Posgrado en Ciencias Biológicas, Unidad de Posgrado, Circuito de Posgrados, Ciudad Universitaria, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.
Departamento de Microbiología y Parasitologia, Facultad de Medicina, Ciudad Universitaria, Universidad Nacional Autónoma de México, Ciudad de México, Mexico.
PeerJ. 2024 Apr 30;12:e17252. doi: 10.7717/peerj.17252. eCollection 2024.
Periodontitis is a chronic infectious disease, characterized by an exacerbated inflammatory response and a progressive loss of the supporting tissues of the teeth. is a key etiologic agent in periodontitis. Cystatin C is an antimicrobial salivary peptide that inhibits the growth of . This study aimed to evaluate the antimicrobial activity of this peptide and its effect on cytokine production, nitric oxide (NO) release, reactive oxygen species (ROS) production, and programmed cell death in human macrophages infected with
Monocyte-derived macrophages generated from peripheral blood were infected with (MOI 1:10) and stimulated with cystatin C (2.75 µg/ml) for 24 h. The intracellular localization of and cystatin C was determined by immunofluorescence and transmission electron microscopy (TEM). The intracellular antimicrobial activity of cystatin C in macrophages was assessed by counting Colony Forming Units (CFU). ELISA assay was performed to assess inflammatory (TNFα, IL-1β) and anti-inflammatory (IL-10) cytokines. The production of nitrites and ROS was analyzed by Griess reaction and incubation with 2',7'-dichlorodihydrofluorescein diacetate (HDCFDA), respectively. Programmed cell death was assessed with the TUNEL assay, Annexin-V, and caspase activity was also determined.
Our results showed that cystatin C inhibits the extracellular growth of . In addition, this peptide is internalized in the infected macrophage, decreases the intracellular bacterial load, and reduces the production of inflammatory cytokines and NO. Interestingly, peptide treatment increased ROS production and substantially decreased bacterial-induced macrophage apoptosis.
Cystatin C has antimicrobial and immuno-regulatory activity in macrophages infected with These findings highlight the importance of understanding the properties of cystatin C for its possible therapeutic use against oral infections such as periodontitis.
牙周炎是一种慢性传染病，其特征为炎症反应加剧和牙齿支持组织的进行性丧失。 是牙周炎的主要病因。半胱氨酸蛋白酶抑制剂 C 是一种抗微生物唾液肽，可抑制 的生长。本研究旨在评估该肽的抗菌活性及其对感染 的人巨噬细胞产生细胞因子、一氧化氮 (NO) 释放、活性氧 (ROS) 产生和程序性细胞死亡的影响。
从外周血中生成单核细胞衍生的巨噬细胞，用 （MOI 1:10）感染，并以半胱氨酸蛋白酶抑制剂 C（2.75 µg/ml）刺激 24 小时。通过免疫荧光和透射电子显微镜（TEM）确定 和半胱氨酸蛋白酶抑制剂 C 的细胞内定位。通过计算集落形成单位（CFU）评估巨噬细胞中半胱氨酸蛋白酶抑制剂 C 的细胞内抗菌活性。通过 ELISA 测定评估炎症（TNFα、IL-1β）和抗炎（IL-10）细胞因子。通过 Griess 反应和用 2',7'-二氯二氢荧光素二乙酸酯（HDCFDA）孵育分别分析亚硝酸盐和 ROS 的产生。通过 TUNEL 测定评估程序性细胞死亡，通过 Annexin-V 和 caspase 活性测定也评估程序性细胞死亡。
我们的结果表明，半胱氨酸蛋白酶抑制剂 C 抑制 的细胞外生长。此外，该肽被内化到感染的巨噬细胞中，降低了细胞内细菌负荷，并减少了炎症细胞因子和 NO 的产生。有趣的是，肽处理增加了 ROS 的产生，并大大降低了细菌诱导的巨噬细胞凋亡。
半胱氨酸蛋白酶抑制剂 C 对感染 的巨噬细胞具有抗菌和免疫调节活性。这些发现强调了了解半胱氨酸蛋白酶抑制剂 C 特性对于其在治疗牙周炎等口腔感染中的可能应用的重要性。
