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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 BAPTA-AM.Cultured HUVECs were pre-treated with serum free medium or BAPTA-AM (5 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), A23187 (1 µM, positive control). The amounts of NO were evaluated by measuring the fluorescence intensity. 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.g008: Volatile oil-induced NO production is blocked by BAPTA-AM.Cultured HUVECs were pre-treated with serum free medium or BAPTA-AM (5 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), A23187 (1 µM, positive control). The amounts of NO were evaluated by measuring the fluorescence intensity. 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: CaM kinase II is a serine/threonine-specific protein kinase that is regulated by the Ca2+/CaM complex. CaM kinase II could mediate the rapid activation of eNOS and vasodilation [16]. Thus, intracellular Ca2+ change and eNOS phosphorylation were investigated after treatment of NRR volatile oil in HUVECs. Fluo-4 AM, a Ca2+ indicator, was applied to monitor the change of Ca2+-induced fluorescence signal in HUVECs. The increase of Ca2+ level was found after treatment of NRR volatile oil (Fig. 6). Pre-treatment of Ca2+ chelator, BAPTA-AM, markedly suppressed Ca2+ increase, both in NRR volatile oil- and A23187-treated HUVECs (Fig. 6). Cultured HUVECs were pre-treated with BAPTA-AM for 3 hours, and then the amount of phosphorylated eNOS was determined after the treatment of NRR volatile oil. The pre-treatment of BAPTA-AM fully reduced the volatile oil-induced eNOS phosphorylation (Fig. 7). In addition, BAPTA-AM pre-treated HUVEC cultures were subjected to the measurement of NO production after treatment of NRR volatile oil. The volatile oil-mediated NO production was suppressed by pre-treatment of BAPTA-AM (Fig. 8). The blockage by BAPTA-AM was also applied to the case of A23187 in inducing eNOS phosphorylation and NO production (Figs. 7 and 8).


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 BAPTA-AM.Cultured HUVECs were pre-treated with serum free medium or BAPTA-AM (5 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), A23187 (1 µM, positive control). The amounts of NO were evaluated by measuring the fluorescence intensity. 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

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

pone.0116761.g008: Volatile oil-induced NO production is blocked by BAPTA-AM.Cultured HUVECs were pre-treated with serum free medium or BAPTA-AM (5 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), A23187 (1 µM, positive control). The amounts of NO were evaluated by measuring the fluorescence intensity. 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: CaM kinase II is a serine/threonine-specific protein kinase that is regulated by the Ca2+/CaM complex. CaM kinase II could mediate the rapid activation of eNOS and vasodilation [16]. Thus, intracellular Ca2+ change and eNOS phosphorylation were investigated after treatment of NRR volatile oil in HUVECs. Fluo-4 AM, a Ca2+ indicator, was applied to monitor the change of Ca2+-induced fluorescence signal in HUVECs. The increase of Ca2+ level was found after treatment of NRR volatile oil (Fig. 6). Pre-treatment of Ca2+ chelator, BAPTA-AM, markedly suppressed Ca2+ increase, both in NRR volatile oil- and A23187-treated HUVECs (Fig. 6). Cultured HUVECs were pre-treated with BAPTA-AM for 3 hours, and then the amount of phosphorylated eNOS was determined after the treatment of NRR volatile oil. The pre-treatment of BAPTA-AM fully reduced the volatile oil-induced eNOS phosphorylation (Fig. 7). In addition, BAPTA-AM pre-treated HUVEC cultures were subjected to the measurement of NO production after treatment of NRR volatile oil. The volatile oil-mediated NO production was suppressed by pre-treatment of BAPTA-AM (Fig. 8). The blockage by BAPTA-AM was also applied to the case of A23187 in inducing eNOS phosphorylation and NO production (Figs. 7 and 8).

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