Seo Taegun, Cha Seho, Woo Kyung Mi, Park Yun-Soo, Cho Yun-Mi, Lee Jeong-Soon, Kim Tae-Il
Department of Life Science, Dongguk University-Seoul, Seoul, Korea.
J Periodontal Implant Sci. 2011 Feb;41(1):17-22. doi: 10.5051/jpis.2011.41.1.17. Epub 2011 Feb 28.
Nitric oxide (NO) has been known as an important regulator of osteoblasts and periodontal ligament cell activity. This study was performed to investigate the relationship between NO-mediated cell death of human periodontal ligament fibroblasts (PDLFs) and N-methyl-D-aspartic acid (NMDA) receptor antagonist (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine hydrogen maleate (MK801).
Human PDLFs were treated with various concentrations (0 to 4 mM) of sodium nitroprusside (SNP) with or without 200 µM MK801 in culture media for 16 hours and the cell medium was then removed and replaced by fresh medium containing MTS reagent for cell proliferation assay. Western blot analysis was performed to investigate the effects of SNP on the expression of Bax, cytochrome c, and caspase-3 proteins. The differences for each value among the sample groups were compared using analysis of variance with 95% confidence intervals.
In the case of SNP treatment, as a NO donor, cell viability was significantly decreased in a concentration-dependent manner. In addition, a synergistic effect was shown when both SNP and NMDA receptor antagonist was added to the medium. SNP treated PDLFs exhibited a round shape in culture conditions and were dramatically reduced in cell number. SNP treatment also increased levels of apoptotic marker protein, such as Bax and cytochrome c, and reduced caspase-3 in PDLFs. Mitogen-activated protein kinase signaling was activated by treatment of SNP and NMDA receptor antagonist.
These results suggest that excessive production of NO may induce apoptosis and that NMDA receptor may modulate NO-induced apoptosis in PDLFs.
一氧化氮（NO）是成骨细胞和牙周膜细胞活性的重要调节因子。本研究旨在探讨NO介导的人牙周膜成纤维细胞（PDLFs）细胞死亡与N-甲基-D-天冬氨酸（NMDA）受体拮抗剂（+）-5-甲基-10,11-二氢-5H-二苯并[a,d]环庚烯-5,10-亚胺马来酸盐（MK801）之间的关系。
在培养基中用不同浓度（0至4 mM）的硝普钠（SNP）处理人PDLFs，同时或不添加200 μM MK801，处理16小时，然后去除细胞培养基，更换为含有MTS试剂的新鲜培养基进行细胞增殖测定。进行蛋白质免疫印迹分析以研究SNP对Bax、细胞色素c和半胱天冬酶-3蛋白表达的影响。使用95%置信区间的方差分析比较样本组间各值的差异。
作为NO供体，SNP处理后，细胞活力以浓度依赖性方式显著降低。此外，当SNP和NMDA受体拮抗剂同时添加到培养基中时，显示出协同作用。在培养条件下，SNP处理的PDLFs呈现圆形，细胞数量显著减少。SNP处理还增加了PDLFs中凋亡标记蛋白如Bax和细胞色素c的水平，并降低了半胱天冬酶-3的水平。丝裂原活化蛋白激酶信号通路通过SNP和NMDA受体拮抗剂的处理而被激活。
这些结果表明，NO的过量产生可能诱导细胞凋亡，并且NMDA受体可能调节PDLFs中NO诱导的细胞凋亡。
