Chen Chun-Yu, Liaw Chih-Chuang, Chen Yi-Hsuan, Chang Wen-Yi, Chung Pei-Jen, Hwang Tsong-Long
Graduate Institute of Natural Products, School of Traditional Medicine, College of Medicine, Chang Gung University, Kweishan 333, Taoyuan, Taiwan; Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Kweishan 333, Taoyuan, Taiwan; Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan.
Free Radic Biol Med. 2014 Jul;72:222-31. doi: 10.1016/j.freeradbiomed.2014.04.018. Epub 2014 Apr 18.
Neutrophils have a crucial role in the immune system and are the first line of defense against pathogenic invaders. Neutrophil activation is required for their defensive function and can be induced by diverse stimuli, through either binding to cell surface receptors or direct intracellular target molecule stimulation. In this study, we found that 4″a,5″,6″,7″,8″,8″a-hexahydro-5,3',4'-trihydroxy-5″,5″,8″a-trimethyl-4H-chromeno [2″,3″:7,6]flavone (ugonin U), a flavonoid isolated from Helminthostachys zeylanica (L) Hook, significantly induced superoxide production and release in a time- and concentration-dependent manner. A series of experiments was performed to dissect the mechanism of ugonin U-induced respiratory burst in human neutrophils. Our results demonstrated that ugonin U induced a slow increase in intracellular Ca(2+), which was necessary for ugonin U-stimulated superoxide release. Use of a formyl peptide receptor (FPR) blocker, G protein inhibitor, and protein tyrosine kinase (PTK) inhibitor proved that FPR, G proteins, and PTKs were not associated with ugonin U-induced respiratory burst. Additionally, immunoblotting results revealed that ugonin U did not affect the phosphorylation of mitogen-activated protein kinases and protein tyrosine. Nevertheless, a phospholipase C (PLC) inhibitor and an inositol triphosphate (IP3) receptor antagonist considerably suppressed ugonin U-stimulated Ca(2+) mobilization and subsequent superoxide release. Ugonin U also induced an increase in intracellular IP3 formation, which could be blocked using a PLC inhibitor. In conclusion, our study reveals that ugonin U represents the first identified natural flavonoid compound to directly stimulate PLC. Moreover, ugonin U induces respiratory burst via the PLC/IP3/Ca(2+) pathway in human neutrophils.
中性粒细胞在免疫系统中发挥着关键作用，是抵御病原体入侵的第一道防线。中性粒细胞的激活是其防御功能所必需的，可通过与细胞表面受体结合或直接刺激细胞内靶分子等多种刺激诱导。在本研究中，我们发现从七指蕨中分离出的一种黄酮类化合物4″a,5″,6″,7″,8″,8″a-六氢-5,3',4'-三羟基-5″,5″,8″a-三甲基-4H-色烯并[2″,3″:7,6]黄酮（乌骨宁U）能以时间和浓度依赖性方式显著诱导超氧化物的产生和释放。我们进行了一系列实验来剖析乌骨宁U诱导人中性粒细胞呼吸爆发的机制。我们的结果表明，乌骨宁U诱导细胞内Ca(2+)缓慢增加，这是乌骨宁U刺激超氧化物释放所必需的。使用甲酰肽受体（FPR）阻滞剂、G蛋白抑制剂和蛋白酪氨酸激酶（PTK）抑制剂证明，FPR、G蛋白和PTK与乌骨宁U诱导的呼吸爆发无关。此外，免疫印迹结果显示，乌骨宁U不影响丝裂原活化蛋白激酶和蛋白酪氨酸的磷酸化。然而，磷脂酶C（PLC）抑制剂和肌醇三磷酸（IP3）受体拮抗剂可显著抑制乌骨宁U刺激的Ca(2+)动员及随后的超氧化物释放。乌骨宁U还诱导细胞内IP3形成增加，这可通过PLC抑制剂阻断。总之，我们的研究表明，乌骨宁U是首个被鉴定出的直接刺激PLC的天然黄酮类化合物。此外，乌骨宁U通过PLC/IP3/Ca(2+)途径在人中性粒细胞中诱导呼吸爆发。
