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The volatile oil of Nardostachyos Radix et Rhizoma induces endothelial nitric oxide synthase activity in HUVEC cells.

Maiwulanjiang M, Bi CW, Lee PS, Xin G, Miernisha A, Lau KM, Xiong A, Li N, Dong TT, Aisa HA, Tsim KW - PLoS ONE (2015)

Bottom Line: In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002.This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation.In HUVECs, application of NRR volatile oil elevated the intracellular Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science and Centre for Chinese Medicine, The Hong Kong University of Science and Technology, Hong Kong, China.

ABSTRACT
Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production.

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Volatile oil-induced NO production is blocked by LY294002.Cultured HUVECs were pre-treated with serum free medium or LY294002 (1 µM) for 3 hours, and then labeled with fluorescent NO indicator DAF-FM DA for 30 min. Fluorimetric measurement was performed after the treatment of NRR volatile oil (25 µg/mL), VEGF (100 ng/mL, positive control). The amounts of NO were evaluated by measuring the fluorescence intensity (upper panel). Micrographs were taken by the confocal microscope. Bar = 100 µm (upper panel). Quantification of NO production was displayed as a ratio of fluorescence intensity at 10 min (F10) to the control at time 0 (F0) in the cultures (lower panel). Mean ± SEM, n = 3, each with triplicate samples. **p<0.01.
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pone.0116761.g005: Volatile oil-induced NO production is blocked by LY294002.Cultured HUVECs were pre-treated with serum free medium or LY294002 (1 µM) for 3 hours, and then labeled with fluorescent NO indicator DAF-FM DA for 30 min. Fluorimetric measurement was performed after the treatment of NRR volatile oil (25 µg/mL), VEGF (100 ng/mL, positive control). The amounts of NO were evaluated by measuring the fluorescence intensity (upper panel). Micrographs were taken by the confocal microscope. Bar = 100 µm (upper panel). Quantification of NO production was displayed as a ratio of fluorescence intensity at 10 min (F10) to the control at time 0 (F0) in the cultures (lower panel). Mean ± SEM, n = 3, each with triplicate samples. **p<0.01.

Mentions: The activated PI3K/Akt signaling leads to phosphorylation of eNOS and increases the production of NO [15]. Here, the phosphorylation of Akt in NRR volatile oil-treated HUVECs was evaluated by using specific antibodies, i.e. phospho-Akt Ser473 (~60 kDa) and total Akt (~60 kDa). The phosphorylation of Akt was peaked, at ~7 folds, after the treatment of NRR volatile oil after 5 min. VEGF increased the phosphorylation of Akt by ~3 folds (Fig. 4A). In order to investigate possible connection between Akt pathway and eNOS phosphorylation, HUVEC cells were pre-incubated with a kinase-specific blocker, LY294002, before the application of NRR volatile oil. After the treatment, the cell lysates were subjected to analyze phosphorylation level of Akt Ser473 and eNOS Ser1177. Total Akt and total eNOS were analyzed for normalizing the phospho-proteins. The pre-treatment of LY294002 significantly reduced the phosphorylation level of Akt in volatile oil-treated HUVEC cultures (Fig. 4A). In addition, the phosphorylations of eNOS in NRR volatile oil- and VEGF-treated cultures were significantly reduced by pre-treatment of Akt inhibitor LY294002 (Fig. 4B). In both cases, total protein levels of Akt and eNOS were not altered. To confirm the role of Akt pathway in NRR volatile oil-mediated NO production in HUVECs, LY294002 was applied on HUVECs for 3 hours before determination of NO production after the treatment of volatile oil. Pre-treatment of LY294002 suppressed VEGF and volatile oil-mediated NO production in HUVEC cells (Fig. 5). These results suggested the possible connection between Akt signaling pathway and the volatile oil-mediated NO production in cultured HUVEC cells.


The volatile oil of Nardostachyos Radix et Rhizoma induces endothelial nitric oxide synthase activity in HUVEC cells.

Maiwulanjiang M, Bi CW, Lee PS, Xin G, Miernisha A, Lau KM, Xiong A, Li N, Dong TT, Aisa HA, Tsim KW - PLoS ONE (2015)

Volatile oil-induced NO production is blocked by LY294002.Cultured HUVECs were pre-treated with serum free medium or LY294002 (1 µM) for 3 hours, and then labeled with fluorescent NO indicator DAF-FM DA for 30 min. Fluorimetric measurement was performed after the treatment of NRR volatile oil (25 µg/mL), VEGF (100 ng/mL, positive control). The amounts of NO were evaluated by measuring the fluorescence intensity (upper panel). Micrographs were taken by the confocal microscope. Bar = 100 µm (upper panel). Quantification of NO production was displayed as a ratio of fluorescence intensity at 10 min (F10) to the control at time 0 (F0) in the cultures (lower panel). Mean ± SEM, n = 3, each with triplicate samples. **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359165&req=5

pone.0116761.g005: Volatile oil-induced NO production is blocked by LY294002.Cultured HUVECs were pre-treated with serum free medium or LY294002 (1 µM) for 3 hours, and then labeled with fluorescent NO indicator DAF-FM DA for 30 min. Fluorimetric measurement was performed after the treatment of NRR volatile oil (25 µg/mL), VEGF (100 ng/mL, positive control). The amounts of NO were evaluated by measuring the fluorescence intensity (upper panel). Micrographs were taken by the confocal microscope. Bar = 100 µm (upper panel). Quantification of NO production was displayed as a ratio of fluorescence intensity at 10 min (F10) to the control at time 0 (F0) in the cultures (lower panel). Mean ± SEM, n = 3, each with triplicate samples. **p<0.01.
Mentions: The activated PI3K/Akt signaling leads to phosphorylation of eNOS and increases the production of NO [15]. Here, the phosphorylation of Akt in NRR volatile oil-treated HUVECs was evaluated by using specific antibodies, i.e. phospho-Akt Ser473 (~60 kDa) and total Akt (~60 kDa). The phosphorylation of Akt was peaked, at ~7 folds, after the treatment of NRR volatile oil after 5 min. VEGF increased the phosphorylation of Akt by ~3 folds (Fig. 4A). In order to investigate possible connection between Akt pathway and eNOS phosphorylation, HUVEC cells were pre-incubated with a kinase-specific blocker, LY294002, before the application of NRR volatile oil. After the treatment, the cell lysates were subjected to analyze phosphorylation level of Akt Ser473 and eNOS Ser1177. Total Akt and total eNOS were analyzed for normalizing the phospho-proteins. The pre-treatment of LY294002 significantly reduced the phosphorylation level of Akt in volatile oil-treated HUVEC cultures (Fig. 4A). In addition, the phosphorylations of eNOS in NRR volatile oil- and VEGF-treated cultures were significantly reduced by pre-treatment of Akt inhibitor LY294002 (Fig. 4B). In both cases, total protein levels of Akt and eNOS were not altered. To confirm the role of Akt pathway in NRR volatile oil-mediated NO production in HUVECs, LY294002 was applied on HUVECs for 3 hours before determination of NO production after the treatment of volatile oil. Pre-treatment of LY294002 suppressed VEGF and volatile oil-mediated NO production in HUVEC cells (Fig. 5). These results suggested the possible connection between Akt signaling pathway and the volatile oil-mediated NO production in cultured HUVEC cells.

Bottom Line: In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002.This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation.In HUVECs, application of NRR volatile oil elevated the intracellular Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production.

View Article: PubMed Central - PubMed

Affiliation: Division of Life Science and Centre for Chinese Medicine, The Hong Kong University of Science and Technology, Hong Kong, China.

ABSTRACT
Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of Nardostachys jatamansi DC.) is a widely used medicinal herb. Historically, NRR is being used for the treatment of cardiovascular and neurological diseases. To search for active ingredients of NRR, we investigated the vascular benefit of NRR volatile oil in (i) the vasodilation in rat aorta ring, and (ii) the release of nitric oxide (NO) and the phosphorylation of endothelial NO synthase (eNOS) in cultured human umbilical vein endothelial cells (HUVECs). By measuring the fluorescence signal in cultures, application of NRR volatile oil resulted in a rapid activation of NO release as well as the phosphorylation of eNOS: both inductions were markedly reduced by L-NAME. In parallel, the phosphorylation level of Akt kinase was markedly increased by the oil treatment, which was partially attenuated by PI3K/Akt inhibitor LY294002. This inhibitor also blocked the NRR-induced NO production and eNOS phosphorylation. In HUVECs, application of NRR volatile oil elevated the intracellular Ca(2+) level, and BAPTA-AM, a Ca(2+) chelator, reduced the Ca(2+) surge: the blockage were also applied to NRR-induced eNOS phosphorylation and NO production. These findings suggested the volatile oil of NRR was the major ingredient in triggering the vascular dilatation, and which was mediated via the NO production.

Show MeSH
Related in: MedlinePlus