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

February The Volatile Oil of Nardostachyos Radix et Rhizoma Induces Endothelial Nitric Oxide Synthase Activity in HUVEC Cells Maitinuer Maiwulanjiang 0 1 Cathy W. C. Bi 0 1 Pinky S. C. Lee 0 1 Guizhong Xin 0 1 Abudureyimu Miernisha 0 1 Kei M. Lau 0 1 Aizhen Xiong 0 1 Ning Li 0 1 Tina T. X. Dong 0 1 Haji A. Aisa 0 1 Karl W. K. Tsim 0 1 0 1 Division of Life Science and Centre for Chinese Medicine, The Hong Kong University of Science and Technology , Hong Kong , China , 2 Xinjiang Key Laboratory of Plant Resources and Natural Products Chemistry, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences , Urumqi , China 1 Academic Editor: Yu Huang, The Chinese University of Hong Kong , HONG KONG 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 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. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), GRF (661110, 662911, 660411, 663012, 662713), TUYF12SC03, The Hong Kong Jockey Club Charities Trust and Foundation of The Awareness of Nature (TAON12SC01) to Karl Tsim. M. Maiwulanjiang received a Kanya Lee Scholarship from Lees Pharmaceutical C. Ltd. The funders had no roles in Nardostachyos Radix et Rhizoma, the root and rhizome of Nardostachys jatamasi DC., is widely distributed in hilly areas of China, India, Pakistan and Nepal [13]. The water decoction of NRR has been used as folk medicine for the treatment of cardiovascular and neuronal diseases [46]. In Xinjiang of China, NRR is frequently used as a herbal decoction for cardiovascular disease, e.g. arrhythmia, coronary heart diseases and atherosclerosis [78]. In traditional Uyghur medicine, Song Bu Li decoction is prepared from water extract and volatile oil of NRR, and this study design, data collection and analysis, decision to publish, or preparation of the manuscript. herbal decoction is mainly used for cardiovascular dysfunction [9]. NRR is highly rich in volatile oil, about 2% by total weight [6], and which plays synergistic role with NRR water extract in activities of neuroprotection [6]. In addition, NRR volatile oil was shown to protect cultured H9c2 cardiomyocytes from oxidative stress-induced cell death [10]. However, the mechanisms by which the volatile oil exerts its role in cardiovascular and circulation system are unknown. The endothelial dysfunction is the major step in a chain of events that leads to atherosclerosis and coronary heart disease. Endothelial cell dysfunction together with low levels of nitric oxide (NO) and endothelium-derived relaxation factor were found in the causes of atherosclerosis [11]. The endothelium is a thin layer of cell that lines the interior surface of blood vessels, forming an interface between blood cell and blood vessel. The endothelium of blood vessel released NO to signal the surrounding smooth muscle to relax for increasing blood flow [12]. NO is synthesized endogenously from L-arginine, oxygen and NADPH by endothelial NO synthase (eNOS): the NO production is governed by multiple signaling, including the regulation of eNOS expression and phosphorylation [13]. The phosphorylation at Ser1177 of eNOS plays an important role in the regulation eNOS activity and NO production [14], and PI3K/ Akt signaling is able to phosphorylate eNOS and increase NO production [15]. On the other hand, an increased intracellular free Ca2+ could activate eNOS via a Ca2+/CaM-dependent protein kinase (CaM kinase). Similar to Akt phosphorylation, CaM kinase could mediate the rapid activation of eNOS and vasodilation [16]. Here, we aimed to reveal the signaling triggered by NRR volatile oil in cultured human umbilical vein endothelial cells (HUVECs), including (i) the activation of NO production and eNOS phosphorylation; (ii) the phosphorylation of Akt kinase; and (iii) the surge of intracellular Ca2+. Materials and Methods Plant materials and chemicals NRR, the root and rhizome of N. jatamansi, was purchased from Hong Kong herbal market. No specific permission was required for this action. In addition, NRR is not classified as an endangered species. The authentication of plant material was performed by Dr. Tina T. X. Dong according to their morphological characteristics. The corresponding vouchers for NRR, as forms of the whole plant, were deposited in Center for Chinese Medicine, Hong Kong University of Science and Technology. 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT), A23187, L-NAME, BAPTA-AM (all at >98% purity) were purchased from Sigma Chemical Co. (St. Louis, MO). LY294002 was purchased from Cell Signaling Technologies (Danvers, MA), Ultra-pure water was prepared from a Milli-Q purification system (Millipore, Molsheim, France). Preparation of NRR volatile oil The total volatile oil from NRR was obtained by water distillation method. Briefly, 50 g of NRR herb was minced and soaked in Milli-Q water in proportion of 1:10 (w/v) overnight. The mixture was submitted to hydro-distillation in a Clevenger-type apparatus for 4 hours. Volatile oil was dried over anhydrous sodium sulfate. The yield of volatile oil was ~1 mL (~2%, v/w), and the oil was stored at -20C until analyze. For cell culture, the volatile oil stock solution was prepared in DMSO solution with the concentration of 180 mg/mL. The volatile oil solution was further diluted with cell culture medium during the cell experiments. Male SpragueDawley rats (~250300 g) were sacrificed by cervical dislocation and bled. The thoracic aorta was excised. After surrounding connective tissue had been carefully cleaned off, four 3-mm-wide (...truncated)