The Volatile Oil of Nardostachyos Radix et Rhizoma Induces Endothelial Nitric Oxide Synthase Activity in HUVEC Cells

<div><p>Nardostahyos Radix et Rhizoma (NRR; the root and rhizome of <i>Nardostachys jatamansi</i> 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 Ca²⁺ level, and BAPTA-AM, a Ca²⁺ chelator, reduced the Ca²⁺ 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.</p></div

NRR volatile oil induces Ca²⁺ mobilization in HUVECs.

<p>Cultured HUVECs were labeled with fluorescent Ca²⁺ indicator Fluo-4 AM for 30 min. Fluorimetric measurement was performed after the treatment of NRR volatile oil (25 µg/mL), A23187 (1 µM, positive control) or control (untreated culture) (upper panel). Bar = 100 µm. Quantification of Ca²⁺ mobilization was displayed as a ratio of fluorescence intensity at 5 min (F5) to the control at time 0 (F0) in the cultures (lower panel). Mean ± SEM, <i>n</i> = 3, each with triplicate samples. **<i>p</i><0.01.</p

Major chemical composition of volatile oil from NRR.

<p>Major chemical composition of volatile oil from NRR.</p

NRR volatile oil-induced eNOS phosphorylation is mediated by PI3K/Akt signaling pathway.

<p>Cultured HUVECs were pre-treated with serum free medium or LY294002 (3 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 (0–20 min). The cell lysates were obtained for western blotting. <b>(A)</b> Phospho-Akt Ser⁴⁷³ (~60 kDa) and total Akt (~60 kDa) were revealed by using specific antibodies. <b>(B)</b> Phospho-eNOS Ser¹¹⁷⁷ (~135 kDa) and total eNOS (~135 kDa) were revealed. The quantification from the blot in (A) and (B) was performed by a densitometer (lower panel). Data were expressed as x Basal where the control was set as 1. Mean ± SEM, <i>n</i> = 3, each with triplicate samples. **<i>p</i><0.01.</p

Volatile oil-induced NO production is blocked by LY294002.

<p>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, <i>n</i> = 3, each with triplicate samples. **<i>p</i><0.01.</p

NRR volatile oil induces vasodilation of rat aortic ring.

<p><b>(A):</b> Rat aortic ring was isolated with or without intact endothelium, the vasoconstriction was induced by the applied phenylephrine (Phe, 0.5 µM); acetylcholine (ACh, 1 µM) was then added (left panel). <b>(B):</b> The contraction of aortic ring was tested similar to (A). Different concentrations of NRR volatile oil (1, 3, 10, 30 and 100 µg/mL) were added to induce the relaxation. Also, L-NAME (100 µM) was applied for 30 min, and then different concentrations of NRR volatile oil were added. Values are expressed as percentage of Phe tone as comparing to the control resting tension (right panel). Mean ± SEM, <i>n</i> = 3.</p

The FSS-induced NO production is mediated by eNOS phosphorylation at S1177.

<p>(<b>A</b>): Cultured HUVECs were seeded in 12-well plates. The cultures were serum starved for 3 hours before the pre-treatment with L-NAME (100 µM) for another 3 hours. The cells were then treated with FSS (1 mg/ml), or VEGF (20 ng/ml, positive control), or control (without drug treatment), at different time points. Total and phosphorylated eNOS was revealed by using specific antibodies (left panel). The quantification from the blots was shown by a densitometer (right panel). Data are expressed as × Basal where control was set as 1, Mean±SEM, <i>n = 4</i>. ** p<0.01. (<b>B</b>): Cultured HUVECs were labeled with fluorescent NO indicator DAF-FM DA for 30 min. Then, the cells were washed with 1× NPSS (pH = 7.4), and then fluorimetric measurements were performed after the treatment of FSS (1 mg/ml), or control (without drug treatment). For the pre-treatment of L-NAME, the cells were pre-treated with L-NAME (100 µM, eNOS blocker) for 30 min, after labeling with DAF-FM DA, NO production was induced by FSS (1 mg/ml). The amount of NO was evaluated by measuring the fluorescence intensity excited at 495 nm and emitted at 515 nm. Micrographs were taken by the confocal microscope (left panel). Values are expressed as percentage of relaxation as comparing to the control resting tension (right panel). Mean ± SEM, <i>n</i> = 4. Bar = 50 µm.</p

FSS-induced NO production is mediated by the phosphorylation of Akt at S473.

<p>Cultured HUVECs were pre-treated with triciribine (1 µM) for 3 hours before the application of FSS (1 mg/ml), or VEGF (20 ng/ml, positive control), or control (without drug treatment) for different time points. The cell lysates were obtained for western blotting. (<b>A</b>): Phospho-Akt S473 (∼60 kDa) and total Akt (∼60 kDa) were revealed by using specific antibodies of phospho-Akt S473 and total Akt. (<b>B</b>): Phospho-eNOS (∼135 kDa) and total eNOS (∼135 kDa) were revealed by using specific antibodies of phosphor-eNOS and total eNOS. (<b>C</b>): The quantification from the blots in (A) and (B) was performed by a densitometer. Data are expressed as × Basal where the control was set as 1. Mean±SEM, <i>n = 3.</i> * p<0.05, ** p<0.01.</p

FSS blocks homocysteine-induced ROS formation.

<p>(<b>A</b>): Cultured HUVECs were pre-treated with FSS (1 mg/ml), or tempol (1 µM), or control (without drug treatment) for 30 min, and then add homocysteine (300 µM, a ROS inducer) for 30 min to induce the ROS formation. The cells were then labeled with DCFH-DA (1 µM) for 30 min, then the cells were washed with 1× NPSS (pH = 7.4) and the photos were taken by using laser confocal fluorescent microscopy, which the fluorescence intensity excited at 505 nm and emitted at 535 nm (left panel). Quantitation of fluorescent was calculated from the labeled HUVECs (right panel). Values are expressed as the percentage of change in the intensity of fluorescent, as compared to control cultures. Mean ± SEM, <i>n</i> = 3. (<b>B</b>): Cultured HUVECs were seeded in cell culture flask. The cultures were pre-treated with homocysteine (300 µM) for 30 min. The cells were then treated with FSS (1 mg/ml), or tempol (1 µM, positive control), or control (without drug treatment) for another 30 min. The cells in a concentration of 5×10⁴ per well on a 96-well white plate were then subjected to chemiluminescence determination. Values are expressed as percentage of increase to basal reading (control group). Mean ± SEM, <i>n</i> = 4. (<b>C</b>): Cultured HUVECs were seeded in 6-well plates. The cultures were pre-treated with homocysteine (300 µM) for 30 min. The cells were then treated with FSS (1 mg/ml), or tempol (1 µM, positive control), or control (without drug treatment) for another 30 min. eNOS protein was revealed by specific antibodies. The amount of eNOS dimer was increased by the application of FSS, or tempol, in homocysteine-treated cells.</p