Metabolic characterization of a potent natural neuroprotective agent dendrobine in vitro and in rats.

Dendrobine is the main sesquiterpene alkaloid of Dendrobium nobile Lindl, which exhibits potent neuroprotective activity. However, its metabolism and disposition are little known. In this study, we investigated the metabolic characteristics of dendrobine in vitro and in rats. The metabolic stability and temporal profile of metabolites formation of dendrobine were assayed in human/rat liver microsomal and S9 fractions. Dendrobine metabolites were separated and identified mainly by UPLC-Q/Orbitrap MS. After oral administration of dendrobine (50 mg/kg) to rats, the accumulative excretion rate of dendrobine in feces, urine, and bile was 0.27%, 0.52%, and 0.031%, respectively, and low systematic exposure of dendrobine (AUC0-∞ = 629.2 ± 56.4 ng·h/mL) was observed. We demonstrated that the elimination of dendrobine was very rapid in liver microsomal incubation (the in vitro elimination t1/2 in rat and human liver microsomes was 1.35 and 5.61 min, respectively). Dendrobine underwent rapid and extensive metabolism; cytochrome P450, especially CYP3A4, CYP2B6, and CYP2C19, were mainly responsible for its metabolism. Aldehyde dehydrogenase, alcohol dehydrogenase and aldehyde oxidase were involved in the formation of carboxylic acid metabolites. By the aid of in-source fragmentation screening, hydrogen/deuterium exchange experiment, post-acquisition processing software, and available reference standards, 50 metabolites were identified and characterized in liver microsomal incubation and in rats. The major metabolic pathways of dendrobine were N-demethylation, N-oxidation, and dehydrogenation, followed by hydroxylation and glucuronidation. Collectively, the metabolic fate of dendrobine elucidated in this study not only yields benefits for its subsequent metabolism study but also facilitates to better understanding the mode of action of dendrobine and evaluating the pharmacologic efficiency of the high exposure metabolites.

Notch1-ADAM8 positive feed-back loop regulates the degradation of chondrogenic extracellular matrix and osteoarthritis progression.

BACKGROUND: Osteoarthritis (OA) is one of the most prevalent joint disease, and there are still no effective therapeutic agents or clinical methods for the cure of this disease to date. The degradation of cartilage extracellular matrix (ECM) is a major cause of OA. METHOD: IL-1ß was used to induce chondrogenic degradation. Q-PCR and Western blotting were used to detect mRNA and protein level, respectively. ELISA was used to detect the secreted TNF-α and IL-6 level. Immunofluorescence was used to detect the protein level of Aggrecan, Collagen II and ki67. TUNEL and flow cytometry were used to examine cell apoptosis of chondrocytes. ChIP and luciferase assay were used to study molecular gene regulation. Osteoarthritic animal model and Safranin-O staining were used to determine the in vivo OA phenotype. RESULTS: The expression of ADAM8 was up-regulated in osteoarthritic chondrocytes. Knockdown of ADAM8 suppressed the OA phenotype in the in vitro OA cell model. ADAM8 regulated OA progression through the activation of EGFR/ERK/NF-κB signaling pathway. Inhibition of Notch signaling suppressed OA phenotype in the in vitro OA cell model. Notch signaling regulated the gene expression of ADAM8 directly via Hes1. Notch1-ADAM8 positive feedback loop promoted the progression of OA in vivo. CONCLUSION: Notch1-ADAM8 feed-back loop regulates the degradation of chondrogenic extracellular matrix and osteoarthritis progression.

Oleanolic acid reprograms the liver to protect against hepatotoxicants, but is hepatotoxic at high doses.

Oleanolic acid (OA) is a triterpenoid that exists widely in fruits, vegetables and medicinal herbs. OA is included in some dietary supplements and is used as a complementary and alternative medicine (CAM) in China, India, Asia, the USA and European countries. OA is effective in protecting against various hepatotoxicants, and one of the protective mechanisms is reprogramming the liver to activate the nuclear factor erythroid 2-related factor 2 (Nrf2). OA derivatives, such as CDDO-Im and CDDO-Me, are even more potent Nrf2 activators. OA has recently been shown to also activate the Takeda G-protein-coupled receptor (TGR5). However, whereas a low dose of OA is hepatoprotective, higher doses and long-term use of OA can produce liver injury, characterized by cholestasis. This paradoxical hepatotoxic effect occurs not only for OA, but also for other OA-type triterpenoids. Dose and length of time of OA exposure differentiate the ability of OA to produce hepatoprotection vs hepatotoxicity. Hepatotoxicity produced by herbs is increasingly recognized and is of global concern. Given the appealing nature of OA in dietary supplements and its use as an alternative medicine around the world, as well as the development of OA derivatives (CDDO-Im and CDDO-Me) as therapeutics, it is important to understand not only that they program the liver to protect against hepatotoxic chemicals, but also how they produce hepatotoxicity.

[An HPLC-MS/MS method for the determination of trans-ferulic acid in human plasma].

In this study, we developed a sensitive and rapid HPLC-MS/MS method for the determination of trans-ferulic acid(trans-FA) in plasma samples, and investigated the pharmacokinetics characteristics in healthy volunteers. The plasma samples were extracted with acetic ether, and then separated on a Hedera ODS-2 column with a mobile phase of methanol and 5 mmol·L(-1) ammonium acetate buffer solution containing 0.05% acetic acid(34∶66) at a flow rate of 0.4 m L·min(-1). Electrospray ionization source was applied and operated in the positive ion mode using MRM. The method exhibited a good linearity over the concentration range of 0.1-5 ng·m L(-1)(r ≥ 0.999 2). The values on both the occasions(intra- and inter-day) were all within 9.2%, and the accuracy was 95.4%-111.4%. No matrix effect and carry-over effect were observed. Trans-FA was stable in human plasma under different storage conditions. The developed HPLC-MS/MS method is rapid, sensitive, accurate, and reproducible, and suitable for the pharmacokinetic study of trans-FA in healthy Chinese volunteers.

[An LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in human urine and application to urinary excretion study].

ABSTRACT: The study aims to develop an LC-MS/MS method for the simultaneous determination of amygdalin and paeoniflorin in urine samples, and to investigate their urinary excretion characteristics in healthy volunteers after intravenous infusion administration of Huoxue-Tongluo lyophilized powder for injection (HTLPI). The urine samples were extracted by methanol, and then separated on a Hedera ODS-2 column with a mobile phase of acetonitrile and 5 mmol · L(-1) ammonium acetate buffer solution containing 0.05% formic acid (20:80). Electrospray ionization source was applied and operated in the positive ion mode using MRM. The method exhibited good linearity over the concentration range of 0.03 -40 µg · mL(-1). The values on both the occasions (intra- and inter-day) were all within 15% at three concentration levels. No matrix effect and carry-over effect were observed. Amygdalin and paeoniflorin were stable in human urine under different storage conditions. Approximately 79.6% of the administered amount of amygdalin was excreted unchanged in urine within 24 h and which was 48.4% for paeoniflorin. The developed LC-MS/MS method can be applied to evaluate the urinary excretion of amygdalin and paeoniflorin.

Cinnabar protects serum-nutrient starvation induced apoptosis by improving intracellular oxidative stress and inhibiting the expression of CHOP and PERK.

Cinnabar has been used for treatment of various disorders for thousands of years. The medical use of cinnabar, however, has been controversial because of its heavy metal mercury content. A large quantity of studies indicate that the toxicity of cinnabar is far below other inorganic or organic mercury-containing compounds. Yet, the underlying molecular basis has remained unresolved. Here, we investigated the beneficial effects of cinnabar on serum-nutrient starvation-elicited cell injury. Our findings showed that treatment of human renal proximal tubular cells (HK-2) with 4 nM cinnabar effectively inhibited nutrient deprivation induced apoptosis, reduced intracellular reactive oxygen species generation and increased GSH content, which was contrary to the exacerbated apoptotic cell death and oxidative stress in cells treated with HgCl2 at equal mercury concentration. In addition, cinnabar exerted robust antioxidative and antiapoptotic effects in cells under dual challenges of nutrient deprivation and treatment of H2O2. The protein expression levels of both CHOP and PERK were remarkably down-regulated in the cells treated with cinnabar compared to the control cells or cells treated with HgCl2. Overall, our data indicates that cinnabar at low concentration exerts anti-oxidative stress and anti-apoptosis effects by inhibiting the expression of the endoplasmic reticulum stress pathway proteins CHOP and PERK.

Sodium ferulate inhibits myocardial hypertrophy induced by abdominal coarctation in rats: Involvement of cardiac PKC and MAPK signaling pathways.

Sodium ferulate (SF) is the sodium salt of ferulic acid which is an active ingredient of Radix Angelica Sinensis and Ligusticum chuanxiong hort. Here, we investigated SF inhibition in a rat model of myocardial hypertrophy induced by coarctation of the abdominal aorta. Following coarctation, rats were given SF (20, 40, and 80 mg/kg/day) for 25 consecutive days. We characterized myocardial hypertrophy using myocardial hypertrophic parameters, histopathology, and gene expression of atrial natriuretic factor (ANF) -a gene related to myocardial hypertrophy. We detected the levels of angiotensin II (Ang II) and endothelin-1 (ET-1), protein kinase C beta (PKC-ß), Raf-1, extracellular regulated protein kinase 1/2 (ERK1/2), and mitogen-activated protein kinase phosphatase-1 (MKP-1) in myocardium. Notably, coarctation of the abdominal aorta increases myocardial hypertrophic parameters, cardiac myocyte diameter, the concentration of Ang II and ET-1 in myocardium, and gene expression of ANF. SF significantly ameliorates myocardial hypertrophy caused by coarctation of the abdominal aorta; reduces concentrations of Ang II and ET-1; suppresses the overexpression of ANF, PKC-ß, Raf-1, and ERK1/2; and increases the expression of MKP-1. These results indicate that SF alleviates myocardial hypertrophy induced by coarctation of the abdominal aorta, and these protective effects could be related to the inhibition of PKC and mitogen-activated protein kinase (MAPK) signaling pathways.

Adulthood Exposure to Lipopolysaccharide Exacerbates the Neurotoxic and Inflammatory Effects of Rotenone in the Substantia Nigra.

Parkinson's disease (PD) is the second most neurodegenerative disorder with a regional decrease of dopamine (DA) neurons in the substantia nigra (SN). Despite intense exploration, the etiology of PD progressive process remains unclear. This study was to investigate the synergistic effects of systemic inflammation of lipopolysaccharide (LPS) and neurotoxicity of rotenone (ROT) on exacerbating DA neuron lesion. Male SD adulthood rats received a single intraperitoneal injection of LPS. Seven months later, rats were subcutaneously given ROT five times a week for consecutive 4 weeks. Rat behavior changes were assessed via rotarod and open-field tests. Brain SN was immunostained to evaluate DA neuronal loss and microglia activation. Striatum DA and its metabolites levels were determined by high performance liquid chromatography (HPLC) coupled with electrochemistry. The protein levels of α-synuclein (α-Syn), inflammatory factors and mitogen-activated protein kinase (MAPK) pathway activation were detected by western blotting analysis. Results indicated that no significant difference between the control and LPS alone groups was shown. Compared with ROT alone group, LPS combined with ROT significantly reduced motor activity and induced SN DA neurons loss accompanied by the decreased contents of striatum DA and its metabolites. Furthermore, LPS together with ROT enhanced microglia activation and the increased expressions of α-Syn and inflammatory factors and also MAPK signaling pathway activation. However, LPS alone had no significant effects on the above parameters. These findings suggest that adulthood exposure to LPS exacerbates the neurotoxic and inflammatory effects of ROT in the SN.