The Effect Of Maggot Excretions/Secretions On The Anti-Pseudomonas Aeruginosa Effect Of Neutrophils In Patients With Diabetic Foot Ulcer

Diabetic foot ulcer(DFU)is one of the serious complications of diabetes.It mainly refers to foot ulcers in diabetic patients with or without peripheral neuropathy or peripheral vascular disease of the lower extremities.The ulcers can involve bone and muscle,DFU Often combined with different degrees of infection,wound healing is difficult,and there may be a risk of disability or even death.Therefore,DFU anti-infection therapy is particularly important.Maggot excretions/secretions(ES)are compounds secreted or excreted by sterile parasitic larvae of the Lucca sericata.ES contain a variety of antibacterial peptides and cell growth factors,which can promote ulcer wound healing in patients with DFU.Staphylococcus aureus was the majority of Gram-positive bacteria and Pseudomonas aeruginosa was the majority of Gram-negative bacteria in DFU infected wounds.Neutrophils play a very important role in the innate immunity of the body.The main steps of neutrophils sterilization are neutrophils phagocytosis,degranulation,generation of Radical Oxygen Specises(ROS)and Neutrophil Extracellular Traps(NETs).At present,Pseudomonas aeruginosa can resist the killing of neutrophils,and antibacterial substances in ES have been confirmed,but no studies have been conducted to clarify whether ES plays a positive role in the anti-Pseudomonas aeruginosa function of neutrophils in DFU patients.ObjectiveThe phagocytosis and bactericidal effects of ES-stimulated neutrophils in patients with DFU and the expression of related cytokines,enzymes and bactericidal substances in the process of neutrophil sterilization were determined,and the effect of ES on the bactericidal function of neutrophils in DFU patients was preliminarily explored.Method1.ES is excreted and secreted by 3-day-old sterile larvae of 15-day-old Lucilia sericata in the same basket.2.Collect peripheral venous blood from newly admitted non-acute septic diabetic foot ulcer patients who have not used intravenous antibiotics,Wagner grade 3 to 4(ulcers involving bone tissue,local gangrene)within two weeks.3.Wright’s staining method stains the phagocytosis of neutrophils in DFU patients infected with Pseudomonas aeruginosa with or without ES stimulation,observes and counts the phagocytic rate and phagocytic index of neutrophils under a microscope,and detects ES Phagocytosis of neutrophils in DFU patients infected with Pseudomonas aeruginosa after stimulation.4.The plate colony counting method counts the number of viable colonies of neutrophil co-culture and Pseudomonas aeruginosa dilution in DFU patients with or without ES stimulation of Pseudomonas aeruginosa infection,and detects the aeruginosa after ES stimulation The bactericidal effect of neutrophils in patients with DFU infected by monospora.5.Real-time fluorescent quantitative PCR method to detect the relative expression of m RNA of the cytokines IL-1β,IL-6,IL-10,TNF-α,neutrophil respiratory burst enzymes myeloperoxidase deficiency(MPO),nicotinamide adenine dinucleotide phosphate(NADPH)oxidase,inducible nitric oxide synthase(i NOs),neutrophil-related bactericidal substance lysozyme,human neutrophil peptide-1(HNP-1),human neutrophil peptide-3(HNP-3)and human neutrophil peptide-4(HNP-4)in neutrophils of DFU patients infected with Pseudomonas aeruginosa after ES stimulation.Enzyme linked immunosorbent Assay(ELISA)to detect the protein level of neutrophil cytokines IL-1β,IL-6,IL-10,TNF-α in DFU patients infected with Pseudomonas aeruginosa after ES stimulation concentration.6.Immunofluorescence and Western blotting method to detect the m RNA expression and protein level of lysozyme in neutrophils of DFU patients infected with Pseudomonas aeruginosa after ES stimulation.Result1.After ES stimulates neutrophils in DFU patients infected with Pseudomonas aeruginosa,the phagocytosis and bactericidal effects of neutrophils are enhanced.2.After ES stimulates neutrophils infected by Pseudomonas aeruginosa in patients with DFU,the expression of IL-1β and IL-6 m RNA and the production of IL-1β and IL-6in neutrophils increase;the expression of IL-10 and TNF-α m RNA and the production of IL-10 and TNF-αdid not change significantly.3.After ES stimulated the neutrophils of DFU patients infected with Pseudomonas aeruginosa,the expression of NADPH oxidase gp 91 phox,p 47 phox,p 40 phox,p 22 phox,p 67 phox mRNA in neutrophils increased.4.After ES stimulated neutrophils infected by Pseudomonas aeruginosa in patients with DFU,the expression of HNP-1,HNP-3,HNP-4 m RNA in neutrophils increased;MPO,iNOs mRNA expression did not change significantly.5.After ES stimulated the neutrophils of DFU patients infected with Pseudomonas aeruginosa,the expression of lysozyme mRNA in neutrophils increased,and the production of lysozyme increased.ConclusionES can enhance the phagocytosis and bactericidal effect of neutrophils on Pseudomonas aeruginosa in DFU patients within a certain period of time.ES can promote the production of neutrophils’ immune defense related cytokines,bactericidal related enzymes and antimicrobial peptides,and enhance the resistance to Pseudomonas aeruginosa infection in DFU patients.