Neuroprotective effects of Cyperus rotundus on SIN-1 induced nitric oxide generation and protein nitration: ameliorative effect against apoptosis mediated neuronal cell damage.

Nitrosylation of tyrosine (3-nitro tyrosine, 3-NT) has been implicated in the pathophysiology of various disorders particularly neurodegenerative conditions and aging. Cyperus rotundus rhizome is being used as a traditional folk medicine to alleviate a variety of disorders including neuronal stress. The herb has recently found applications in food and confectionary industries also. In current study, we have explored the protective effects of C. rotundus rhizome extract (CRE) through its oxido-nitrosative and anti apoptotic mechanism to attenuate peroxynitrite (ONOO(-)) induced neurotoxicity using human neuroblastoma SH-SY5Y cells. Our results elucidate that pre-treatment of neurons with CRE ameliorates the mitochondrial and plasma membrane damage induced by 500 µM SIN-1 to 80% and 24% as evidenced by MTT and LDH assays. CRE inhibited NO generation by downregulating i-NOS expression. SIN-1 induced depletion of antioxidant enzyme status was also replenished by CRE which was confirmed by immunoblot analysis of SOD and CAT. The CRE pre-treatment efficiently potentiated the SIN-1 induced apoptotic biomarkers such as bcl-2 and caspase-3 which orchestrate the proteolytic damage of the cell. The ONOO(-) induced damage to cellular, nuclear and mitochondrial integrity was also restored by CRE. Furthermore, CRE pre-treatment also regulated the 3-NT formation which shows the potential of plant extract against tyrosine nitration. Taken together, our findings suggest that CRE might be developed as a preventive agent against ONOO(-) induced apoptosis.

The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity: involvement of its flavonoid constituents and protein kinase C.

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. Numerous studies have shown that the Ginkgo biloba extract EGb 761 is a NO scavenger with neuroprotective properties. However, the mechanisms underlying its neuroprotective ability remain to be fully established. Thus, we investigated the effect of different constituents of EGb 761, i.e., flavonoids and terpenoids, against toxicity induced by NO generators on cells of the hippocampus, a brain area particularly susceptible to neurodegenerative damage. Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP; 100 microM) or 3-morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free radical accumulation. These SNP-induced events were blocked by either EGb 761 (10-100 microg/ml) or its flavonoid fraction CP 205 (25 microg/ml), as well as by inhibitors of protein kinase C (PKC; chelerythrine) and L-type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3-[(4-octadecyl)benzoyl]acrylic acid (OBAA)] and C (U-73122), failed to display any significant effects. Moreover, EGb 761 (50 microm) CP 205 (25 microg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 microg/ml) was shown to block the activation of PKC induced by SNP (100 microM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also via their ability to inhibit NO-stimulated PKC activity.

SIN-1-induced cytotoxicity in cultured endothelial cells involves reactive oxygen species and nitric oxide: protective effect of sepiapterin.

The purpose of this study was to examine whether tetrahydrobiopterin (BH4), one of the cofactors of nitric oxide (NO) synthase, attenuates endothelial cell death induced by 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), which is known to produce both superoxide and NO. Endothelial cell death was assessed by the release of intracellular lactate dehydrogenase (LDH). Addition of SIN-1 (500, 1,000 microM) to endothelial cells induced cell death from 6 h after its addition. The SIN-1-induced endothelial cell death was strongly reduced by treatment with carboxy-PTIO, a NO scavenger, or superoxide dismutase (SOD). Iron chelators and hydroxyl radical scavengers also reduced the SIN-1-induced endothelial cell death. Interestingly, the SIN-1-induced endothelial cell death was also reduced by treatment with catalase. Thus NO, superoxide, hydroxyl radical, and hydrogen peroxide are likely to be implicated in SIN-1-induced endothelial cell death. Moreover, pretreatment with sepiapterin, a precursor of BH4 synthesis, reduced the SIN-1-induced endothelial cell death and increased the intracellular BH4 content. Both the protective effect of sepiapterin and the increase in intracellular BH4 content were prevented by co-pretreatment with N-acetylserotonin (NAS), an inhibitor of BH4 synthesis. The protective effect of sepiapterin also was observed when up-take of trypan blue was used as another marker of cell death. These findings suggest that BH4 has a protective effect against endothelial cell death caused by the presence of NO and superoxide. The protective effect of BH4 may at least partly involve scavenging of superoxide or hydrogen peroxide or both, because we and other groups previously found that BH4 has a scavenging activity for reactive oxygen species.