DataSheet1_The effects of antimicrobial peptides buCaTHL4B and Im-4 on infectious root canal biofilms.PDF

PurposeThe primary cause of pulp and periapical diseases is the invasion of bacteria into the root canal, which results from the continuous destruction of dental hard tissues. Effective management of infections during root canal therapy necessitates effectively irrigation. This study aims to investigate the effects of two antimicrobial peptides (AMPs), buCaTHL4B and Im-4, on root canal biofilms in vitro.MethodsTwo-species biofilms (Enterococcus faecalis and Fusobacterium nucleatum) were selected and anaerobically cultivated. The following treatments were applied: 10 μg/mL buCaTHL4B, 10 μg/mL Im-4, 5 μg/mL buCaTHL4B, 5 μg/mL Im-4, 1 μg/mL buCaTHL4B, 1 μg/mL Im-4, 1% NaOCl, and sterile water. Each group was treated for 3 min. Subsequently, the two strains were co-cultured with 10 μg/mL buCaTHL4B, 10 μg/mL Im-4, 1% NaOCl, and sterile water for 24, 48, and 72 h. The biofilms were examined using confocal laser scanning microscopy (CLSM) with fluorescent staining, and the percentages of dead bacteria were calculated. Quantitative real-time PCR (qRT-PCR) was employed to assess the variations in bacterial proportions during biofilm formation.ResultsCompared to 1% NaOCl, 10 μg/mL buCaTHL4B or Im-4 exhibited significantly greater bactericidal effects on the two-species biofilms (p < 0.05), leading to their selection for subsequent experiments. Over a 48-hour period, 10 μg/mL Im-4 demonstrated a stronger antibiofilm effect than buCaTHL4B (p < 0.05). Following a 24-hour biofilm formation period, the proportion of F. nucleatum decreased while the proportion of E. faecalis increased in the sterile water group. In the buCaTHL4B and 1% NaOCl groups, the proportion of F. nucleatum was lower than that of E. faecalis (p < 0.05), whereas in the Im-4 group, the proportion of F. nucleatum was higher than that of E. faecalis (p < 0.05). The proportions of bacteria in the two AMPs groups gradually stabilized after 24 h of treatment.ConclusionbuCaTHL4B and Im-4 exhibited remarkable antibacterial and anti-biofilm capabilities against pathogenic root canal biofilms in vitro, indicating their potential as promising additives to optimize the effectiveness of root canal treatment as alternative irrigants.

本研究旨在探讨两种抗菌肽（buCaTHL4B和Im-4）对体外根管生物膜的影响。主要病因为细菌侵入牙根管，导致牙硬组织的持续破坏。在根管治疗中，有效管理感染需要实施有效的冲洗。本研究选取了两种生物膜（粪肠球菌和核梭杆菌）进行厌氧培养，并对其施加了包括10μg/mL的buCaTHL4B、10μg/mL的Im-4、5μg/mL的buCaTHL4B、5μg/mL的Im-4、1μg/mL的buCaTHL4B、1μg/mL的Im-4、1%的NaOCl和无菌水等不同处理，每组处理3分钟。随后，将两种菌株与10μg/mL的buCaTHL4B、10μg/mL的Im-4、1%的NaOCl和无菌水共培养24小时、48小时和72小时。利用共聚焦激光扫描显微镜（CLSM）结合荧光染色对生物膜进行检查，并计算细菌死亡率。采用定量实时PCR（qRT-PCR）技术评估生物膜形成过程中细菌比例的变化。结果表明，与1%的NaOCl相比，10μg/mL的buCaTHL4B或Im-4对两种生物膜表现出显著更强的杀菌效果（p < 0.05），从而被选中进行后续实验。在48小时内，10μg/mL的Im-4显示出比buCaTHL4B更强的抗生物膜效果（p < 0.05）。在无菌水组中，24小时的生物膜形成后，核梭杆菌的比例下降，粪肠球菌的比例上升。在buCaTHL4B和1%的NaOCl组中，核梭杆菌的比例低于粪肠球菌（p < 0.05），而在Im-4组中，核梭杆菌的比例高于粪肠球菌（p < 0.05）。两种抗菌肽组的细菌比例在治疗后24小时逐渐稳定。结论指出，buCaTHL4B和Im-4在体外对致病性根管生物膜表现出显著的抗菌和抗生物膜活性，表明它们作为根管治疗中替代冲洗剂的潜在添加剂，有望优化根管治疗的疗效。