Department of Biological Science, College of Medical and Life Sciences, Silla University, Busan, Korea.
J Periodontol. 2013 Apr;84(4):545-55. doi: 10.1902/jop.2012.120180. Epub 2012 Jul 6.
Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage-like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action.
NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real-time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) mRNA expression. iNOS and HO-1 protein expression and phosphorylation of c-Jun N-terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox-sensitive fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate.
Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS-stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO-1 expression in LPS-activated cells. Inhibition of HO-1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS-induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS-induced NO.
Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS-stimulated RAW264.7 cells at the translational level via HO-1-mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.
一氧化氮（NO）可能成为开发牙周病治疗新疗法的潜在靶点，因为这种分子在牙周炎中观察到的组织破坏中发挥着重要作用。在这项研究中，作者研究了山奈酚对脂多糖（LPS）刺激的鼠巨噬细胞样 RAW264.7 细胞产生 NO 的影响，LPS 来自中间普氏菌，这是一种与牙周病有关的病原体，并试图确定其作用机制。
通过测量培养上清液中亚硝酸盐的积累来测定 NO 的产生。通过实时聚合酶链反应定量测定诱导型一氧化氮合酶（iNOS）和血红素加氧酶-1（HO-1）mRNA 的表达。通过免疫印迹分析来表征 iNOS 和 HO-1 蛋白表达以及 c-Jun N-末端激酶和 p38 的磷酸化。通过使用氧化还原敏感荧光探针 2'，7'-二氯二氢荧光素二乙酸酯来测量活性氧（ROS）的产生。
山奈酚显著抑制了中间普氏菌 LPS 刺激的 RAW246.7 细胞中 NO 的产生和 iNOS 蛋白的表达，而不影响 iNOS mRNA 的表达。山奈酚上调了 LPS 激活细胞中的 HO-1 表达。HO-1 活性的抑制物锡原卟啉 IX（SnPP）消除了山奈酚对 NO 产生的抑制作用。此外，山奈酚显著减弱了中间普氏菌 LPS 诱导的细胞内 ROS 的增加，而 SnPP 阻断了这种减少。抗氧化剂的处理下调了 LPS 诱导的 NO 的产生。
山奈酚通过 HO-1 介导的 ROS 减少在中间普氏菌 LPS 刺激的 RAW264.7 细胞中抑制 NO 的产生和 iNOS 蛋白的表达，并且可能是治疗牙周病中宿主反应的有效调节剂。
