双抗生素糊剂的体外抗菌和免疫调节特性
Antimicrobial and immunomodulatory in vitro profile of double antibiotic paste.
机构信息
Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília, Brasília, Brazil.
Curso de Farmácia, Universidade Católica de Brasília, Brasília, Brazil.
出版信息
Int Endod J. 2021 Oct;54(10):1850-1860. doi: 10.1111/iej.13576. Epub 2021 Jul 9.
AIM
To evaluate the antimicrobial and immunomodulatory activity of double antibiotic paste (DAP) in an in vitro infection model.
METHODOLOGY
The minimum inhibitory and bactericidal concentrations (MIC and MBC) and the antibiofilm activities (TTC assay) of DAP and its components (ciprofloxacin and metronidazole) were evaluated against Staphylococcus aureus and Enterococcus faecalis compared with triple antibiotic paste (TAP). The cellular viability of RAW 264.7 macrophages (24 and 72 h) and L929 fibroblasts (48 and 72 h) was evaluated by MTT. Furthermore, the production of TNF-α, IL-12, IL-6, IL-1α, IL-10 and NO (on RAW 264.7), besides IL-6, TGF-β and NO (on L929), stimulated with DAP in baseline and associated with heat-killed microbial-antigen conditions was measured by ELISA and Griess reaction. Data were analysed using the one-way ANOVA test with Bonferroni's corrections.
RESULTS
The MBC of pharmacopoeia DAP was similar to TAP for E. faecalis (0.25 μg. mL ) and lower for S. aureus (DAP 1 μg. mL and TAP 2 μg. mL ; p < .001). Ciprofloxacin was the most effective antibiofilm drug from the pastes (35% of reduction for E. faecalis and S. aureus; p < .0001), and both pastes had a similar antibiofilm eradication against both biofilm species (29% and 35% for S. aureus and 76% and 85% for E. faecalis; p < .0001). DAP was cytotoxic against the tested cells. DAP significantly upregulated IL-1α (p < .001), IL-6 (p < .0001), TNF-α (p < .01) and IL-12 (p < .05; in the absence of antigens) and significantly reduced IL-6 (p < .0001; in the presence of HK-S. aureus) and IL-10 (p < .05; in the presence of both antigens) on macrophages. Furthermore, DAP upregulated IL-6 (p < .001) and NO (p < .05; in the absence of antigens), IL-6 (p < .001; in the presence of HK-S. aureus) and reduced NO (p < .001; in the presence of HK-S. aureus).
CONCLUSIONS
Double antibiotic paste and TAP had similar antimicrobial activity against S. aureus and E. faecalis. DAP upregulated pro-inflammatory cytokines mainly in the absence of antigens and had pro- and anti-inflammatory activity in RAW 264.7 macrophages and L929 fibroblasts in the presence of antigens involved in pulp infections.
目的
在体外感染模型中评估双抗生素糊剂（DAP）的抗菌和免疫调节活性。
方法
比较 DAP 及其成分（环丙沙星和甲硝唑）与三抗生素糊剂（TAP）对金黄色葡萄球菌和粪肠球菌的最小抑菌浓度（MIC）和杀菌浓度（MBC）以及抗生物膜活性（TTC 测定）。通过 MTT 评估 RAW 264.7 巨噬细胞（24 和 72 小时）和 L929 成纤维细胞（48 和 72 小时）的细胞活力。此外，通过 ELISA 和 Griess 反应测量 DAP 在基线时和与热灭活微生物抗原条件下刺激下产生的 TNF-α、IL-12、IL-6、IL-1α、IL-10 和 NO（在 RAW 264.7 上）以及 IL-6、TGF-β和 NO（在 L929 上）的产生。使用单因素方差分析和 Bonferroni 校正分析数据。
结果
药典 DAP 的 MBC 与 TAP 对粪肠球菌相似（0.25μg.ml），对金黄色葡萄球菌较低（DAP 1μg.ml 和 TAP 2μg.ml；p<0.001）。环丙沙星是糊剂中最有效的抗生物膜药物（粪肠球菌和金黄色葡萄球菌减少 35%；p<0.0001），两种糊剂对两种生物膜物种的抗生物膜清除作用相似（金黄色葡萄球菌和粪肠球菌分别为 29%和 35%和 76%和 85%；p<0.0001）。DAP 对测试细胞具有细胞毒性。DAP 显著上调 IL-1α（p<0.001）、IL-6（p<0.0001）、TNF-α（p<0.01）和 IL-12（p<0.05；在没有抗原的情况下），并显著下调 IL-6（p<0.0001；在存在热灭活金黄色葡萄球菌的情况下）和 IL-10（p<0.05；在存在两种抗原的情况下）在巨噬细胞上。此外，DAP 上调了 IL-6（p<0.001）和 NO（p<0.05；在没有抗原的情况下），IL-6（p<0.001；在存在热灭活金黄色葡萄球菌的情况下）并降低了 NO（p<0.001；在存在热灭活金黄色葡萄球菌的情况下）。
结论
双抗生素糊剂和 TAP 对金黄色葡萄球菌和粪肠球菌具有相似的抗菌活性。DAP 主要在没有抗原的情况下上调促炎细胞因子，并在存在与牙髓感染相关的抗原的情况下在 RAW 264.7 巨噬细胞和 L929 成纤维细胞中具有促炎和抗炎活性。
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