Svensson D, Westman J, Wickström C, Jönsson D, Herwald H, Nilsson B-O
Department of Experimental Medical Science, Lund University, Lund, Sweden.
J Periodontal Res. 2015 Feb;50(1):80-8. doi: 10.1111/jre.12184. Epub 2014 Apr 4.
High levels of the antimicrobial peptide, LL-37, are detected in gingival crevicular fluid from patients with chronic periodontitis. LL-37 not only shows antimicrobial activity but also affects host-cell viability. The objective of the present study was to identify endogenous mechanisms that antagonize the detrimental effects of LL-37 on osteoblast viability, focusing on the human peptide p33 expressed on the surface of various cell types.
Human osteoblast-like MG63 cells and human hFOB1.19 osteoblasts were treated with or without LL-37 in the presence or absence of p33. Recombinant human p33 was expressed in an Escherichia coli expression system. Lactate dehydrogenase (LDH) was assessed using an enzymatic spectrophotometric assay. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation. Cell number was assessed by counting cells in a Bürker chamber. Intracellular Ca(2+) was monitored by recording Fluo 4-AM fluorescence using a laser scanning confocal microscope. Cellular expression of p33 was determined by western blotting.
LL-37 caused a concentration-dependent release of LDH from human osteoblasts, showing a half-maximal response value (EC50 ) of 4 μm and a rapid and sustained rise in the intracellular Ca(2+) concentration of osteoblasts, suggesting that LL-37 forms pores in the cell membrane. p33 (10 μm) inhibited the LL-37-induced LDH release and LL-37-evoked rise in intracellular Ca(2+) concentration, suggesting that p33 prevents LL-37-induced permeabilization of the cell membrane. Moreover, p33 blocked LL-37-induced attenuation of osteoblast numbers. Also, mucin antagonized, at concentrations representative for nonstimulated whole saliva, LL-37-evoked LDH release, whilst cationic endogenous polyamines had no impact on LL-37-induced LDH release from osteoblasts.
The endogenous peptide p33 prevents LL-37-induced reduction of human osteoblast viability. Importantly, this mechanism may protect the osteoblasts from LL-37-induced cell damage in patients suffering from chronic periodontitis associated with high levels of LL-37 locally.
在慢性牙周炎患者的龈沟液中检测到高水平的抗菌肽LL-37。LL-37不仅具有抗菌活性，还会影响宿主细胞的活力。本研究的目的是确定拮抗LL-37对成骨细胞活力有害影响的内源性机制，重点关注在各种细胞类型表面表达的人肽p33。
在有或无p33存在的情况下，用或不用LL-37处理人成骨样MG63细胞和人hFOB1.19成骨细胞。重组人p33在大肠杆菌表达系统中表达。使用酶分光光度法评估乳酸脱氢酶（LDH）。通过测量[³H]-胸苷掺入量来测定DNA合成。通过在 Bürker 计数板中计数细胞来评估细胞数量。使用激光扫描共聚焦显微镜记录Fluo 4-AM荧光来监测细胞内Ca²⁺。通过蛋白质印迹法测定p33的细胞表达。
LL-37导致人成骨细胞中LDH浓度依赖性释放，半最大反应值（EC50）为4μm，且成骨细胞内Ca²⁺浓度迅速且持续升高，表明LL-37在细胞膜上形成孔道。p33（10μm）抑制LL-37诱导的LDH释放以及LL-37引起的细胞内Ca²⁺浓度升高，表明p33可防止LL-37诱导的细胞膜通透性增加。此外，p33阻断了LL-37诱导的成骨细胞数量减少。同样，在代表未刺激全唾液的浓度下，粘蛋白拮抗LL-37引起的LDH释放，而阳离子内源性多胺对LL-37诱导的成骨细胞LDH释放没有影响。
内源性肽p33可防止LL-37诱导的人成骨细胞活力降低。重要的是，这种机制可能保护成骨细胞免受局部LL-37水平升高的慢性牙周炎患者中LL-37诱导的细胞损伤。
