Li F F, Zhang J, Gao C C, Wang T Y, Zhi L M, Wang J, Wang A P
Department of Endocrinology, the Second Hospital of Anhui Medical University, Hefei 230601, China.
Diabetes Foot Center, Department of Endocrinology, Air Force Hospital of Eastern Theater Command, Nanjing 210002, China.
Zhonghua Shao Shang Za Zhi. 2021 May 20;37(5):413-419. doi: 10.3760/cma.j.cn501120-20210312-00085.
To investigate the effects of medical maggot excretions/secretions (ES) on neutrophils phagocytosis and bactericidal effect in patients with diabetic foot ulcer (DFU). The experimental research method was used. Thirty DFU patients (16 males and 14 females, aged (64±7) years)who were admitted to the Diabetes Foot Center, the Department of Endocrinology of Air Force Hospital of Eastern Theater Command from June to December 2020 and met the inclusion criteria were recruited. Discontinuous percoll gradient centrifugation method was used to separate the neutrophils. Cells from each patient were enrolled into normal saline group and maggot ES group (30 wells in each group), respectively; sterile normal saline and ES with a final mass concentration of 357 μg/mL (the same as below) were added, respectively. After 1 and 2 hour(s) of culture, the phagocytosis rate and phagocytic index of cells were observed and counted under Wright's staining. Ten patients were selected, then the cells of each patient were enrolled into +neutrophils group and +neutrophils+maggot ES group (10 wells in each group) and were treated corresponding, respectively. alone group and +maggot ES group (10 wells in each group) were set up respectively; +RPMI 1640 culture medium+sterile normal saline and +RPMI 1640 culture medium+maggot ES were added, respectively. After 2 hours of culture, the number of viable bacteria colony was counted by plate colony number method. Six, six, and three patients were selected respectively, and the cells of each patient were respectively enrolled into maggot ES group and normal saline group (6, 6, and 3 wells in each group, respectively) and treated accordingly. After 6 hours of culture, real-time fluorescent quantitative reverse transcription polymerase chain reaction was used to detect the mRNA expressions of interleukin 1β (IL-1β), IL-6, and lysozyme in cells, the content of IL-1β and IL-6 in cell culture supernatant were determined by enzyme-linked immunosorbent assay, and the positive cells expressing lysozyme were observed with immunofluorescence method. Data were statistically analyzed with one-way analysis of variance, paired sample test, least significant difference test, and Wilcoxon rank sum test. After 1 hour of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (53.5% (49.7%, 58.0%) and 3.18 (2.96, 3.32)) were similar to 52.0% (47.5%, 55.2%) and 3.15 (2.96, 3.25) of normal saline group (=-1.701, -1.092, >0.05). After 2 hours of culture, the phagocytosis rate and phagocytic index of cells in maggot ES group (70.0% (66.7%, 72.0%) and 4.47 (4.22, 4.96)) were significantly higher than 58.0% (55.0%, 60.0%) and 4.11 (3.52, 4.24) in normal saline group (=-4.786, -4.279, <0.01). After 2 hours of culture, the number of viable bacteria colony in +neutrophils group was significantly lower than that in alone group (<0.01), and the number of viable bacteria colony in +neutrophils+maggot ES group was significantly lower than that in +maggot ES group and +neutrophils group (<0.01). After 6 hours of culture, the mRNA expressions of IL-1β, IL-6, and lysozyme of cells in maggot ES group were significantly higher those in normal saline group (=-3.279, -4.273, -4.763, <0.05 or <0.01); the concent of IL-1β and IL-6 in cell culture supernatant of maggot ES group were significantly higher than those of normal saline group (=-9.526, -6.447, <0.01); there were significantly more positive cells expressing lysozyme in maggot ES group than in normal saline group. Maggot ES can enhance the phagocytosis and bactericidal effect of neutrophils on Pseudomonas aeruginosa by promoting the production of neutrophils immune defense related cytokines and lysozyme in DFU patients.
探讨医用蛆虫分泌物/排泄物（ES）对糖尿病足溃疡（DFU）患者中性粒细胞吞噬及杀菌作用的影响。采用实验研究方法。选取2020年6月至12月东部战区空军医院内分泌科糖尿病足中心收治的符合纳入标准的30例DFU患者（男16例，女14例，年龄（64±7）岁）。采用间断性Percoll梯度离心法分离中性粒细胞。将每位患者的细胞分别纳入生理盐水组和蛆虫ES组（每组30孔）；分别加入无菌生理盐水和终质量浓度为357μg/mL的ES（下同）。培养1、2小时后，经瑞氏染色观察并计数细胞的吞噬率和吞噬指数。选取10例患者，再将每位患者的细胞分别纳入+中性粒细胞组和+中性粒细胞+蛆虫ES组（每组10孔）并分别进行相应处理。分别设置单独组和+蛆虫ES组（每组10孔）；分别加入+RPMI 1640培养基+无菌生理盐水和+RPMI 1640培养基+蛆虫ES。培养2小时后，采用平板菌落计数法计数活菌菌落数。分别选取6例、6例和3例患者，再将每位患者的细胞分别纳入蛆虫ES组和生理盐水组（每组分别为6孔、6孔和3孔）并进行相应处理。培养6小时后，采用实时荧光定量逆转录聚合酶链反应检测细胞中白细胞介素1β（IL-1β）、IL-6和溶菌酶的mRNA表达，采用酶联免疫吸附测定法测定细胞培养上清液中IL-1β和IL-6的含量，采用免疫荧光法观察表达溶菌酶的阳性细胞。数据采用单因素方差分析、配对样本t检验、最小显著差检验和Wilcoxon秩和检验进行统计学分析。培养1小时后，蛆虫ES组细胞的吞噬率和吞噬指数分别为53.5%（49.7%，58.0%）和3.18（2.96，3.32），与生理盐水组的52.0%（47.5%，55.2%）和3.15（2.96，3.25）相似（t=-1.701，-1.092，P>0.05）。培养2小时后，蛆虫ES组细胞的吞噬率和吞噬指数分别为70.0%（66.7%，72.0%）和4.47（4.22，4.96），显著高于生理盐水组的58.0%（55.0%，60.0%）和4.11（3.52，4.24）（t=-4.786，-4.279，P<0.01）。培养2小时后，+中性粒细胞组的活菌菌落数显著低于单独组（P<0.01），+中性粒细胞+蛆虫ES组的活菌菌落数显著低于+蛆虫ES组和+中性粒细胞组（P<0.01）。培养6小时后，蛆虫ES组细胞中IL-1β、IL-6和溶菌酶的mRNA表达显著高于生理盐水组（t=-3.279，-4.273，-4.763，P<0.05或P<0.01）；蛆虫ES组细胞培养上清液中IL-1β和IL-6的含量显著高于生理盐水组（t=-9.526，-6.447，P<0.01）；蛆虫ES组表达溶菌酶的阳性细胞显著多于生理盐水组。蛆虫ES可通过促进DFU患者中性粒细胞免疫防御相关细胞因子和溶菌酶的产生，增强中性粒细胞对铜绿假单胞菌的吞噬及杀菌作用。
