Effect of scutellarin on nitric oxide production in early stages of neuron damage induced by hydrogen peroxide
Introduction
Flavonoids are a group of naturally occurring compounds, which are widely distributed in nature. They exhibit a variety of biological activities such as antioxidant and cardiovascular protection actions [1], [2]. Scutellarin (Fig. 1) is a flavonoid derived from the traditional Chinese medicine Erigeron breviscapus (Vant). In recent years, many studies have provided evidence for the neuroprotective effects of E. breviscapus [3]. The result of our previous studies showed that scutellarin is an effective radical scavenger in vitro (including hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide) [4]. Our studies also demonstrate that scutellarin exert a potent protective effect against oxidative damage in synaptosomes induced by superoxide [5].
An imbalance between antioxidants and reactive oxygen species (ROS) results in oxidative stress, leading to cellular oxidative damage [6], [7]. Hydrogen peroxide is a common reactive oxygen species, which was produced during the degradation of fatty acids and other molecules that produce as a by-product [8]. NO is another reactive nitrogen species which has been identified as a neuronal messenger in the central nervous system (CNS) and takes an active part in the regulation of physiologically significant functions of the cardiovascular, immune, and CNS [9], [10]. NO is synthesized from l-arginine by a reaction catalyzed by the NO synthases (NOS) [11]. In order to explore whether the generation of NO could be affected by hydrogen peroxide and the free radical scavenger scutellarin could suppress this affection, we used a selective NO electrode to directly assess the dynamics of NO release during the early stage of neuron injury induced by hydrogen peroxide. The alteration of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) activities, MDA level were also studied.
Section snippets
Chemicals
Scutellarin (purity >96%, HPLC) was provided by Yuxi Pharmaceutical Co. Ltd. (Yunnan, China). S-nitroso-N-acetylpenicillamine (SNAP, purity 98%) was received from World Precision Instruments, Inc. (WPI; Sarasota, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma (St. Louis, MO, USA). NOS kits and MDA kits were purchased from Jiancheng Bioenineering Institute (Nanjing, China). Dulbecco's modified Eagle's medium (DMEM), fetal calf serum and horse
Inhibitory effect of scutellarin on NO release
For calibration, 20, 40, 80, 160 μl of SNAP solution (0.02 mg ml−1) were used as a generator of NO. The relationship between the output current of the electrode and the NO concentration was y = 18.781x + 2.5021, R2 = 0.9955.
Previous studies suggested that scutellarin is an efficient free radical scavenger in vitro [4], [5]. Based on these studies, we hypothesized that scutellarin could inhibit the release of hydrogen peroxide-induced NO production in neuronal cells. As shown in Fig. 2A, NO production
Discussion
The main use of scutellarin in clinic is to treat cerebral ischemic injury. More and more evidence is presented indicating that, NO, as an important intra- and intercellular messenger molecule, plays an important role in the mechanisms of cerebral ischemia. On the one hand, the production of NO in blood vessels causes vasodilation and improves blood flow in the penumbral region of brain infarcts. On the other hand, brain ischemia leads to the generation of superoxide. NO promotes oxidative
Acknowledgements
Financial support of this work by the Ministry of Science and Technology of the People's Republic of China (2001BA310A07) is gratefully acknowledged.
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