Metabolomic and Genomic Evidence for Compromised Bile Acid Homeostasis by Senecionine, a Hepatotoxic Pyrrolizidine Alkaloid

Pyrrolizidine alkaloids (PAs) are among the most hepatotoxic natural products that produce irreversible injury to humans via the consumption of herbal medicine and honey, and through tea preparation. Toxicity and death caused by PA exposure have been reported worldwide. Metabolomics and genomics provide scientific and systematic views of a living organism and have become powerful techniques for toxicology research. In this study, senecionine hepatotoxicity on rats was determined via a combination of metabolomic and genomic analyses. From the global analysis generated from two omics data, the compromised bile acid homeostasis in vivo was innovatively demonstrated and confirmed. Serum profiling of bile acids was altered with significantly elevated conjugated bile acids after senecionine exposure, which was in accordance with toxicity. Similarly, the hepatic mRNA levels of several key genes associated with bile acid metabolism were significantly changed. This process included cholesterol 7-α hydroxylase, bile acid CoA-amino acid <i>N</i>-acetyltransferase, sodium taurocholate cotransporting polypeptide, organic anion-transporting polypeptides, and multidrug-resistance-associated protein 3. In conclusion, a cross-omics study provides a comprehensive analysis method for studying the toxicity caused by senecionine, which is a hepatotoxic PA. Moreover, the change in bile acid metabolism and the respective transporters may provide a new PA toxicity mechanism

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

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

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

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