Objective To investigate the mechanism of maggot debridement therapy (MDT) in promoting wound angiogenesis in patients with diabetic foot ulcer (DFU).
Methods (1) From June 2018 to June 2019, the patients admitted to Nanjing Junxie Hospital who met the inclusion criteria were recruited, including 12 DFU patients given MDT for three days [6 males and 6 females, aged (56±12) years] and 12 acute trauma patients without diabetes mellitus [6 males and 6 females, aged (53±10) years], who were enrolled into DFU group and non-diabetic trauma group respectively. Before and after application of MDT, the wound characteristics of patients in DFU group were observed and the wound tissue samples were taken. The wound tissue in non-diabetic trauma group was taken at patient′s first visit before debridement. The expression of angiogenesis marker CD31 in the wound tissue of patients in DFU group was detected by immunohistochemistry before and after application of MDT. Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) were used respectively to detect the protein and mRNA expressions of fatty acid synthase (FAS) in wound tissue of patients in DFU group before and after application of MDT and in non-diabetic trauma group before debridement. (2) Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell culture medium containing 10% fetal bovine serum. The 3rd to 6th passages of cells in logarithmic growth phase were used in the following experiments. Excretions/secretions (ES) were extracted from 3-day-old sterile Lucilia sericata larvae for subsequent experiments. Three batches of cells were divided into phosphate buffer solution (PBS) control group, high glucose alone group, high glucose+ 5 μg/mL maggot ES group, and high glucose+ 10 μg/mL maggot ES group, which were treated with PBS, glucose in final molarity concentration of 20 mmol/L, glucose in final molarity concentration of 20 mmol/L+ maggot ES in final mass concentration of 5 μg/mL, and glucose in final molarity concentration of 20 mmol/L+ maggot ES in final mass concentration of 10 μg/mL respectively. The total volume of reagents in each group was the same. After 48 hours of culture, Western blotting, real-time fluorescent quantitative RT-PCR and immunofluorescence method were used to detect the protein and mRNA expressions of FAS in each batch of cells and the expression and localization of FAS protein in cells respectively. The number of samples for mRNA expression was 3. (3) Two batches of cells were divided into small interference RNA (siRNA) alone group, siRNA control+ maggot ES group and siRNA-FAS+ maggot ES group, which were transfected with 100 μmol/L (final molarity concentration) insignificant control siRNA, insignificant control siRNA, and siRNA-FAS for 4-6 h respectively, and then they were routinely cultured for 24 h with PBS added, maggot ES in final mass concentration of 10 μg/mL, and maggot ES in final mass concentration of 10 μg/mL respectively. The total volume of reagents in each group was the same. One batch of cells was used for scratch test, the scratch width was observed at 24 hour after scratching to detect the cell migration ability; one batch of cells was subjected to tube forming experiment, and the formation of cell tubules was observed after 24 hours of culture. The number of samples was 3 in scratch test and tube forming experiments. Data were statistically analyzed with
t test, one-way analysis of variance, least significant difference test, analysis of variance for repeated measurement, and Bonferroni method.
Results (1) Compared with those before application of MDT, fresh granulation tissue significantly increased and necrotic tissue decreased obviously in wound, and the expression of CD31 significantly increased in wound tissue of patients in DFU group after application of MDT. The expression of FAS protein in wound tissue of patients in DFU group before application of MDT was significantly lower than that in non-diabetic trauma group before debridement, and the expression of FAS protein in wound tissue of patients in DFU group after application of MDT was significantly higher than that before application of MDT. The expression of FAS mRNA in wound tissue of patients in DFU group before application of MDT was 1.00±0.17, which was significantly less than 3.87±1.02 in non-diabetic trauma group before debridement (
t=9.808,
P<0.01). The expression of FAS mRNA in wound tissue of patients in DFU group after application of MDT was 1.85±0.31, which was significantly higher than that before application of MDT (
t=-10.853,
P<0.01). (2) After 48 hours of culture, Western blotting detection showed that the expression of FAS protein in cells in high glucose alone group was significantly less than that in PBS control group, and the expressions of FAS protein in cells in high glucose+ 5 μg/mL maggot ES group and high glucose+ 10 μg/mL maggot ES group were significantly higher than the expression in high glucose alone group. Real-time fluorescent quantitative RT-PCR determination showed that the expression of FAS mRNA in cells in high glucose alone group was 0.392±0.073, which was significantly lower than 1.000±0.085 in PBS control group (
P<0.01); there was statistically significant difference between the expression of FAS mRNA in cells in high glucose+ 5 μg/mL maggot ES group (0.561±0.047) and that in high glucose+ 10 μg/mL maggot ES group (0.687±0.013) (
P<0.05), both of which were significantly higher than the expression in high glucose alone group (
P<0.01). The results of immunofluorescence detection showed that FAS protein was mainly located in the cytoplasm of cells in each group, and its expression was similar to that detected by Western blotting. (3) At 24 hour after scratch, the uncured widths of cell scratch in siRNA control+ maggot ES group and siRNA-FAS+ maggot ES group were significantly narrower than the uncured width in siRNA alone control group (
P<0.01), and the uncured width of cell scratch in siRNA-FAS+ maggot ES group was significantly wider than that in siRNA control+ maggot ES group (
P<0.01). After 24 hours of culture, the numbers of tubules in siRNA+ maggot ES group and siRNA-FAS+ maggot ES group were significantly more than the number in siRNA alone control group (
P<0.05 or
P<0.01), and the number of tubules in siRNA-FAS+ maggot ES group was obviously less than that in siRNA control+ maggot ES group (
P<0.05).
Conclusions MDT up-regulates the expression of FAS through maggot ES, which promotes the activity of vascular endothelial cells, thus promoting the wound angiogenesis in patients with DFU.
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