The possible role of quercetin, a naturally occurring plant flavonoid, in protecting against oxygen-glucose deprivation (OGD)-, excitotoxins-, and free radical-induced neuronal injury in mouse cortical cell cultures was investigated. Pre- and co-treatment with quercetin (100 microM) inhibited 50 min OGD-, 20 microM N-methyl-D-aspartate (NMDA)-, and 50 microM kainate-induced neurotoxicity by 36, 22, and 61%, respectively. Quercetin significantly ameliorated free radical-induced neuronal injury caused by buthionine sulfoximine, sodium nitroprusside, ZnCl(2), and FeCl(2). These results suggest that quercetin may contribute a neuroprotective action against ischemic neural injury, partially via antioxidant actions.

Amyloid beta (Aβ) peptide, one of the most important pathogenic traits of Alzheimer’s disease (AD), invokes a cascade of oxidative damage and eventually leads to neuronal death. In the present study, baicalein, wogonin, and oroxylin A, main active flavones in Scutellaria baicalensis, were evaluated for their neuroprotective effects against Aβ25–35-stimulated damage. All tested compounds decreased Aβ25–35-induced ROS generation and cell cycle arrest. In particular, baicalein exhibited the strongest antioxidant activity. In addition, these compounds suppressed apoptosis by attenuating mitochondrial dysfunction such as loss of membrane potential, Ca2+ accumulation and Bax/Bcl-2 ratio. Furthermore, all tested flavones inhibited the expression of iNOS and COX-2, which resulted in suppressing inflammatory cytokines including TNF-α, NO, and PGE2. Noticeably, all compounds exhibited the anti-inflammatory effects through downregulating NF-κB/MAPK pathway. Especially, oroxylin A was effective against both p65 and IκBα, while wogonin and baicalein were suppressed phospho-p65 and phospho-IκBα, respectively. Taken together, baicalein, wogonin, and oroxylin A can effectively relieve Aβ25–35-stimulated neuronal apoptosis and inflammation in PC12 cells via downregulating NF-κB/MAPK signaling pathway.

Reactive oxygen species (ROS) regulate various biological processes by modifying reactive cysteine residues in the proteins participating in the relevant signaling pathways. Identification of ROS target proteins requires specific reagents that identify ROS-sensitive cysteine sulfhydryls that differ from the known alkylating agents, iodoacetamide and N-ethylmaleimide, which react nonspecifically with oxidized cysteines including sulfenic and sulfinic acid. We designed and synthesized a novel reagent, methyl-3-nitro-4-(piperidin-1-ylsulfonyl)benzoate (NPSB-1), that selectively and specifically reacts with the sulfhydryl of cysteines in model compounds. We validated the specificity of this reagent by allowing it to react with recombinant proteins followed by peptide sequencing with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS), and mutant studies employed it to identify cellular proteins containing redox-sensitive cysteine residues. We also obtained proteins from cells treated with various concentrations of hydrogen peroxide, labeled them with biotinylated NPSB-1 (NPSB-B), pulled them down with streptavidin beads, and identified them with MS/MS. We grouped these proteins into four families: (1) those having reactive cysteine residues easily oxidized by hydrogen peroxide, (2) those with cysteines reactive only under mild oxidative stress, (3) those with cysteines reactive only after exposure to oxidative stress, and (4) those with cysteines that are reactive regardless of oxidative stress. These results confirm that NPSBs can serve as novel chemical probes for specifically capturing reactive cysteine residues and as powerful tools for measuring their oxidative sensitivity and can help to understand the function of cysteine modifications in ROS-mediated signaling pathways.

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Rutin is the major polyphenol found in buckwheat and can downregulate inflammatory responses in macrophages. However, the underlying mechanism is unclear. Overproduction of nitric oxide (NO) by inducible nitric synthase (iNOS) is closely correlated with inflammation and the pathology of a variety of diseases. It has been reported that rutin inhibited various pro-inflammatory mediators, including cytokine signaling in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, and suppressed the production of NO and the expression of cyclooxygenase-2 (COX-2) and iNOS protein in LPS-stimulated macrophages. These results suggest that rutin exerts anti-inflammatory effects by suppressing the expression of COX-2 and iNOS in RAW 264.7 macrophage cells. Therefore, rutin can be considered as a functional food for the prevention of various diseases.