Department of Oral Pathology and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 130-701, Republic of Korea.
Arch Oral Biol. 2011 Oct;56(10):1091-7. doi: 10.1016/j.archoralbio.2011.03.016. Epub 2011 Apr 17.
Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs).
Human gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed.
Exposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage.
These results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs.
香烟烟雾是一种由超过 4700 种化学物质组成的复杂混合物，包括自由基和氧化剂，这是一个在世界范围内对健康造成严重问题的现象。尼古丁是烟草中的主要成分，被认为是导致牙龈炎和牙周炎的原因。它会引起细胞内氧化应激，被认为是生物分子损伤的重要因素。本研究旨在阐明尼古丁对人牙龈成纤维细胞（HGFs）的细胞毒性途径。
使用尼古丁刺激的人牙龈成纤维细胞作为体外模型。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定法检测细胞活力，并使用 2,7-二氯荧光素二乙酸酯（DCF-DA）评估活性氧（ROS）的产生。通过末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸（dUTP）缺口末端标记（TUNEL）测定法检测形态变化，并用 4,6-二脒基-2-苯基吲哚（DAPI）染色。为了阐明细胞外信号调节激酶（ERK）、P38 和 c-Jun N-末端激酶（JNK）的作用，进行了 Western blot 和 caspase-3（CASP3）活性测定。
暴露于人牙龈成纤维细胞的尼古丁会随时间和剂量依赖性降低细胞活力，并增加 ROS 的产生。它还显示出增加凋亡的形态学证据，导致 JNK 和 ERK 的短暂激活，同时激活 P38，并通过 CASP3 激活和多聚 ADP 核糖聚合酶（PARP）切割来刺激凋亡。
这些结果表明，尼古丁通过 ROS 生成和 caspase-3 依赖途径诱导 HGFs 中的细胞凋亡。
