, 15 tube of the elution, no significant activity was found in other tubes. The results of SDS-PAGE and silver stain of the elution in tube 13-15 showed that there was a clear protein bandage (MW<14.4KDa), no other protein bandage was found in the elution of tube 13-15. The experiment approved that DEAE-Sepharose ion exchange followed by Superdex-75 gel filtration could purify the antibacterial substance effectively.4. Molecular structure and biological informatics study of antibacterial substance of WuGuChongIn the study, the objective protein bandage was sliced and dealed with Trypsin, the fragments after zymohydrolysis were achieved. The nano-ESI-MS/MS instrument was used to assay the fragments. We did the study of molecular structure and biological informatics of antibacterial substance of WuGuChong with mass spectrum, searched the homologous substance and achieved effective biological information of the antibacterial substance. The results showed that three fragments were found as follows: ENAGEDPGLAR; LGKDAVEDLESVGK; DAVEDLESVGK. Antibacterial protein of the secretion of WuGuChong contained the information of those three fragments. Checked NCBInr database with Bioworks software, achieved the information of homologous proteins: dermcidin preproprotein [Homo sapiens]; dermcidin preproprotein [Macaca mulatta]; protein phosphatase 1 inhib. After identification and analysis of biological informatics, the experiment approved that the antibacterial substance of the secretion of WuGuChong was a kind of antibacterial peptide, there was a higher degree of similarity existed between WuGuChong and Dermcidin preproprotein.Part 3. The study of antibacterial and promoting healing function about WuGuChong on infective wound1. The antibacterial and healing function of WuGuChong on ulcer wound of DM ratThe"Fu Xiaobing"model of ulcer wound of DM rats was made. The ulcer wound of DM rats in maggot secretion group spread maggot secretions, but no secretion on ulcer wound was found in control group. Morphological and immunohistological detection were used to observe the expression of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) in ulcer tissue. It was found that the secretion of WuGuChong could elevate the expressions of bFGF and CTFG in ulcers of DM rats , the expression level was higher than that in contrast group (P<0.01), WuGuChong could promote the wound's healing.In this study, the secretion from the wound of experimental and contrast group was checked with bacterial culture, the results showed that no Staphylococcus aureus infection was seen in experimental group, Staphylococcus aureus infection occurred in contrast group, the rate of Staphylococcus aureus infection was 60%. The experiment approved that the secretion of WuGuChong could prevent bacterial infection on wound.2. The clinical research about antibacterium and biological debridement of WuGuChong on infective woundIn the clinical research, WuGuChong therapeutics was used to repair different infective wounds, the cases as follows: 6 cases suffered from foot ulcer of diabetes mellitus, 5 cases suffered from pressure ulcer after spinal cord injury. The changes of wound surface were observed, bacterial culture was tested. The research showed that negative results were found in the bacterial culture(average 11.2days), the suppurative and necrotic tissue in the wound was declined, and a great amount fresh granulation tissue was generated(average 2.6 days), ideal wound healing occurred in all cases(average 25.6days). The clinical research approved that WuGuChong could resist bacterial infection of wounds, the biological debridement therapy of WuGuChong was valuable and safe in the debridement of intractable chronic wounds. WuGuChong could reach necrotic tissue in all areas of the wound and clean minutes areas without damaging healthy tissue.