Search

Superhydrophobic surfaces with high adhesion provide high potential for underwater applications. Inspired by Salvinia leaf, here, we have reported a simple method for fabricating adhesive Salvinia-like micropillars via photolithography and spontaneous adsorption of organic molecules from the atmosphere. With continuous hydrocarbon adsorption on sputtered cerium dioxide (CeO2) films, the surface gradually evolved and eventually became chemically heterogeneous. Huge wetting contrast from superhydrophilic to superhydrophobic over exposure time was observed ; meanwhile, the wetting mode changed from the Wenzel (W) state to Cassie- Baxter (C-B) state. As a result, hydrophobic hydrocarbons (C−C/C−H) and trapped air between adjacent pillars contributed to the high apparent contact angle (CA), while the hydrophilic domains of C−O/OC−O and CeO2 on the top layer made the surface highly adhesive with water droplets. In comparison with traditional fluorinated superhydrophobic surfaces, CeO2-coated surfaces showed high adhesive force with water droplets and can be used as a “mechanical hand” for water droplet transport. The adsorption-induced Salvinia-like micropillars with high adhesion may find many other droplet-based applications in microfluidic fields.

Background It is well known that nonalcoholic fatty liver disease (NAFLD) is associated with insulin resistance (IR). LB100, a serine/threonine protein phosphatase 2A (PP2A) inhibitor, is closely related to IR. However, there is little data regarding its direct influence on NAFLD. Aim To elucidate the effect and underlying mechanism of LB100 in NAFLD. Methods After 10 wk of high fat diet (HFD) feeding, male C57BL/6 mice were injected intraperitoneally with vehicle or LB100 for an additional 6 wk (three times a week). The L02 cell line was treated with LB100 and free fatty acids (FFAs) for 24 h. Hematoxylin and eosin and oil red O staining were performed for histological examination. Western blot analysis was used to detect the protein expression of Sirtuin 1 (Sirt1), total and phosphorylated AMP-activated protein kinase α (AMPKα), and the proteins involved in lipogenesis and fatty acid oxidation. The mRNA levels were determined by qPCR. Pharmacological inhibition of AMPK was performed to further examine the exact mechanism of LB100 in NAFLD. Results LB100 significantly ameliorated HFD-induced obesity, hepatic lipid accumulation and hepatic injury in mice. In addition, LB100 significantly downregulated the protein levels of acetyl-CoA carboxylase, sterol regulatory element-binding protein 1 and its lipogenesis target genes, including stearoyl-CoA desaturase-1 and fatty acid synthase, and upregulated the levels of proteins involved in fatty acid β-oxidation, such as peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), carnitine palmitoyltransferase 1α, acyl-CoA oxidase 1 and uncoupling protein 2, as well as the upstream mediators Sirt1 and AMPKα in the livers of HFD-fed mice. In vitro, LB100 alleviated FFA-induced lipid accumulation in L02 cells through the AMPK/Sirt1 signaling pathway. Further studies showed that the curative effect of LB100 on lipid accumulation was abolished by inhibiting AMPKα in L02 cells. Conclusion PP2A inhibition by LB100 significantly ameliorates hepatic steatosis by regulating hepatic lipogenesis and fatty acid oxidation via the AMPK/Sirt1 pathway. LB100 may be a potential therapeutic agent for NAFLD.

BACKGROUND Allyl isothiocyanate (AITC), a classic anti-inflammatory and antitumorigenic agent, was recently identified as a potential treatment for obesity and insulin resistance. However, little is known about its direct impact on the liver.

Cytochrome P450 3A (CYP3A) activity is inhibited, and its expression is suppressed during many diseases, including nonalcoholic fatty liver disease (NAFLD). However, the mechanism is controversial. Here, we report that PXR may not take part in the downregulation of CYP3A during NAFLD. Hepatic CYP3A11 (major subtype of mouse CYP3A) mRNA and protein expression was significantly decreased in both mice fed a high-fat diet (HFD) for 8 weeks and palmitate (PA)-treated mouse primary hepatocytes. Similarly, in HepG2 cells, PA treatment significantly suppressed the CYP3A4 (major subtype of human CYP3A) mRNA level and promoter transcription activity. However, Western blotting analysis found an induction of PXR nuclear translocation during NAFLD in both in vivo and in vitro models. Moreover, immunofluorescence determination also found nuclear translocation effect of PXR by PA stimulation in HepG2 cells. In addition, the siRNA knockdown of PXR did not affect the suppressive effects of PA on the CYP3A4 promoter transcription activity and mRNA levels in HepG2 cells. Similarly, PXR knockdown also did not affect the suppressive effects of PA on CYP3A11 mRNA and protein expression levels in mouse primary hepatoctyes. Taken together, the results showed that the suppressive effect of CYP3A transcription was independent of PXR regulation.

Background and Purpose Currently, ursodeoxycholic acid is the only FDA-approved drug for limited cholestatic liver diseases, and thus the development of new therapeutic approaches is of great importance. This study aimed to evaluate the anti-cholestasis effect of Schisandrol B (SolB), a bioactive compound isolated from Schisandra sphenanthera. Experimental Approach Hepatoprotective effect of SolB against lithocholic acid (LCA)-induced intrahepatic extrahepatic cholestasis was evaluated in mice. Metabolomic analysis and gene analysis were performed to reveal the involvement of PXR (pregnane X receptor). Further molecular docking, cell-based reporter gene analysis and knockout mice were used to demonstrate the vital role of PXR pathway on SolB's anti-cholestasis effect. Key Results SolB was found to significantly protect against LCA-induced intrahepatic and BDL-induced extrahepatic cholestasis. Furthermore, therapeutic treatment of SolB significantly decreased the mortality of cholestatic mice. Metabolomics and gene analysis revealed that SolB accelerates the metabolism of bile acid, promotes bile acid efflux into the intestine, and induces hepatic expression of the PXR-target genes Cyp3a11, Ugt1a1, and Oatp2, which are involved in bile acid homeostasis. Further mechanistic study show that SolB can activate human PXR and up-regulate PXR target genes in human cell lines. Additionally, SolB could not significantly protect Pxr-null mice from liver injury induced by intrahepatic cholestasis thus providing genetic evidence that the effect of SolB is PXR-dependent. Conclusion and Implications These findings provide direct evidence for the hepatoprotection of SolB against cholestasis by activating PXR. Therefore, SolB may be an effective approach for the prevention and treatment of cholestatic liver diseases.

We previously showed that the vitamin D receptor (VDR) plays a crucial role in acute inflammatory bowel disease and that intestinal fibrosis is a common complication of Crohn’s disease (CD). Epithelial–mesenchymal transition (EMT) is an important hallmark of fibrogenesis through which epithelial cells lose their epithelial phenotype and transform into mesenchymal cells. It is known that the VDR plays an essential role in epithelial integrity and mitochondrial function, but its role in intestinal fibrosis remains unknown. Here, we investigated whether the VDR is involved in epithelial mitochondrial dysfunction that results in EMT in intestinal fibrosis. Using human CD samples, intestine-specific VDR-KO mice, and fibroblast cellular models, we showed that the expression of the VDR was significantly lower in intestinal stenotic areas than in nonstenotic areas in patients with chronic CD. Genetic deletion of the VDR in the intestinal epithelium exacerbated intestinal fibrosis in mice administered with dextran sulfate sodium or 2,4,6-trinitrobenzene sulfonic acid, two experimental colitis inducers. In addition, we found that vitamin D dietary intervention regulated intestinal fibrosis by modulating the intestinal expression of the VDR. Mechanistically, knocking down the VDR in both CCD-18Co cells and human primary colonic fibroblasts promoted fibroblast activation, whereas VDR overexpression or VDR agonist administration inhibited fibroblast activation. Further analysis illustrated that the VDR inhibited EMT in the HT29 cell model and that mitochondrial dysfunction mediated epithelial integrity and barrier function in VDR-deficient epithelial cells. Together, our data for the first time demonstrate that VDR activation alleviates intestinal fibrosis by inhibiting fibroblast activation and epithelial mitochondria-mediated EMT.

A heterometallic organic framework {Pb[Pd(bpydc)Cl2]DMF}n (1) (H2bpydc = 2,2′-bipyridine-5,5′-dicarboxylic acid) was synthesized via a one-pot solvothermal method and characterized using thermogravimetric analysis, X-ray photoelectron spectroscopy as well as powder and single-crystal X-ray diffraction. The crystal structure of 1 indicates that, in metalloligand Pd(bpydc)Cl2, every Pd atom adopts a square planar coordination mode with two chloride ions and two nitrogen atoms from bpydc, and the carboxyl groups of Pd(bpydc)Cl2 connect Pb atoms to form a one-dimensional chain along the crystallographic a-axis, which is interlinked via metalloligands to form a two-dimensional layer structure. This complex is highly active, stable and recyclable as a catalyst for the Suzuki–Miyaura and Heck reactions of a wide range of aryl halides including electron-rich and electron-poor aryl iodides/bromides, affording the corresponding products in good to excellent yields. Copyright © 2016 John Wiley & Sons, Ltd.

Two Lewis acid–base bifunctional monometallic aluminum–salen complexes were prepared based on a new type of salen ligand with two N-methylhomopiperazine moieties at the 3,3′-position. The Al(salen) complexes proved to be efficient and recyclable homogeneous catalysts towards the organic solvent-free synthesis of cyclic carbonates from epoxides and CO2 in the absence of a co-catalyst, in which >90% yield of cyclic carbonate could be obtained under relatively mild conditions. The catalysts can be easily recovered and reused five times without significant loss of activity and selectivity. Furthermore, the Lewis acid–base cooperative activation mechanism by the bifunctional Al(salen) complexes was proposed according to experimental data.

Acetaminophen (APAP) hepatotoxicity is the most common cause of drug-induced liver injury and N-acetylcysteine (NAC) is the primary antidote of APAP poisoning. Wuzhi tablet (WZ), the active constituents well identified and quantified, is a preparation of an ethanol extract of Schisandra sphenanthera and exerts a protective effect toward APAP-induced hepatotoxicity in mice. However, the clinical use of WZ to rescue APAP-induced acute liver injury and the mechanisms involved in the therapeutic effect of WZ remain unclear. Therefore, the effect of WZ on APAP hepatotoxicity was compared with NAC in mice, and molecular pathways contributing to its therapeutic action were investigated. Administration of WZ 4 hours after APAP treatment significantly attenuated APAP hepatotoxicity and exerted much better therapeutic effect than NAC, as revealed by morphologic, histologic, and biochemical assessments. Both WZ and NAC prevented APAP-induced c-Jun N-terminal protein kinase activation and mitochondrial glutathione depletion in livers. The protein expression of nuclear factor erythroid 2-related factor 2 target genes including Gclc, Gclm, Ho-1, and Nqo1 was increased by WZ administration. Furthermore, p53 and p21 levels were upregulated upon APAP exposure, which were completely reversed by postdosing of WZ 4 hours after APAP treatment over 48 hours. In comparison with NAC, WZ significantly increased the expression of cyclin D1, cyclin D-dependent kinase 4, proliferating cell nuclear antigen, and augmenter of liver regeneration in APAP-injured livers. This study demonstrated that WZ possessed a therapeutic efficacy against APAP-induced liver injury by inhibiting oxidative stress and stimulating a regenerative response after liver injury. Thus WZ may represent a new therapy for APAP-induced acute liver injury.

Carnitine palmitoyltransferase 1C (CPT1C), an enzyme located in the outer mitochondria membrane, has a crucial role in fatty acid transport and oxidation. It is also involved in cell proliferation and is a potential driver for cancer cell senescence. However, its upstream regulatory mechanism is unknown. Peroxisome proliferator activated receptor α (PPARα) is a ligand-activated transcription factor that regulates lipid metabolism and tumor progression. The current study aimed to elucidate whether and how PPARα regulates CPT1C and then affects cancer cell proliferation and senescence. Here, for the first time we report that PPARα directly activated CPT1C transcription and CPT1C was a novel target gene of PPARα, as revealed by dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Moreover, regulation of CPT1C by PPARα was p53-independent. We further confirmed that depletion of PPARα resulted in low CPT1C expression and then inhibited proliferation and induced senescence of MDA-MB-231 and PANC-1 tumor cell lines in a CPT1C-dependent manner, while forced PPARα overexpression promoted cell proliferation and reversed cellular senescence. Taken together, these results indicate that CPT1C is a novel PPARα target gene that regulates cancer cell proliferation and senescence. The PPARα-CPT1C axis may be a new target for the intervention of cancer cellular proliferation and senescence.

Two Lewis acid–base bifunctional monometallic aluminum–salen complexes were prepared based on a new type of salen ligand with two N-methylhomopiperazine moieties at the 3,3′-position. The Al(salen) complexes proved to be efficient and recyclable homogeneous catalysts towards the organic solvent-free synthesis of cyclic carbonates from epoxides and CO2 in the absence of a co-catalyst, in which >90% yield of cyclic carbonate could be obtained under relatively mild conditions. The catalysts can be easily recovered and reused five times without significant loss of activity and selectivity. Furthermore, the Lewis acid–base cooperative activation mechanism by the bifunctional Al(salen) complexes was proposed according to experimental data.

It has been documented that due to limited attentional resources, the size of the attentional focus is inversely correlated with processing efficiency. Moreover, by adopting a variety of two-dimensional size illusions induced by pictorial depth cues (e.g., the Ponzo illusion), previous studies have revealed that the perceived, rather than the retinal, size of an object determines its detection. It remains unclear, however, whether and how the retinal versus perceived size of a cue influences the process of attentional orienting to subsequent targets, and whether the corresponding influencing processes differ between two-dimensional (2-D) and three-dimensional (3-D) space. In the present study, we incorporated the dot probe paradigm with either a 2-D Ponzo illusion, induced by pictorial depth cues, or a virtual 3-D world in which the Ponzo illusion turned into visual reality. By varying the retinal size of the cue while keeping its perceived size constant (Exp. 1), we found that a cue with smaller retinal size significantly facilitated attentional orienting as compared to a cue with larger retinal size, and that the effects were comparable between 2-D and 3-D displays. Furthermore, when the pictorial background was removed and the cue display was positioned in either the farther or the closer depth plane (Exp. 2), or when both the depth and the background were removed (Exp. 3), the retinal size, rather than the depth, of the cue still affected attentional orienting. Taken together, our results suggest that the retinal size of a cue plays the crucial role in the visuospatial orienting of attention in both 2-D and 3-D.

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain-containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor-responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

Wuzhi tablet (WZ, registration no. in China: Z20025766) is a preparation of an ethanol herb extract of Wuweizi (Schisandra sphenanthera) containing 7.5 mg Schisantherin A per tablet. It was reported recently that WZ could significantly increase the blood concentrations of tacrolimus, which might be due to the inhibitory effect of WZ and its ingredients on P-gp and/or CYP450 activity. Paclitaxel is a substrate of the efflux transporter P-gp, and is mainly metabolized by CYP450 enzymes in the liver. Therefore, the purpose of this study was to investigate whether and how WZ affects the pharmacokinetics of paclitaxel in rats. After pretreatment with WZ, there were significant increases in the AUC0-24h of oral paclitaxel (from 280.8 ± 97.3 to 543.5 ± 115.2 h ng/mL; p

Obstructive cholestasis is characterized by impairment of hepatic canalicular bile efflux and there are no clinically effective drugs to cure except surgeries. Previously we revealed that oleanolic acid (OA) protected against lithocholic acid (LCA)-induced intrahepatic cholestasis in mice. Cholestasis caused by LCA is characterized by segmental bile duct obstruction, whether OA possesses the beneficial effect on completed obstructive cholestasis induced by bile duct ligation (BDL) remains unknown. In this study, we demonstrated that BDL-induced mice liver pathological change, and increase in serum levels of ALT, AST and ALP were all significantly reduced by OA (20 mg/kg, i.p.). Meanwhile, OA also lowered total bilirubin and total bile acids levels in serum, as well as total bile acids level in liver, in contrast, urinary total bile acids output was remarkably up-regulated by OA. Gene expression analysis showed that OA caused significant increased mRNA expression of MRP3 and MRP4 located at hepatic basolateral membrane, and restoration of MRP2 and BSEP located at hepatic cannalicular membrane. Furthermore, significant NRF2 protein accumulation in nucleus was also observed in OA treated mice. In mice primary cultured hepatocytes, the effects of OA on MRP2, MRP3 and MRP4 expression were directly proved to be mediated via NRF2 activation, and BSEP downregulation induced by OA was in part due to FXR antagonism. Luciferase assay performed in Hep G2 cells also illustrated that OA was a partial FXR antagonist. Taken together, we conclude that OA attenuates obstructive cholestasis in BDL mice, possibly via activation of NRF2-MRPs and FXR antagonism.

Resveratrol (RES) has been shown to possess many pharmacological activities including protective effect against liver damage induced by hepatotoxins. In the present study, the hepato-protective effect of RES against acetaminophen (APAP)-induced liver injury in mice and the involved mechanisms was investigated. This study clearly demonstrated that administration of RES three days before APAP treatment significantly alleviated APAP-induced hepatotoxicity, as evidenced by morphological, histopathological, and biochemical assessments such as GSH content and serum ALT/AST activity. Treatment with RES resulted in significant inhibition of CYP2E1, CYP3A11, and CYP1A2 activities, and then caused significant inhibition of the bioactivation of APAP into toxic metabolite NAPQI. Pretreatment with RES significantly reduced APAP-induced JNK activation to protect against mitochondrial injury. Additionally, RES treatment significantly induced SIRT1 and then negatively regulated p53 signaling to induce cell proliferation-associated proteins including cyclin D1, CDK4, and PCNA to promote hepatocyte proliferation. This study demonstrated that RES prevents APAP-induced hepatotoxicity by inhibition of CYP-mediated APAP bioactivation and regulation of SIRT1, p53, cyclin D1 and PCNA to facilitate liver regeneration following APAP-induced liver injury.

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra fructus is widely-used traditional Chinese medicine which possesses hepato-protective potential. Schisandrin A (SinA), Schisandrin B (SinB), Schisandrin C (SinC), Schisandrol A (SolA), Schisandrol B (SolB), and Schisantherin A (SthA) are the major bioactive lignans. Most recently, we found SolB exerts significant hepato-protection against APAP-induced liver injury. In this study, the protective effects of the other five schisandra lignans against APAP-induced acute hepatotoxicity in mice were investigated and compared with that of SolB. The results of morphological and biochemical assessment clearly demonstrated significant protective effects of SinA, SinB, SinC, SolA, SolB, and SthA against APAP-induced liver injury. Among these schisandra lignans, SinC and SolB exerted the strongest hepato-protective effects against APAP-induced hepatotoxicity. Six lignans pretreatment before APAP dosing could prevent the depletions of total liver glutathione (GSH) and mitochondrial GSH caused by APAP. Additionally, the lignans treatment inhibited the enzymatic activities of three CYP450 isoforms (CYP2E1, CYP1A2, and CYP3A11) related to APAP bioactivation, and further decreased the formation of APAP toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI) in mouse microsomal incubation system. This study demonstrated that SinA, SinB, SinC, SolA, SolB and SthA exhibited significant protective actions toward APAP-induced liver injury, which was partially associated with the inhibition of CYP-mediated APAP bioactivation.

Schisandra sphenanthera is widely used as a tonic and restorative in many countries to enhance the function of liver and other organs. Wuzhi tablet (WZ) is a preparation of an ethanol extract of Schisandra sphenanthera. Our previous study demonstrated that WZ exerted a protective effect toward acetaminophen (APAP)-induced hepatotoxicity. However, the molecular mechanisms of this protection remain unclear. This study aimed to determine what molecular pathways contributed to the hepatoprotective effects of WZ against APAP toxicity. Administration of WZ 3 days before APAP treatment significantly attenuated APAP hepatotoxicity in a dose-dependent manner and reduced APAP-induced JNK activation. Treatment with WZ resulted in potent inhibition of CYP2E1, CYP3A11, and CYP1A2 activities and then caused significant inhibition of the formation of the oxidized APAP metabolite N-acetyl-p-benzoquinone imine–reduced glutathione. The expression of NRF2 was increased after APAP and/or WZ treatment, whereas KEAP1 levels were decreased. The protein expression of NRF2 target genes including Gclc, Gclm, Ho-1, and Nqo1 was significantly increased by WZ treatment. Furthermore, APAP increased the levels of p53 and its downstream gene p21 to trigger cell cycle arrest and apoptosis, whereas WZ pretreatment could inhibit p53/p21 signaling to induce cell proliferation-associated proteins including cyclin D1, CDK4, PCNA, and ALR to promote hepatocyte proliferation. This study demonstrated that WZ prevented APAP-induced liver injury by inhibition of cytochrome P450–mediated APAP bioactivation, activation of the NRF2–antioxidant response element pathway to induce detoxification and antioxidation, and regulation of the p53, p21, cyclin D1, CDK4, PCNA, and ALR to facilitate liver regeneration after APAP-induced liver injury.

Enabled by liquid chromatography/tandem mass spectrometry (LC/MS/MS) based multiple-component assays, cassette dosing is an effective tool to significantly improve the throughput for pharmacokinetic (PK) screening. However, this higher throughput approach also carries its limitations. In addition to potential drug–drug interactions, the multiple-component assays used to analyze cassette-dosing samples are also subject to potentially serious errors. In this work, a systematic approach has been taken to investigate each critical step in the development of a specific, accurate, and rapid multiple-component assay. These steps include pool selection, sample preparation, chromatographic separation and assay qualification. Based on the results, effective strategies for the development of multiple-component assays are proposed which involve proactive pool assignment, on-line extraction, monolithic-column separation and cassette/discrete response comparison. The effectiveness of these strategies was evaluated by comparing the pharmacokinetic results between discrete and cassette studies for over 20 compounds. Statistically good correlation between discrete and cassette data was established.

Single-dose pharmacokinetics of 1-aminobenzotriazole (ABT), a potent nonspecific inhibitor of cytochromes P450 (P450s), were characterized after oral administration to mice and guinea pigs at doses of 50, 100, and 150 mg/kg using serial sampling in both species. Only 30-microl blood samples were drawn from jugular vein-cannulated mice using Microvette capillary tubes containing lithium heparin. A comparison of the pharmacokinetics of antipyrine (AP) administered i.v. at 20 mg/kg to mice followed by serial and terminal sampling techniques yielded similar results. The ABT concentrations in plasma were sustained at high levels (5-100 microM) for at least 12 h in both species. Pretreatment of animals with ABT 2 h prior to AP administration decreased the plasma AP clearance by about 95% in mice at all ABT doses studied and 84, 95, and 95% in guinea pigs at a dose of 50, 100, and 150 mg/kg ABT, respectively. In vitro, the dissociation constants (KI) for ABT as the P450 mechanism-based inactivator were determined to be 45.6 and 193 microM, and the maximal inactivation rate constants (kinact) were determined to be 0.089 and 0.075 min(-1) for the mouse and guinea pig liver microsomes, respectively. The projected P450 inactivations at the plasma Cmax of ABT agreed with the inhibitions of P450-mediated AP clearance observed in vivo. For mechanistic studies in vivo overall, a 2-h prior oral pretreatment with ABT at 50 mg/kg in mice and 100 mg/kg in guinea pigs would provide significant systemic concentrations of the inhibitor over 24 h and inhibition of P450-dependent clearance of test compounds.

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra sphenanthera is a traditional hepato-protective Chinese medicine and Schisandrol B (SolB) is one of its major active constituents. In this study, the protective effect of SolB against APAP-induced acute hepatotoxicity in mice and the involved mechanisms were investigated. Morphological and biochemical assessments clearly demonstrated a protective effect of SolB against APAP-induced liver injury. SolB pretreatment significantly attenuated the increases in alanine aminotransferase and aspartate aminotransferase activity, and prevented elevated hepatic malondialdehyde formation and the depletion of mitochondrial glutathione (GSH) in a dose-dependent manner. SolB also dramatically altered APAP metabolic activation by inhibiting the activities of CYP2E1 and CYP3A11, which was evidenced by significant inhibition of the formation of the oxidized APAP metabolite NAPQI–GSH. A molecular docking model also predicted that SolB had potential to interact with the CYP2E1 and CYP3A4 active sites. In addition, SolB abrogated APAP-induced activation of p53 and p21, and increased expression of liver regeneration and antiapoptotic-related proteins such as cyclin D1 (CCND1), PCNA, and BCL-2. This study demonstrated that SolB exhibited a significant protective effect toward APAP-induced liver injury, potentially through inhibition of CYP-mediated APAP bioactivation and regulation of the p53, p21, CCND1, PCNA, and BCL-2 to promote liver regeneration.

The Ru(II)-catalyzed ortho-C–H amination directed by a weakly coordinating amide auxiliary with O-benzoyl hydroxylamines at room temperature has been achieved. This reaction is compatible with heterocycles including pyrazole, thiophene, benzothiophene, furan, benzofuran, and indole.

Oleanolic acid (OA) is a natural triterpenoid and has been demonstrated to protect against varieties of hepatotoxicants. Recently, however, OA at high doses was reported to produce apparent cholestasis in mice. In this study, we characterized the protective effect of OA at low doses against lithocholic acid (LCA)-induced cholestasis in mice and explored further mechanisms. OA cotreatment (5, 10, and 20 mg/kg, i.p.) significantly improved mouse survival rate, attenuated liver necrosis, and decreased serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase; more importantly, serum total bile acids and bilirubin, as well as hepatic total bile acids were also remarkably reduced. Gene and protein expression analysis showed that hepatic expression of multidrug resistance-associated protein 2 (Mrp2), Mrp3, and Mrp4 was significantly increased by OA cotreatment, whereas other bile acid metabolism- and transport-related genes, including Na+/taurocholate cotransporter, organic anion transporter 1b2, bile salt export pump, multidrug resistance protein 3, Cyp3a11, Cyp2b10, Sulfotransferase 2a1 (Sult2a1), and UDP-glucuronosyltransferase 1a1 (Ugt1a1), were only slightly changed. OA also caused increased nuclear factor-E2-related factor (Nrf2) mRNA expression and nuclear protein accumulation, whereas nuclear receptors farnesoid X receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor were not significantly influenced by OA. Luciferase (Luc) assays performed in HepG2 cells illustrated that OA was a strong Nrf2 agonist with moderate PXR and weak FXR agonism. Finally, in mouse primary cultured hepatocytes, OA dose- and time-dependently induced expression of Mrp2, Mrp3, and Mrp4; however, this upregulation was abrogated when Nrf2 was silenced. In conclusion, OA produces a protective effect against LCA-induced hepatotoxicity and cholestasis, possibly due to Nrf2-mediated upregulation of Mrp2, Mrp3, and Mrp4.

The simultaneous dosing of numerous compounds followed by multiple-component analysis using LC-MS-MS (the N-in-1 approach) has significantly improved the throughput of the drug-screening process. However, plasma samples still need to be extracted before LC-MS-MS analysis, which frequently limits the throughput of the assay. In this work, a high-throughput on-line extraction technique has been developed for multiple-component LC-MS-MS assays using a high-flow column-switching technique. In N-in-1 LC-MS-MS assays, high sensitivity is required since the dose level is generally reduced to minimize drug-drug interactions. In addition, good chromatographic separation is essential to minimize interference and suppression effects. The direct plasma sample injection method developed in this work has successfully met the two requirements for multiple-component LC-MS-MS assays in high-throughput pharmacokinetic screening. Plasma samples containing a large number of potential drug candidates were directly injected onto an extraction column operated under a flow rate sufficiently high to exhibit a turbulent-flow profile. The extracted analytes were then eluted onto an analytical column via column switching for LC-MS-MS analysis. The use of turbulent flow resulted in a faster and more rugged extraction with reduced carryover compared with results obtained under laminar-flow conditions. Meanwhile, the use of a column-switching method maintained the chromatographic resolving power and high sensitivity of the LC-MS-MS assay. Separation efficiency, dynamic range, accuracy, and precision comparable with those of solid-phase extraction have been achieved with the turbulent-flow column-switching technique. As a result, this technique has been successfully and routinely used for high-throughput pharmacokinetic screening.

The Ru(II)-catalyzed ortho-C–H amination directed by a weakly coordinating amide auxiliary with O-benzoyl hydroxylamines at room temperature has been achieved. This reaction is compatible with heterocycles including pyrazole, thiophene, benzothiophene, furan, benzofuran, and indole.

Taxus yunnanensis (T. yunnanensis) is endemic to China and has been used in traditional medicine for the treatment of cancer, diabetic ailments and others. Paclitaxel is a representative antitumor compound in the Taxus species. The pharmacokinetic behavior of paclitaxel after oral administration of the crude extract of T. yunnanensis has not been investigated. This study attempts to compare the pharmacokinetics of paclitaxel after an oral administration of the crude extract of the twigs and leaves of T. yunnanensis and pure paclitaxel. A UPLC and a UPLC/MS/MS analysis method were developed for the determination of paclitaxel in T. yunnanensis extract and in the comparative pharmacokinetic study. Caco-2 cells were used to investigate the transport profile of paclitaxel in vitro. In the pharmacokinetic study, rats were randomly grouped and administered with T. yunnanensis extract or pure paclitaxel. The results showed that the AUC and C(max) of paclitaxel in rats receiving the T. yunnanensis extract were significantly increased than those receiving the pure paclitaxel, and the in vitro Caco-2 cell monolayer transport study found that the coexisting constituents in the extract of T. yunnanensis could inhibit the efflux of paclitaxel. These findings suggested that the oral absorption and bioavailability of paclitaxel in T. yunnanensis extract were remarkably higher when compared with the pure paclitaxel, and the coexisting constituents in the T. yunnanensis extract might play an important role for the enhancement of the oral absorption and bioavailability of paclitaxel.

The melanocortin 4 receptor (MC4R) plays an important role in body weight regulation and energy homeostasis. Administration of peptidic MC4R antagonists (usually by intracerebro ventricular injection) has been shown in the literature to increase body weight and/or food intake in several rodent models. We report here the identification of a novel nonpeptidic MC4R antagonist and its effects on tumor-induced weight loss in mice following peripheral administration.

In this work, monolithic columns were used as a fast separation tool for multiple-component quantitative liquid chromatography-tandem mass spectrometry (LC-MS-MS) assays of drug candidates in biological fluids. A considerably reduced runtime was achieved while maintaining good chromatographic separations. This significantly improved separation speed demanded higher throughput on sample extraction. To this end, monolithic separations were coupled on-line with high-flow extraction, which allowed for the fast extraction and separation of samples containing multiple analytes. An evaluation of this system was performed using a mixture of fenfluramine, temazepam, oxazepam, and tamoxifen in plasma. A total cycle time of 1.2 min was achieved which included both sample extraction and subsequent monolithic column separation via column switching. A total of over 400 plasma samples were analyzed in less than 10 h. The sensitivity and responses were reproducible throughout the run. The system has been routinely used in the authors' laboratory for high-throughput quantitation of compounds in biological fluids in support of drug discovery programs. The assay for samples from a 9-in-1 dog pharmacokinetic study is shown as an example to demonstrate the capability of this system.

Recently direct plasma injection LC/MS/MS technique has been increasingly used in pharmaceutical research and development due to the demand for higher throughput of sample analyses. In this work, two on-line extraction methods including high flow LC/MS/MS and high flow column switching LC/MS/MS were investigated. The evaluations were conducted and focused on their performances with respect to peak responses, separation efficiency, and signal to-noise ratio in a multiple-component LC/MS/MS assay. Two HPLC pumps were used-with one for high flow delivery and one for gradient elution. A CTC autosampler was used to inject plasma samples. High flow LC was achieved by the use of 4 ml/min flow rate on a 1 x 50 mm Waters Oasis column. A 2 x 100 mm YMC column was coupled via a column-switching valve. The extracted analytes were analyzed in multiple-reaction-monitoring (MRM) mode using a triple quadrupole MS/MS. As a rapid and simple procedure, vortex-mixing plasma and internal standard directly in sample vials completed sample preparation. The high flow column switching method (two-column system) provided sharper peak shape than the conventional high flow method. This effect increased analyte signal-to-noise ratio and sensitivity. Narrower peak width resulted in much better separation efficiency, which was required for multiple compound (N-in-1) analysis. A 2 mm I.D. column resulted in better peak shape and resolution than using a smaller I.D. column. The selected method achieved acceptable recoveries for most of the compounds tested, and it was successfully applied to a 10-in-1 pharmacokinetic (PK) study. The results showed that the dynamic range, lower limit of quantitation, assay accuracy and precision were acceptable for all compounds. Rapid sample preparation eliminated labor intensive and time consuming processes and improved productivity. This high throughput on-line extraction high flow column switching method has been proven particularly useful for multiple component analysis in PK studies.

In this work, an on-line four-column high-flow parallel extraction device was set up and interfaced to a four-column parallel separation system with a four-sprayer tandem mass spectrometric detector. This system allowed for the direct and simultaneous sampling, extraction, separation, and detection of four samples in biological fluids, resulting in significantly increased throughput. The performance of this system was evaluated using rat plasma samples spiked with methotrexate as a test compound. The results showed that the relative standard deviation for responses across the four parallel channels was less than 10% for a concentration range from 10 to 2500 nM, resulting in comparable sensitivity (slope of curve) for the analyte on all channels. A ruggedness test was performed using a mixed set of new and used extraction columns and the relative standard deviation in analyte response was found to be below 15%. The inter-channel crosstalk was measured to be about 0.1%. A comparison between the data generated on this system and on a conventional system for a rat pharmacokinetic study showed no more than 20% difference in measured concentrations. This parallel system is a viable tool for high-throughput bioanalysis in a drug discovery environment.

With the ever-increasing workload from a variety of in vitro and in vivo screening procedures, new analytical methodologies to perform bioanalysis in an accurate and high-throughput manner are in great demand. In this work, monolithic columns were used instead of conventional particulate HPLC columns to perform chromatographic separations. Because the pressure drop on a monolithic column was considerably lower than that on a particulate column, a high flow rate (6 mL/min) was used for a 4.6 × 50 mm monolithic column with a total backpressure of about 61 bar measured using acetonitrile/water (50 : 50). The capability of using a regular column length at high flow rates, combined with the extremely small dependency of separation efficiency on linear flow velocity, allowed for the generation of sufficient chromatographic resolving power in a significantly reduced runtime. As demonstrated in this work, a plasma extract of a mixture of tempazepam, tamoxifen, fenfluramine, and alprozolam were baseline separated within a total analysis time of one minute. An average peak width at half maximum of approximately one second was noted using a generic broad gradient. It was also found that the separation efficiency and signal/noise (S/N) ratios for this separation remained almost constant at flow rates of 1, 3, and 6 mL/min, respectively. The ruggedness of the separation was evaluated by injecting 600 plasma extracts containing the replicates of a standard curve of the above mixture during an overnight run. The chromatographic retention time, separation quality, peak response and sensitivity were highly reproducible throughout the run. This high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been used routinely in the authors' laboratory to support drug discovery programs. Copyright © 2001 John Wiley & Sons, Ltd.