Song Bu Li Decoction, a Traditional Uyghur Medicine, Protects Cell Death by Regulation of Oxidative Stress and Differentiation in Cultured PC12 Cells

Song Bu Li decoction (SBL) is a traditional Uyghur medicinal herbal preparation, containing Nardostachyos Radix et Rhizoma. Recently, SBL is being used to treat neurological disorders (insomnia and neurasthenia) and heart disorders (arrhythmia and palpitation). Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness. Here, we aimed to evaluate the protective and differentiating activities of SBL in cultured PC12 cells. The pretreatment of SBL protected the cell against tBHP-induced cell death in a dose-dependent manner. In parallel, SBL suppressed intracellular reactive oxygen species (ROS) formation. The transcriptional activity of antioxidant response element (ARE), as well as the key antioxidative stress proteins, was induced in dose-dependent manner by SBL in the cultures. In cultured PC12 cells, the expression of neurofilament, a protein marker for neuronal differentiation, was markedly induced by applied herbal extract. Moreover, the nerve growth factor- (NGF-) induced neurite outgrowth in cultured PC12 cells was significantly potentiated by the cotreatment of SBL. In accord, the expression of neurofilament was increased in the treatment of SBL. These results therefore suggested a possible role of SBL by its effect on neuron differentiation and protection against oxidative stress

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

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-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

Major chemical composition of volatile oil from NRR.

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

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

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