Limits...
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.

Show MeSH

Related in: MedlinePlus

Phosphorylation of eNOS by NRR volatile oil in cultured HUVEC cells.Cultured HUVECs were pre-treated with serum free medium or L-NAME (100 µM) for 3 hours, and treated with NRR volatile oil (25 µg/mL), VEGF (10 ng/mL, positive control) or control (serum free medium) for different time points (0 to 20 min). Phospho-eNOS Ser1177 (~135 kDa) and total eNOS (~135 kDa) were revealed by using specific antibodies (upper panel). Quantification of phospho-eNOS protein (at 5 min) from the blot was calculated by a densitometer (lower panel). Data were expressed as x Basal where the control (untreated culture) was set as 1, Mean ± SEM, n = 4, each with triplicate samples. **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359165&req=5

pone.0116761.g003: Phosphorylation of eNOS by NRR volatile oil in cultured HUVEC cells.Cultured HUVECs were pre-treated with serum free medium or L-NAME (100 µM) for 3 hours, and treated with NRR volatile oil (25 µg/mL), VEGF (10 ng/mL, positive control) or control (serum free medium) for different time points (0 to 20 min). Phospho-eNOS Ser1177 (~135 kDa) and total eNOS (~135 kDa) were revealed by using specific antibodies (upper panel). Quantification of phospho-eNOS protein (at 5 min) from the blot was calculated by a densitometer (lower panel). Data were expressed as x Basal where the control (untreated culture) was set as 1, Mean ± SEM, n = 4, each with triplicate samples. **p<0.01.

Mentions: The production of NO in HUVEC cells was determined after treatment of NRR volatile oil. Endogenous NO production was monitored using specific dye DAF-FM DM. Application of NRR volatile oil triggered a progressive rise in intracellular NO production in cultured HUVECs, as reflected by an increase of fluorescence intensity, which peaked at around 8 min after the drug treatment (Fig. 2). A23187, a calcium ionophore, was employed as a positive control to evoke NO production (Fig. 2). The maximal NO production was better in the scenario of NRR oil treatment. Additionally, L-NAME, an eNOS blocker, also markedly attenuated the volatile oil-mediated NO production in HUVEC cells (Fig. 2), suggesting that NRR volatile oil-induced NO production was mediated by eNOS in HUVECs. To investigate the role of eNOS and its upstream regulators, the phosphorylation of eNOS was first determined to reflect the eNOS activity. The phosphorylation of eNOS at Ser1177 (~135 kDa) in HUVECs was observed by 5 min treatment of the oil. Vascular endothelial growth factor (VEGF), a positive control, markedly elevated the phosphorylation of eNOS by ~8 folds (Fig. 3). The phosphorylation of eNOS after treatment of VEGF and NRR volatile oil was reduced by pre-treatment of L-NAME in HUVECs (Fig. 3).


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)

Phosphorylation of eNOS by NRR volatile oil in cultured HUVEC cells.Cultured HUVECs were pre-treated with serum free medium or L-NAME (100 µM) for 3 hours, and treated with NRR volatile oil (25 µg/mL), VEGF (10 ng/mL, positive control) or control (serum free medium) for different time points (0 to 20 min). Phospho-eNOS Ser1177 (~135 kDa) and total eNOS (~135 kDa) were revealed by using specific antibodies (upper panel). Quantification of phospho-eNOS protein (at 5 min) from the blot was calculated by a densitometer (lower panel). Data were expressed as x Basal where the control (untreated culture) was set as 1, Mean ± SEM, n = 4, 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.g003: Phosphorylation of eNOS by NRR volatile oil in cultured HUVEC cells.Cultured HUVECs were pre-treated with serum free medium or L-NAME (100 µM) for 3 hours, and treated with NRR volatile oil (25 µg/mL), VEGF (10 ng/mL, positive control) or control (serum free medium) for different time points (0 to 20 min). Phospho-eNOS Ser1177 (~135 kDa) and total eNOS (~135 kDa) were revealed by using specific antibodies (upper panel). Quantification of phospho-eNOS protein (at 5 min) from the blot was calculated by a densitometer (lower panel). Data were expressed as x Basal where the control (untreated culture) was set as 1, Mean ± SEM, n = 4, each with triplicate samples. **p<0.01.
Mentions: The production of NO in HUVEC cells was determined after treatment of NRR volatile oil. Endogenous NO production was monitored using specific dye DAF-FM DM. Application of NRR volatile oil triggered a progressive rise in intracellular NO production in cultured HUVECs, as reflected by an increase of fluorescence intensity, which peaked at around 8 min after the drug treatment (Fig. 2). A23187, a calcium ionophore, was employed as a positive control to evoke NO production (Fig. 2). The maximal NO production was better in the scenario of NRR oil treatment. Additionally, L-NAME, an eNOS blocker, also markedly attenuated the volatile oil-mediated NO production in HUVEC cells (Fig. 2), suggesting that NRR volatile oil-induced NO production was mediated by eNOS in HUVECs. To investigate the role of eNOS and its upstream regulators, the phosphorylation of eNOS was first determined to reflect the eNOS activity. The phosphorylation of eNOS at Ser1177 (~135 kDa) in HUVECs was observed by 5 min treatment of the oil. Vascular endothelial growth factor (VEGF), a positive control, markedly elevated the phosphorylation of eNOS by ~8 folds (Fig. 3). The phosphorylation of eNOS after treatment of VEGF and NRR volatile oil was reduced by pre-treatment of L-NAME in HUVECs (Fig. 3).

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