The bioinformatics and metabolomics research on anti-hypoxic molecular mechanisms of Salidroside via regulating the PTEN mediated PI3K/Akt/NF-κB signaling pathway.

Salidroside (SAL), a major bioactive compound of Rhodiola crenulata, has significant anti-hypoxia effect, however, its underlying molecular mechanism has not been elucidated. In order to explore the protective mechanism of SAL, the lactate dehydrogenase (LDH), reactive oxygen species (ROS), superoxide dismutase (SOD) and hypoxia-induced factor 1α (HIF-1α) were measured to establish the PC12 cell hypoxic model. Cell staining and cell viability analyses were performed to evaluate the protective effects of SAL. The metabolomics and bioinformatics methods were used to explore the protective effects of salidroside under hypoxia condition. The metabolite-protein interaction networks were further established and the protein expression level was examined by Western blotting. The results showed that 59 endogenous metabolites changed and the expression of the hub proteins of CK2, p-PTEN/PTEN, PI3K, p-Akt/Akt, NF-κB p65 and Bcl-2 were increased, suggesting that SAL could increase the expression of CK2, which induced the phosphorylation and inactivation of PTEN, reduced the inhibitory effect on PI3K signaling pathways and activated the PI3K/Akt/NF-κB survival signaling pathway. Our study provided an important insight to reveal the protective molecular mechanism of SAL as a novel drug candidate.

Cytosolic ß-glucosidase inhibition and renal blood flow suppression are leading causes for the enhanced systemic exposure of salidroside in hypoxic rats.

The promising benefits of salidroside (SAL) in alleviating high altitude sickness boost investigations on its pharmacokinetics and biological activity. However, the transportation and disposition process of SAL under hypoxic conditions has never been explored. The current study was proposed to investigate the pharmacokinetics of SAL in hypoxic rats and to explore the underlying mechanisms for the distinct metabolic fate of SAL under hypoxia. Pharmacokinetic studies on SAL was conducted in both hypoxic and normoxic rats. The transport properties of SAL were investigated on both hypoxic and normoxic Caco-2 monolayer models. Enzymes involved in SAL metabolism were identified and the effects of hypoxia on these enzymes were assessed by real-time PCR, western blotting analyses, and rat liver homogenate incubation. The renal clearance (CLr) of SAL, effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) in both hypoxic and normoxic rats were also determined for renal function assessment. It was found that the systemic exposure of SAL in hypoxic rats was remarkably higher than that in normoxic rats. The barrier function of Caco-2 monolayer was weakened under hypoxia due to the impaired brush border microvilli and decreased expression of tight junction protein. Hepatic metabolism of SAL in hypoxic rats was attenuated due to the reduced activity of cytosolic ß-glucosidase (CBG). Moreover, CLr of SAL was reduced in hypoxic rats due to the suppressed ERPF. Our findings suggest the potential need for dose-adjustment of SAL or its structural analogs under hypoxic conditions.

Mechanistic study on the pharmacokinetic process of salidroside in hypoxic rats / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of hypoxia on the pharmacokinetic process of salidrosidein rats and to explore its underlying mechanisms. METHODS The Caco-2 cell monolayerwas exposed to 1% oxygen (O2) concentration for 24 h to build the hypoxiccell model. The transportation mode of salidroside was investigated with the aid of this hypoxia model by detecting the apparent permeability coefficient(Papp). Healthy Sprague Dawley (SD) rats were exposed to 9% O2 for 72 h for the construction of hypoxic rat model. Liver sample was subsequently collected from the hypoxic rats with an aim to identify enzymes responsible for salidroside metabolism. The expression levels of sali?droside-transporting and salidroside-metabolizing enzymes, including Sodium-dependent glucose cotrans?porters (SGLT1), β-glucosidase (GBA3)and sulfotransferase (SULT2A1), were thereafter detected by RT-PCR and Western blot. The metabolic activity of GBA3 and SULT2A1 was monitored by rat liver microsome incubation.In addition, the renal function of rats under hypoxia was assessed by detecting concentrations of blood urea nitrogen and creatinine. RESULTS The AUC and t1/2 values of salidroside in hypoxic rats were more than doubled, while the in vivo clearance was significantly reduced. Mechanistic study demonstrated that the PappA- B/PappB- A eualsto 10.3, indicating the potential active transport of salidrosile. The expression of SGLT1 and GBA3 was significantly decreased, which indicated a reduced metabolism of salidroside under hypoxia. Moreover, rat under hypoxia was found to suffer from renal dysfunction, with an abnormal value of blood urea nitrogen. CONCLUSION Due to the reduced metabolism and the abnormal renal function under hypoxia, the systemic exposure of salidroside in rats was signifi?cantly enhanced.

Inhibitory effects of lapachol on rat C6 glioma in vitro and in vivo by targeting DNA topoisomeraseⅠ and topoisomeraseⅡ / 中国药理学与毒理学杂志

OBJECTIVE Lapachol is a natural naphthoquinone compound that possesses extensive biological activities. The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo, as well as the potential mechanisms. METHODS The antitumor effect of lapachol was firstly evaluated in the C6 glioma model in Wistar rats. The effects of lapachol on C6 cell proliferation, apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/ phenazinemethosulfate (PMS) assay, hoechst 33358 staining, annexinⅤ-FITC/PI staining, and comet assay. Effects of lapachol on topoisomerase I (TOP I) and topoi?somerase Ⅱ (TOP Ⅱ) activities were detected by TOP Ⅰ and TOP Ⅱ mediated supercoiled pBR322 DNA relaxation assays and molecular docking. TOPⅠ and TOPⅡ expression levels in C6 cells were also determined. RESULTS High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats (P<0.05). It was showed that lapachol could inhibit proliferation, induce apoptosis and DNA damage of C6 cells in dose dependent manners. Lapachol could inhibit the activities of both TOPⅠ and Ⅱ. Lapachol-TOPⅠ showed relatively stronger interaction than that of lapachol-TOPⅡ in molecular docking study. Also, lapachol could inhibit TOPⅡ expression levels, but not TOPⅠ expression levels. CONCLUSION These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro, which might be related with inhibiting TOPⅠ and TOPⅡ activities, as well as TOPⅡ expression.

Glucuronidation is the dominating in-vivo metabolism pathway of herbacetin:elucidation of herbacetin pharmacokinetics after intravenous and oral administration in rats / 中国药理学与毒理学杂志

OBJECTIVE To map a comprehensive metabolic pathway of herbacetin in rats, specifically, to elucidate the biotransformation of herbacetin in vivo and to simultaneously monitor the pharmacokinetic process of both parent drug and its major metabolites. METHODS liquid chromatography/ion trap mass spectrometry (LC/MSn) and ultra-liquid chromatography coupled with mass spectrometry (UPLC/MS) were combined in the current study for qualitative and quantitative determinations of herbacetin and its metabolites in bile, urine and feces after both oral and intravenous administration of herbacetin to rats. Enzyme kinetic studies on the intestinal and hepatic metabolism of herbacetin were further conducted to elucidate metabolic profiles of herbacetin in rat tissues and organs. Additionally, plasma concentration profiles of herbacetin and its metabolites in rats were obtained to characterize the overall pharmacokinetic behavior of herbacetin. RESULTS It was found that herbacetin was excreted primarily from rat urine in the form of glucuronide-conjugations. Subsequent in vitro enzyme kinetic studies and in vivo pharmacokinetic investigations suggested an extensive hepatic metabolism of herbacetin and the high exposure of herbacetin- glucuronides in systemic circulation. The clearance, half- life and bioavailability of herbacetin in rats were determined as (16.4±1.92)mL·kg-1·min-1, (11.9±2.7)min, and 1.32%, respectively. On basis of these findings, a comprehensive metabolic pathway of herbacetin in rats was composed. In addition, a physiology based pharmacokinetic (PBPK) model was successfully developed with the aid of the GastroPlus to simulate the pharmacokinetic process of herbacetin in rats. Application of the PBPK modeling can provide a useful starting point to understand and extrapolate pharmacokinetic parameters among different species, populations, and disease states. CONCLUSION After oral administration, herbacetin was subjected to colonic degradation and extensive first pass metabolism, with glucuronidation as its dominating in vivo metabolic pathway.

Pharmacokinetics and pharmacodynamics of cisatracurium in children undergoing cleft lip or cleft palate repair surgery / 中国药理学与毒理学杂志

OBJECTIVE The aim of the study was to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) profile of cisatracurium in 0-2 years and 2-5 years old children patients with cheilopalatognathus, to find if there are some connections between the different muscle relaxation action and different PK procedure . METHODS 14 children patients were divided into two groups, ≤2 years and 2-5 years group, venous samples were taken before injection of a 0.15 mg·kg- 1 dose of cisatracurium and then at 2, 5, 10, 30, 60, 90, and 120 min. Cisatracurium plasma concentrations were determined by ultra- performance liquid chromatography/electrospray ionization/triple quadrupole tandem mass spectrometer system (UPLC/MS/MS). The degree of neuromuscular block was measured by train of four (TOF) testing. An indirect PK-PD link model with a sigmoid Emax model was established using Win Nonlin software. The model were applied to PK and PD data analysis, respectively. RESULTS The TOF monitor parameters showed that cisatracurium works very quickly, the onset time were (2.64± 0.93) min and (2.59 ± 0.90) min for ≤2 years and 2- 5 years group respectively. Young children ≤2 years have longer muscle blocking duration time (62.5 ± 6.01 min vs 53.86 ± 12.18 min) and slower recovery index (32.14±7.10 min and 27.43±10.63 min) than those children in group of 2-5 years. More children ≤2 years have postoperative complication than that in 2-5 children. PK parameters showed that there were no statistical differences in blood concentration and pharmacokinetic parameters. While the concentration of cisatracurium in muscle site calculated by using PK/PD model were higher and longer for ≤2 year children than that of 2-5 year children. This means that cisatracurium could stay at high concentration for a longer time in younger children' muscle tissue. CONCLUSION As a result young children tend to have postoperative complications related to slower muscle recovery action and increased concentration in skeletal muscle. So more careful observation and monitor are needed for younger children, our study could be of use in clinical practice for the administration of cisatracurium to children patients.

Analysis of the key networks, metabolic pathways, and regulation substances of hypoxia based on the omics and zebrafish model / 中国药理学与毒理学杂志

OBJECTIVE Hypoxia is associated with many complicated pathophysiological and biochemical processes that integrated and regulated via the key gene, protein and endogenous metabolite levels. Up to date, the exact molecular mechanism of hypoxia still remains unclear. In this work, we further explore the molecular mechanism of hypoxia and adaption to attenuate the damage in zebrafish model that have potential to resist hypoxic environment. METHODS The hypoxic zebrafish model was established in different concentration of oxygen with 3%,5%,10%,21% in water. The brain tissue was separated and the RNA-seq was used to identify the differentially expressed genes. The related endogenous metabolites profiles were obtained by LC-HDMS, and the multivariate statistics was applied to discover the important metabolites candidates in hypoxic zebrafish. The candidates were searched in HMDB, KEGG and Lipid Maps databases. RESULTS The zebrafish hypoxic model was successfully constructed via the different concentration of oxygen, temperature and hypoxic time. The activities of the related hypoxic metabolic enzymes and factors including HIF-1a, actate dehydrogenase (LDH) and citrate synthase (CS) were evaluated. Significant differences (P<0.05 and fold change >2) in the expression of 422 genes were observed between the normal and 3% hypoxic model. Among them, 201 genes increased depended on the lower concentration of oxygen. 53 metabolites were identified that had significant difference between the hypoxia and control groups (P<0.05, fold change>1.5 and VIP>1.5). The ten key metabolites were increased gradually while six compounds were decreased. The endogenous hypoxic metabolites of phenylalanine, D-glucosamine-6P and several important lipids with the relevant hub genes had similar change in hypoxic model. In addition, the metabolic pathways of phenylalanine, glutamine and glycolipid were influenced in both the levels of genes and metabolites. CONCLUSION The up- regulation of phenylalanine, D- glucosamine- 6P and lipid may have further understanding of protective effect in hypoxia. Our data provided an insight to further reveal the hypoxia and adaptation mechanism.

Simultaneous determination of five novel luteinizing hormone-releasing hormone antagonists by LC-MS and pharmacokinetics in rats following cassette dosing.

Long acting luteinizing hormone-releasing hormone (LHRH) antagonists designed to be protease-resistant were a series of novel decapeptides structurally similar to LHRH. In the present work, a high-throughput method based on a LC-MS/MS has been developed for the simultaneous determination of pharmacokinetics of five LHRH antagonists in rat via cassette dosing. The method was performed under selected reaction monitoring (SRM) in positive ion mode. The analytes were extracted from rat plasma by liquid-liquid extraction with acetonitrile. Chromatographic separation of the analytes was successfully achieved on a Hypersil Gold (100mm×2.1mm, 3µm) using a mobile phase composed of acetonitrile-water (30:70) containing 0.05% (v/v) formic acid. The result showed good linearity and selectivity were obtained for all antagonists. The limits of quantification of the five LHRH antagonists were from 5 to 10ng/mL. The average extract recoveries in the rat plasma were all over 72%. The intra-day and inter-day precisions (R.S.D. %) were all within 10% and the accuracy was ranged from 92.54 to 109.05%. This method has been successfully applied to the pharmacokinetic studies of the five LHRH antagonists. The results indicated that the plasma drug concentrations versus time curves after intravenous injection of five antagonists via cassette dosing were all fitted to a two-compartment model. The pharmacokinetic parameters of five LHRH antagonists suggested that LY616 could be the more stable candidate drugs and optimized as the candidate drug for further study. Our studies enabled high-throughput rapid screening for pharmacokinetic assessment of new peptide candidates, and provided abundant information on the metabolic properties of these LHRH antagonists.

Alpha(1D)-adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes.

AIM: It is unclear why alpha(1D)-adrenergic receptors (alpha(1D)-ARs) play a critical role in the mediation of peripheral vascular resistance and blood pressure in situ but function inefficiently when studied in vitro. The present study examined the causes for these inconsistencies in native alpha(1)-adrenergic functional performance between the vascular smooth muscle and myocytes. METHODS: The alpha(1)-adrenergic mediated contraction, Ca(2+) signaling and the subcellular receptor distribution were evaluated using the Fluo-4, BODIPY-FL prazosin and subtype-specific antibodies. RESULTS: Rat aortic rings and freshly dissociated myocytes displayed contractile and increased intracellular Ca(2+) responses to stimulation with phenylephrine (PE, 10 micromol), respectively. However, the PE-induced responses disappeared completely in cultured aortic myocytes, whereas PE-enhanced Ca(2+) transients were seen in cultured rat cardiac myocytes. Further studies indicated that alpha(1D)-ARs, the major receptor subtype responsible for the alpha(1)-adrenergic regulation of aortic contraction, were distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, similar to the alpha(1A)-AR subcellular localization in the cultured cardiomyocytes. In the cultured aortic myocytes, however, in addition to a marked decrease in their protein expression relative to the aorta, most labeling signals for alpha(1D)-ARs were found in the cytoplasm. Importantly, treating the culture medium with charcoal/dextran caused the reappearance of alpha(1D)-ARs at the cell surface and a partial restoration of the Ca(2+) signal response to PE in approximately 30% of the cultured cells. CONCLUSION: Reduction in alpha(1D)-AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to alpha(1)-adrenergic receptor activation.

[Effects of theanine and houpu extract in 7-day chick social separation-stress procedure].

OBJECTIVE: To examine the abilities of theanine and houpu extracts (HE) to reverse behavioral indexes (separation vocalization, stress-induced analgesia and activity). METHOD: 7-day-old chicks received IP injection of theanine and HE 30 min before being tested in the presence of three social companions or in isolation for 3-min observation period. Dependent measures were: a) Chicks were placed into an infrared ray device to calculate their spontaneous activities by a computer program b) record the separation vocalizations for every chick. c) In the experiment of stress-induced analgesia, 50 uL of formalin (0.1%) was injected into the plantar of the animal foot to index stress-induced analgesia (i. e. foot-lift frequency, foot-lift duration and peck frequency). RESULT: In the experiments, isolated chicks exhibited more vocalizations (P < 0.01) and fewer pain-related behaviors than non-isolated chicks (P < 0.01). Theanine (12.5, 25, 50 mg x kg(-1)) and HE (25 mg x kg(-1)) decrease separately the tendency (dB) of the principal frequency (P < 0.05, P < 0.01); The stress induced analgesia can be reversed by theanine in 25, 50 mg x kg(-1). Both of the materials do not affect the spontaneous activities in this chick model without causing sedation. CONCLUSION: These results suggest that theanine and HE in the dosages may be useful in modulating anxiety states. They are seems no synergism in the chick model.