Ligands and Signaling of Mas-Related G Protein-Coupled Receptor-X2 in Mast Cell Activation.

Mas-related G protein-coupled receptor-X2 (MRGPRX2) is known as a novel receptor to activate mast cells (MCs). MRGPRX2 plays a dual role in promoting MC-dependent host defense and immunomodulation and contributing to the pathogenesis of pseudo-allergic drug reactions, pain, itching, and inflammatory diseases. In this article, we discuss the possible signaling pathways of MCs activation mediated by MRGPRX2 and summarize and classify agonists and inhibitors of MRGPRX2 in MCs activation. MRGPRX2 is a low-affinity and low-selectivity receptor, which allows it to interact with a diverse group of ligands. Diverse MRGPRX2 ligands utilize conserved residues in its transmembrane (TM) domains and carboxyl-terminus Ser/Thr residues to undergo ligand binding and G protein coupling. The coupling likely initiates phosphorylation cascades, induces Ca2+ mobilization, and causes degranulation and generation of cytokines and chemokines via MAPK and NF-κB pathways, resulting in MCs activation. Agonists of MRGPRX2 on MCs are divided into peptides (including antimicrobial peptides, neuropeptides, MC degranulating peptides, peptide hormones) and nonpeptides (including FDA-approved drugs). Inhibitors of MRGPRX2 include non-selective GPCR inhibitors, herbal extracts, small-molecule MRGPRX2 antagonists, and DNA aptamer drugs. Screening and classifying MRGPRX2 ligands and summarizing their signaling pathways would improve our understanding of MRGPRX2-mediated physiological and pathological effects on MCs.

The effects of sodium sulfite on Helicobacter pylori by establishing a hypoxic environment.

Helicobacter pylori (H. pylori) is an obligate microaerobion and does not survive in low oxygen. Sodium sulfite (SS) reacts and consume oxygen in solutions. The present study aimed to investigate the effects of SS on H. pylori. The effects of SS on oxygen concentrations in solutions and on H. pylori in vivo and in vitro were examined, and the mechanisms involved were explored. The results showed that SS decreased the oxygen concentration in water and artificial gastric juice. In Columbia blood agar and special peptone broth, SS concentration-dependently inhibited the proliferation of H. pylori ATCC43504 and Sydney strain-1 in Columbia blood agar or special peptone broth, and dose-dependently decreased the number of H. pylori in Mongolian gerbils and Kunming mouse infection models. The H. pylori was relapsed in 2 weeks withdrawal and the recurrence in the SS group was lower than that in the positive triple drug group. These effects were superior to positive triple drugs. After SS treatments, the cell membrane and cytoplasm structure of H. pylori were disrupted. SS-induced oxygen-free environment initially blocked aerobic respiration, triggered oxidative stress, disturbed energy production. In conclusion, SS consumes oxygen and creates an oxygen-free environment in which H. pylori does not survive. The present study provides a new strategy and perspective for the clinical treatment of H. pylori infectious disease.

Pharmacodynamics, toxicology and toxicokinetics of ropivacaine oil delivery depot.

BACKGROUND: Ropivacaine oil delivery depot (RODD) can be used to treat postoperative incision pain. The aim was to study pharmacodynamics, toxicity and toxicokinetics of RODD. METHODS: The base research of RODD were conducted. Thirty rabbits were randomly divided into saline, solvent, ropivacaine aqueous injection (RAI) 0.9 mg, RODD 0.9 mg and RODD 3 mg groups. The sciatic nerve of rabbits were isolated, dripped with RODD and the effect of nerve block were observed. In toxicity study, the rats were divided into saline, solvent and RODD 75, 150 and 300 mg/kg groups, 30 rats per group. In toxicokinetics, rats were divided into RODD 75, 150 and 300 mg/kg groups, 18 rats per group. The rats were subcutaneously injected drugs. RESULTS: The analgesic duration of RODD 3 mg and RAI 0.9 mg blocking ischiadic nerve lasted about 20 h and 2 h, respectively, and their blocking intensity was similar. The rats in RODD 75 mg/kg did not show any toxicity. Compared with saline group, in RODD 150 mg/kg group neutrophils and mononuclear cells increased, lymphocytes decreased and albumin decreased(P < 0.05), and pathological examination showed some abnormals. In RODD 300 mg/kg group, 10 rats died and showed some abnormalities in central nerve system, hematologic indexes, part of biochemical indexes, and the weights of spleen, liver, and thymus. However, these abnormal was largely recovered on 14 days after the dosing. The results of toxicokinetics of RODD 75 mg/kg group showed that the Cmax was 1.24 ± 0.59 µg/mL and the AUC(0-24 h) was 11.65 ± 1.58 h·µg/mL. CONCLUSIONS: Subcutaneous injection RODD releases ropivacaine slowly, and shows a stable and longer analgesic effect with a large safety range.

Irreversible epidermal growth factor receptor inhibitor Z25h exhibits pronounced inhibition on non-small cell lung adenocarcinoma cell line Hcc827.

The epidermal growth factor receptor (EGFR) signaling is frequently activated in lung cancer. In our previous study, a new class of compounds containing pyrido[3,4-d]pyrimidine scaffold with an acrylamide moiety was designed as irreversible EGFR-tyrosine kinase inhibitors to overcome acquired EGFR-T790M resistance. In this study, we selected the most promising compound Z25h to further investigate its effects and the underlying mechanism against non-small cell lung adenocarcinoma cells in vitro. Four different non-small cell lung adenocarcinoma cell lines were selected to test the antiviability profile of Z25h, and Hcc827 was the most sensitive to the drug treatment. Z25h caused cell cycle arrest at G0-G1 phase, and triggered strong early apoptosis in Hcc827 cells at 0.1 µM and late apoptosis in A549, H1975 and H1299 cells at 10 µM by 48 h treatment. Z25h inhibited the activation of EGFR and its downstream PI3K/AKT/mTOR pathway in the four tested cell lines, leading to the inhibition of cellular biosynthetic and metabolic processes and the promotion of apoptotic process. However, the effect of Z25h on mitogen-activated protein kinase pathway varies from cell lines. In addition, Z25h sensitized H1975 cells to X-ray radiation, and it also enhanced the radiation effect on A549 cells, while no obvious effect of Z25h was observed on the cell viability inhibition of H1299 cells induced by radiation. Hereby, Z25h might be considered as a potential therapeutic drug candidate for non-small cell lung adenocarcinoma treatment.

Secondhand cigarette smoke induces increased expression of contractile endothelin receptors in rat coronary arteries via a MEK1/2 sensitive mechanism.

OBJECTIVES: Cigarette smoke, a strong risk factor for cardiovascular diseases, upregulates contractile endothelin (ET) receptors in coronary arteries. The present study examined the effects of second hand cigarette smoke exposure on the contractile endothelin receptors and the role of the MEK1/2 pathway in rat coronary arteries. Design: Rats were exposed to secondhand smoke (SHS) for 8 weeks followed by intraperitoneal injection of a MEK1/2 inhibitor, U0126 daily for another 4 weeks. Contractile responses of isolated coronary arteries were recorded by a sensitive wire myograph. The receptor protein expression levels were examined by Western blotting. Results: The results showed that SHS in vivo caused increased expression of ET receptors ETA and ETB, and that the MEK1/2 blocker U0126 significantly reversed SHS exposure-increased ETA-mediated contractile responses and protein levels. Similar alterations were observed in ETB receptors. U0126 showed dose-dependent effects on SHS-induced response on contractile property and protein levels of the ETB receptor. However, only the higher dose U0126 (15 mg/kg) had inhibitory effects on the ETA receptor. Conclusions: Taken together, our data show that SHS increases contractile ET receptors and MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA and ETB receptor upregulation in rat coronary arteries.

Hydrogen Peroxide-Mediated Oxygen Enrichment Eradicates Helicobacter pylori In Vitro and In Vivo.

Helicobacter pylori is an important risk factor for gastric ulcers. However, antibacterial therapies increase the resistance rate and decrease the eradication rate of H. pylori Inspired by the microaerophilic characteristics of H. pylori, we aimed at effectively establishing an oxygen-enriched environment to eradicate and prevent the recurrence of H. pylori The effect and the mechanism of an oxygen-enriched environment in eradicating H. pylori and preventing the recurrence were explored in vitro and in vivo During oral administration and after drug withdrawal, H. pylori counts were evaluated by Giemsa staining in animal cohorts. An oxygen-enriched environment in which H. pylori could not survive was successfully established by adding hydrogen peroxide into several solutions and rabbit gastric juice. Hydrogen peroxide effectively killed H. pylori in Columbia blood agar and special peptone broth. Minimum inhibition concentrations and minimum bactericidal concentrations of hydrogen peroxide were both relatively stable after promotion of resistance for 30 generations, indicating that hydrogen peroxide did not easily promote resistance in H. pylori In models of Mongolian gerbils and Kunming mice, hydrogen peroxide has been shown to significantly eradicate and effectively prevent the recurrence of H. pylori without toxicity and damage to the gastric mucosa. The mechanism of hydrogen peroxide causing H. pylori death was related to the disruption of bacterial cell membranes. The oxygen-enriched environment achieved by hydrogen peroxide eradicates and prevents the recurrence of H. pylori by damaging bacterial cell membranes. Hydrogen peroxide thus provides an attractive candidate for anti-H. pylori treatment.

Chronic real-time particulate matter exposure causes rat pulmonary arteriole hyperresponsiveness and remodeling: The role of ETBR-ERK1/2 signaling.

Exposure to air pollution is associated with the incidence of respiratory diseases. The present study evaluated the pulmonary vascular system injury by chronic real-time particulate matter (PM10) exposure and investigated the underlying mechanisms. Rats were exposed to PM10 or filtered air for 2 to 4 months using a whole body exposure system, and intraperitoneally injected with the MEK1/2 inhibitor U0126. Right heart catheterization and myography were performed to detect lung function and pulmonary vascular reactivity, respectively. Western blotting, qRT-PCR, enzyme-linked immunosorbent assay and histological analyses were used to detect the effects and mechanisms by which PM10 exposure-induced pulmonary vascular dysfunction. Functional experiment results showed that PM10 exposure increased the pulmonary artery pressure of rats and caused endothelin B receptor (ETBR)-mediated pulmonary arteriole hyperreactivity. U0126 significantly rescued these pathological changes. PM10 exposure upregulated the contractile ETBR of pulmonary arteriolar smooth muscle, and damaged pulmonary artery endothelial cells to induce the release of more endothelin 1 (ET-1). The upregulated ETBR bound to increased ET-1 induced pulmonary arteriolar hyperresponsiveness and remodeling. U0126 inhibited the PM10 exposure-induced upregulation of ETBR in pulmonary arteriole, ETBR-mediated pulmonary arterial hyperresponsiveness and vascular remodeling. In conclusion, chronic real-time particulate matter exposure can activate the ERK1/2 signaling, thereby inducing the upregulation of contractile ETBR in pulmonary arteriole, which may be involved in pulmonary arteriole hyperresponsiveness and remodeling in rats. These findings provide new mechanistic evidence of PM10 exposure-induced respiratory diseases, and a new possible target for treatment.

Interactions of Tofacitinib and Dexamethasone on Lymphocyte Proliferation.

PURPOSES: Lymphocyte proliferation is a major factor determining the magnitude of the immune response. Both dexamethasone (DEX) and tofacitinib (TOF) exert marked immunosuppressive effects and are mainstay drugs in the treatment of rheumatoid arthritis (RA). This study was aimed to explore the single and combined anti-proliferative action of DEX and TOF on lymphocytes and their sex differences. METHODS: The single-drug effects and dual-drug interactions of TOF and DEX were assessed on the in vitro concanavalin A-stimulated proliferation of lymphocytes isolated from male and female rat and human peripheral blood. RESULTS: DEX was more potent than TOF across species and sex. DEX showed greater inhibition on rat lymphocytes compared to those from humans, which was reflected in both Imax and IC50. The antiproliferative action of TOF was comparable in rats and humans with exception of a higher IC50 in male rats. Both sex- and species-related differences were detected in DEX/TOF interactions with synergistic effects in male lymphocytes, and additive and antagonistic effect for females in humans and rats. CONCLUSION: TOF has a promising steroid-sparing potential with the beneficial effects of the combination therapy more likely in males than females.

Determination of pharmacokinetic parameters of vitamin K1 in rats after an intravenous infusion.

Pharmacokinetic parameters of vitamin K1 have a large range of values in different literature. The aim of this study was to determine the pharmacokinetic parameters of vitamin K1 following post-constant speed intravenous infusion (PCSII) to provide rational pharmacokinetic parameters of vitamin K1 and compare these with results of noncompartmental analysis following intravenous injection (IV). After 15 hours intravenous infusion of vitamin K1 in rats, the logarithmic concentration-time curve of vitamin K1 was fit to a linear equation following PCSII (R2  = 0.9599 ± 0.0096). Then, half-time (T1/2 ), apparent volume of distribution (Vd ), and clearance rate (CL) were estimated successively. T1/2 of vitamin K1 was 4.07 ± 0.41 hour, CL was 89.47 ± 3.60 mL/h, and Vd was 525.38 ± 54.45 mL in rats following PCSII. There was no significant difference in pharmacokinetic parameters of vitamin K1 among different sampling times. For noncompartmental analysis, T1/2 and mean residence time (MRTINF ) for a sampling duration of 6h were shorter than those of 12 hours or 24 hours sampling duration following IV (P < .05, P < .01). In addition, T1/2 of vitamin K1 was obviously different from MRT-equated half-time (T1/2,MRT )(P < .05). Vd and CL of vitamin K1 following PCSII were larger than those following IV based on noncompartmental analysis (P < .01). The results demonstrated that drug distribution in the body was balanced and the Napierian logarithmic concentration-time curve of vitamin K1 fit to a linear equation following PCSII. Vitamin K1 has a long T1/2 and a relatively large Vd following PCSII.

A novel small-molecule PI3K/Akt signaling inhibitor, W934, exhibits potent antitumor efficacy in A549 non-small-cell lung cancer.

Small-molecule targeted antitumor drugs are considered to be a promising treatment that can improve the efficacy and reduce side effects. PI3K/Akt signaling pathway is constantly activated in various cancers. We recently synthesized a series of novel compounds of PI3K/Akt pathway inhibitors and found the most effective analog to be W934. In this study, we explored the in-vitro and in-vivo antitumor effects of W934 on A549 non-small-cell lung cancer cells and HCT116 colorectal cancer cells. In-vitro assays showed that W934 caused an inhibition of PI3Kα kinase. W934 can significantly suppress the viability of A549 and HCT116 cells with IC50 values of 0.25 and 0.23 µmol/l, respectively. Besides, the inhibitory effects on cell migration, invasion and apoptosis were also observed after treatment of W934 for the indicated hours. According to the cell cycle analysis, W934 caused an inhibition of G0-G1 phase progression and correspondingly decreased the percentage of cells in S and G2-M phases. Results of western blotting indicated that W934 concentration dependently suppressed the activation of the PI3K/Akt pathway. Meanwhile, the in-vivo effect was studied in an A549 xenograft mouse model. Oral administration of W934 inhibited the tumor growth in a dose-dependent manner. Hereby, W934 might be considered as a potential therapeutic drug candidate for non-small-cell lung cancer treatment.

PM2.5 upregulates rat mesenteric arteries 5-HT2A receptor via inflammatory-mediated mitogen-activated protein kinases signaling pathway.

Fine particulate matter (PM2.5 ) is an important environmental risk factor for cardiovascular diseases. However, little is known about the effects of PM2.5 on arteries. The present study investigated whether PM2.5 alters 5-hydroxytryptamine (5-HT) receptor expression and inflammatory mediators on rat mesenteric arteries, and examined the underlying mechanisms. Isolated rat mesenteric arteries segments were cultured with PM2.5 in the presence or absence of ERK1/2, JNK, and p38 pathway inhibitors. Contractile reactivity was monitored by a sensitive myograph. The expression of 5-HT2A/1B receptors and inflammatory mediators were studied by a real-time polymerase chain reaction and/or by immunohistochemistry. The phosphorylation of mitogen-activated protein kinases (MAPK) pathway was detected by Western blot. Compared with the fresh or culture alone groups, 1.0 µg/mL PM2.5 cultured for 16 hours significantly enhanced contractile response induced by 5-HT and increased 5-HT2A receptor mRNA and protein expressions, indicating PM2.5 upregulates 5-HT2A receptor. SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly decreased PM2.5 -induced elevated contraction and mRNA and protein expression of 5-HT2A receptor. Cultured with PM2.5 significantly increased the mRNA expression of inflammatory mediators (NOS2, IL-1ß, and TNF-α), while SB203580 decreased mRNA expression level of NOS2, IL-1ß, and TNF-α. SP600125 (JNK inhibitor) decreased mRNA expression level of TNF-α and IL-1ß. After PM2.5 exposure, the phosphorylation of p38 and ERK1/2 protein were increased. SB203580 and U0126 inhibited the PM2.5 caused increased phosphorylation protein of p38 and ERK1/2. In conclusion, PM2.5 induces inflammatory-mediated MAPK pathway in artery which subsequently results in enhanced vascular contraction responding to 5-HT via the upregulated 5-HT2A receptors.

Synthesis and biological evaluation of irreversible EGFR tyrosine kinase inhibitors containing pyrido[3,4-d]pyrimidine scaffold.

In the present study, a new class of compounds containing pyrido[3,4-d]pyrimidine scaffold with an acrylamide moiety was designed as irreversible EGFR-TKIs to overcome acquired EGFR-T790M resistance. The most promising compound 25h inhibited HCC827 and H1975 cells growth with the IC50 values of 0.025 µM and 0.49 µM, respectively. Meanwhile, 25h displayed potent inhibitory activity against the EGFRL858R (IC50 = 1.7 nM) and EGFRL858R/T790M (IC50 = 23.3 nM). 25h could suppress EGFR phosphorylation in HCC827 and H1975 cell lines and significantly induce the apoptosis of HCC827 cells. Additionally, compound 25h could remarkably inhibit cancer growth in established HCC827 xenograft mouse model at 50 mg/kg in vivo. These results indicated that the 2,4-disubstituted 6-(5-substituted pyridin-2-amino)pyrido[3,4-d]pyrimidine derivatives can serve as effective EGFR inhibitors and potent anticancer agents.

Synthesis and evaluation of 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine as new EGFR inhibitors.

In present study, we described the synthesis and biological evaluation of a new class of EGFR inhibitors containing 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine scaffold. Thirty-one compounds were synthesized. Among them, compound C9 displayed the IC50 of 29.4 nM against HCC827 cell line and 1.9 nM against EGFRL858R. Compound C12 showed moderate inhibitory activity against EGFRL858R/T790M/C797S (IC50 = 114 nM). Western bolt assay suggested that compound C9 significantly inhibited EGFR phosphorylation. In vivo test, compound C9 remarkably exhibited inhibitory effect on tumor growth at 5.0 mg/kg by oral administration in established nude mouse HCC827 xenograft model. These results indicate that the 2,9-disubstituted 8-phenylsulfinyl/phenylsulfinyl-9H-purine derivatives can act as potent EGFR(L858R) inhibitors and effective anticancer agents. Additionally, optimization of compound C12 may result in discovering the fourth-generation EGFR-TKIs.

Comprehensive comparison of therapeutic efficacy of radiofrequency target disc decompression and nucleoplasty for lumbar disc herniation: a five year follow-up.

PURPOSE: To compare the therapeutic efficacy of radiofrequency target disc decompression(TDD) and nucleoplasty for lumbar disc herniation. METHODS: Two hundred sixty patients with lumbar disc herniation were divided into two groups: target disc decompression group (group T, n = 147) and nucleoplasty group (group N, n = 113). Visual analogue scale (VAS) and functional rating index (FRI) were measured at one, three, six, 12, 24, and 60 months after the surgery. Hospitalization time, operation time, complications, and recurrence/invalid were compared between the two groups. RESULTS: Compared with the pre-operation, the VAS and FRI in both groups were significantly decreased in post-operation(P < 0.01). The VAS and FRI in group T have no significant difference compared to those in group N. The hospitalization and operation time of group T were significantly longer than those in group N. There was no significant difference of the occurrence of complications and disease recurrence/invalid during the follow-up between the two groups. Logstic regression analysis showed that operation time was an independent factor in the prognosis. Operation time affects the treatment effect. Shorter operation time leads to better therapeutic efficacy, and longer operation time leads to poor therapeutic efficacy. CONCLUSIONS: Both TDD and nucleoplasty can reduce pain in patients with lumbar disc herniation and improve quality of life. Group N had shorter hospitalization and operation time than group T.

Phenylsulfonylfuroxan NO-donor phenols: Synthesis and multifunctional activities evaluation.

Phenylsulfonyfuroxan nitric oxide (NO)-donor phenols were designed, synthesized and evaluated. The compounds were designed through a symbiotic approach using selected phenols and phenylsulfonylfuroxan NO-donor. The antioxidant activities of the hybrid compounds T2-T6 showed to be good in vivo. Compounds T4 and T6 revealed excellent yeast α-glucosidase inhibitory activity and anti-glycosylation activity. All of the compounds exhibited strong NO releasing activity and significant anti-platelet aggregation activity. The inhibition of platelet aggregation was more than 50% at low concentration (1.5µM) and 95% at higher concentration (15µM and 150µM). The vasodilatation experiment demonstrated that the six compounds under test exhibited definite vasodilation activity (pIC50 ranged from 5.698 to 6.383), especially compound T6 (pIC50 was 6.383) which was similar to sodium nitroprusside (pIC50 was 6.786). Both anticoagulant and vasodilatation effects were correlated with their NO releasing activities. These hybrid phenylsulfonyfuroxan-based NO-donor phenols offer a multifunctional prodrug design concept for the development of therapeutic or preventive agents for metabolic syndrome.

Airborne fine particulate matter causes murine bronchial hyperreactivity via MAPK pathway-mediated M3 muscarinic receptor upregulation.

Regarding the human health effects, airborne fine particulate matter 2.5 (PM2.5 ) is an important environmental risk factor. However, the underlying molecular mechanisms are largely unknown. The present study examined the hypothesis that PM2.5 causes bronchial hyperreactivity by upregulated muscarinic receptors via the mitogen-activated protein kinase (MAPK) pathway. The isolated rat bronchi segments were cultured with different concentration of PM2.5 for different time. The contractile response of the bronchi segments were recorded by a sensitive myograph. The mRNA and protein expression levels of M3 muscarinic receptors were studied by quantitative real-time PCR and immunohistochemistry, respectively. The muscarinic receptors agonist, carbachol induced a remarkable contractile response on fresh and DMSO cultured bronchial segments. Compared with the fresh or DMSO culture groups, 1.0 µg/mL of PM2.5 cultured for 24 h significantly enhanced muscarinic receptor-mediated contractile responses in bronchi with a markedly increased maximal contraction. In addition, the expression levels of mRNA and protein for M3 muscarinic receptors in bronchi of PM2.5 group were higher than that of fresh or DMSO culture groups. SB203580 (p38 inhibitor) and U0126 (MEK1/2 inhibitor) significantly inhibited the PM2.5 -induced enhanced contraction and increased mRNA and protein expression of muscarinic receptors. However, JNK inhibitor SP600125 had no effect on PM2.5 -induced muscarinic receptor upregulation and bronchial hyperreactivity. In conclusion, airborne PM2.5 upregulates muscarinic receptors, which causes subsequently bronchial hyperreactivity shown as enhanced contractility in bronchi. This process may be mediated by p38 and MEK1/2 MAPK pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 371-381, 2017.

Establishing a rat model for the study of vitamin K deficiency.

The main vitamin K-deficient model, minidose warfarin, is different from the pathological mechanism of vitamin K deficiency, which is a shortage of vitamin K. The objective of this study was to establish a new method of vitamin K-deficient model combining a vitamin K-deficient diet with the intragastrical administration of gentamicin in rats. The clotting was assayed by an automated coagulation analyser. The plasma PIVKA-II was assayed by ELISA. The vitamin K status was detected by an HPLC-fluorescence system. In the diet- and gentamicin-induced vitamin K-deficient 14-day group, the rats had undetected vitamin K1 and vitamin K2 in the liver and a prolonged APTT. In the 21-day group, there was also a prolonged PT and a decrease of the FIX activities. In the 28-day group, the undetected vitamin K1 and vitamin K2, the prolonged PT and APTT, and the decrease of the FII, FVII, FIX, and FX activities prompted the suggestion that there were serious deficiencies of vitamin K and vitamin K-dependent coagulation in rats. It is suggested that the diet- and gentamicin-induced vitamin K-deficient 14-day or 21-day model can be used for studies related to the status of vitamin K. The vitamin K-deficient 28-day model can be applied to research involving both the status of vitamin K and of vitamin K-dependent coagulation. In conclusion, the combination of a vitamin K-deficient diet with the administration of gentamicin results in a useful model of vitamin K-deficieny.

The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries.

Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries.

Combination of 4-anilinoquinazoline, arylurea and tertiary amine moiety to discover novel anticancer agents.

In present study, 4-anilinoquinazolines scaffold, arylurea and tertiary amine moiety were combined to design, synthesize gefitinib analogs and discover novel anticancer agents. A series of 4-anilinoquinazoline derivatives (1, 2, 3 and 4) bearing arylurea and tertiary amine moiety at its 6-position were synthesized. Their antiproliferative activities in vitro were evaluated via MTT assay against A431 cell and A549 cell. The SAR of the title compounds was discussed. The compounds 2d, 2i and 2j with potent antiproliferative activities were evaluated their inhibitory activity against EGFR-TK. Compound 2j displayed potent inhibitory activity against EGFR-TK. In addition, compound 2j, at 50 mg/kg, can completely inhibit cancer growth in established nude mouse A549 xenograft model in vivo. These results suggest that the 4-anilinoquinazoline derivatives bearing diarylurea and tertiary amino moiety at its 6-position can serve as anticancer agents and EGFR inhibitors.

Modifications of the Method for Calculating Absolute Drug Bioavailability.

PURPOSE: Absolute bioavailability (F) is calculated as the ratio of the area under the plasma drug concentration-time curve (AUC) between extravascular administration and intravenous injection. However, as distribution of a drug after intravenous administration does not reach an equilibrium in the body during the distribution phase, the plasma drug concentration at this phase does not reflect the total amount of drug in the body. The goal of this paper was to analyze the insufficiencies of the method for calculating on absolute bioavailability and to propose a modification to improve the calculation. METHODS: Literature reporting absolute bioavailability published during 1983-2014 was searched for ten drug candidates. Plasma drug concentrations representing the amount of drug in the body were then calculated at each time point during the distribution phase according to the plasma drug concentration-time relationship during the elimination phase. RESULTS: The AUC values based on the distribution equilibrium drug concentrations following intravenous injection were 75%±11% of the actually measured drug concentrations in the literature. The absolute bioavailability values in the literature were 76%±12% of the actual bioavailability based on the AUCs from distribution-equilibrium drug concentrations. CONCLUSIONS: The present method underestimates the absolute drug bioavailability and should be modified to represent the data more accurately. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.