Comparison of growth features and cancer stem cell prevalence in intrahepatic and extrahepatic cholangiocarcinoma cell lines.

Aim of the study: Intra- and extrahepatic cholangiocarcinoma (I-CCA and E-CCA respectively) exhibit different growth features that contribute to different clinical outcomes. Cancer stem cells (CSCs) influence tumor growth and thereby may be responsible for these differences. The aim of this study was to document and compare the growth features of human I-CCA and E-CCA cell lines and determine whether any differences observed could be explained by differences in the prevalence and/or stem cell surface marker (SCSM) expression profiles of CSCs within the tumor cell lines. Material and methods: Six CCA cells lines, three I-CCA and three E-CCA, were studied. Tumor cell growth features including cell proliferation, colony/spheroid formation, migration and invasion were documented. CSC prevalence and SCSM expression profiles were examined by flow cytometry. Results: I-CCA cells had significantly increased proliferative activity, shorter doubling times and were more invasive than E-CCA cells, while colony/spheroid formation and migration were similar in the two cell populations. There were no significant differences in CSC prevalence rates or SCSM expression profiles. Conclusions: These findings suggest that I-CCA cells proliferate at a more rapid rate and are more invasive than E-CCA cells but the differences cannot be explained by differences in the prevalence or SCSM expression profiles of CSCs within the tumor cell population.

Therapeutic impact of orally administered cannabinoid oil extracts in an experimental autoimmune encephalomyelitis animal model of multiple sclerosis.

There is a growing surge of investigative research involving the beneficial use of cannabinoids as novel interventional alternatives for multiple sclerosis (MS) and associated neuropathic pain (NPP). Using an experimental autoimmune encephalomyelitis (EAE) animal model of MS, we demonstrate the therapeutic effectiveness of two cannabinoid oil extract formulations (10:10 & 1:20 - tetrahydrocannabinol/cannabidiol) treatment. Our research findings confirm that cannabinoid treatment produces significant improvements in neurological disability scoring and behavioral assessments of NPP that directly result from their ability to reduce tumor necrosis factor alpha (TNF-α) production and enhance brain derived neurotrophic factor (BDNF) production. Henceforth, this research represents a critical step in advancing the literature by scientifically validating the merit for medical cannabinoid use and sets the foundation for future clinical trials.

The Antioxidant Activity of Recombinant Rat Hepatic Fatty Acid Binding Protein T94A Variant.

PURPOSE: Liver fatty acid binding protein (FABP1) is a cytoplasmic polypeptide that transports substrates throughout the cytosol and functions as an antioxidant. A common polymorphic variant, FABP1 T94A has a minor allele frequency of 26-38%, 8.3±1.9% homozygous in the human population. The purpose of this study was to mutate and isolate recombinant rat FABP1 to the T94A variant to evaluate the mutant's antioxidant activity using in vitro studies. METHODS: Site-directed mutagenesis was used to generate a mutation in rat cDNA within a pGEX-6p-2 vector. This plasmid was transformed into competent cells and cultured for expression of FABP1 T94A mutant. The mutated protein was purified using GSTrap Fastflow columns within an ÄKTA FPLC system. A 2,7-dichlorofluorescein (DCF) assay was used to screen the T94A variant antioxidant activity. Additionally, Thiobarbituric Acid Reactive Substances (TBARS) assay was used in determining T94A mutant antioxidant activity in hydrophilic and lipophilic environments through the use of the azo compounds AAPH and MeO-AMVN, respectively and in the presence and absence of the long-chain fatty acid palmitate and α-bromo palmitate. RESULTS: Although the FABP1 T94A (20 µM) mutant significantly reduced DCF fluorescence compared to control (no protein; P< 0.001), there were no significant difference when compared to the wild-type (WT) FABP1. T94A was able to diminish the formation of malondialdehyde (MDA) in both lipophilic and hydrophilic systems. There were significant differences between T94A mutant and WT FABP1 at concentrations 1 and 10 µM (P< 0.05) in the hydrophilic milieu, however, this was not seen at 20 µM and also not seen in the lipophilic milieu at all concentrations. When T94A was pre-incubated with the long-chain fatty acids palmitate or α -bromo palmitate, MDA formation was decreased in both lipid peroxidation systems. There were no statistical differences between the WT FABP1 and T94A bound with fatty acids in both lipid peroxidation systems, however, there was a slight statistical difference when the T94A and WT FABP1 bound α-Br-PA in the AAPH lipid peroxidation system only. CONCLUSIONS: The T94A has antioxidant activity in both hydrophilic and lipophilic environments. The T94A variant of FABP1 does not have a loss of function in regard to acting as an antioxidant but the extent of function may be influenced by ligand binding. We conclude that populations having the minor T94A allele frequency would have similar ROS scavenging potential as those with nascent FABP1.

Pharmaceutical Characterization of MyoNovin, a Novel Skeletal Muscle Regenerator: in silico, in vitro and in vivo Studies.

PURPOSE: MyoNovin is a novel skeletal muscle-regenerating compound developed through synthesis of two nitro groups onto a guaifenesin backbone to deliver nitric oxide to skeletal muscle with a potential to treat muscle atrophy. The purpose of this study was to utilize in silico, in vitro, and in vivo approaches to characterize MyoNovin and examine its safety, biodistribution, and feasibility for drug delivery. METHODS: In silico software packages were used to predict the physicochemical and biopharmaceutical properties of MyoNovin. In vitro cardiotoxicity was assessed using human cardiomyocytes (RL-14) while effects on CYP3A4 metabolic enzyme and antioxidant activity were examined using commercial kits. A novel HPLC assay was developed to measure MyoNovin concentration in serum, and delineate initial pharmacokinetic and acute toxicity after intravenous administration (20 mg/kg) to male Sprague-Dawley rats. RESULTS: MyoNovin showed relatively high lipophilicity with a LogP value of 3.49, a 20-fold higher skin permeability (19.89 cm/s*107) compared to guaifenesin (0.66 cm/s*107), and ~10-fold higher effective jejunal permeability (2.24 cm/s*104) compared to guaifenesin (0.26 cm/s*104). In vitro, MyoNovinwas not cytotoxic to cardiomyocytes at concentrations below 8 µM and did not inhibit CYP3A4 or show antioxidant activity. In vivo, MyoNovin had a short half-life (t1/2) of 0.16 h, and a volume of distribution Vss of 0.62 L/kg. Biomarkers of MyoNovincardiac and renal toxicity did not differ significantly from baseline control levels. CONCLUSIONS: The predicted high lipophilicity and skin permeability of MyoNovin render it a potential candidate for transdermal administration while its favourable intestinal permeation suggests it may be suitable for oral administration. Pharmacokinetics following IV administration of MyoNovin were delineated for the first time in a rat model. Preliminary single 20 mg/kg dose assessment of MyoNovin suggest no influenceon cardiac troponin or ß-N-Acetylglucosaminidase. 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.

Inhibition of BMP4 and Alpha Smooth Muscle Actin Expression in LX-2 Hepatic Stellate Cells by BMP4-siRNA Lipid Based Nanoparticle.

PURPOSE: To develop and characterize vitamin A (VA)-coupled liposomes for the targeted delivery of BMP4-siRNA to hepatic stellate cells (HSC). VA was selected to increase the uptake by HSC based on their function in the storage of VA. METHODS: DOTAP/DOPE liposomes were prepared by film hydration method and their surfaces were decorated with VA. The cytotoxicity of VA-conjugated liposomes was evaluated by the WST-1 assay. Inhibition of BMP4 and α-SMA was determined by PCR and ELISA. RESULTS: VA-coated lipoplexes exhibited an average particle sizes less than 200 nm and zeta potential around +25 mV both determined using ZetaPALS. Inclusion of VA to liposomal surfaces significantly enhanced their cellular uptake without affecting cytotoxicity. VA-coupled liposomes carrying BMP4-siRNA resulted in a significant reduction in BMP4 and α-SMA at both mRNA and protein levels.  Conclusion: VA-coated liposomes were successfully designed to deliver BMP4-siRNA to specifically target HSC. The novel delivery system discussed herein may serve as a potential therapeutic strategy for the treatment of liver fibrosis in the future. 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.

Review of Recent Patents and Developments in Skeletal Muscle Regeneration.

Skeletal muscle is a highly-specialized tissue that is capable of contractile function and able to withstand and adapt to daily mechanical and physiological stress. Musculoskeletal disorders, including muscular dystrophies, result in chronic pain and disability, reduced quality of life, burden on family, and increased healthcare costs. Although several mechanisms have been identified for muscle injury and regeneration, mechanisms of these diseases are poorly understood, and targeted and effective pharmacologic treatment(s) are not available. More research is needed in this area in order to develop effective treatment regimens. The purpose of this review is to discuss known mechanisms of muscle injury and repair, and highlight some recent patents and research developments for treatment of skeletal muscle disorders.

Pharmacokinetic and Toxicodynamic Characterization of a Novel Doxorubicin Derivative.

Doxorubicin (Dox) is an effective anti-cancer medication with poor oral bioavailability and systemic toxicities. DoxQ was developed by conjugating Dox to the lymphatically absorbed antioxidant quercetin to improve Dox's bioavailability and tolerability. The purpose of this study was to characterize the pharmacokinetics and safety of Dox after intravenous (IV) and oral (PO) administration of DoxQ or Dox (10 mg/kg) and investigate the intestinal lymphatic delivery of Dox after PO DoxQ administration in male Sprague-Dawley rats. Drug concentrations in serum, urine, and lymph were quantified by HPLC with fluorescence detection. DoxQ intact IV showed a 5-fold increase in the area under the curve (AUC)-18.6 ± 1.98 compared to 3.97 ± 0.71 µg * h/mL after Dox-and a significant reduction in the volume of distribution (Vss): 0.138 ± 0.015 versus 6.35 ± 1.06 L/kg. The fraction excreted unchanged in urine (fe) of IV DoxQ and Dox was ~5% and ~11%, respectively. Cumulative amounts of Dox in the mesenteric lymph fluid after oral DoxQ were twice as high as Dox in a mesenteric lymph duct cannulation rat model. Oral DoxQ increased AUC of Dox by ~1.5-fold compared to after oral Dox. Concentrations of ß-N-Acetylglucosaminidase (NAG) but not cardiac troponin (cTnI) were lower after IV DoxQ than Dox. DoxQ altered the pharmacokinetic disposition of Dox, improved its renal safety and oral bioavailability, and is in part transported through intestinal lymphatics.

Clofibrate Attenuates ROS Production by Lipid Overload in Cultured Rat Hepatoma Cells.

PURPOSE: To investigate the effect of clofibrate on inducing liver fatty acid binding protein (FABP1) following a high-fat load in a hepatocyte cell culture model. METHODS: Rat hepatoma cells (CRL-1548) were treated with a fatty acid (FA) mixture consisting of oleate:palmitate (2:1) in the presence of 3% albumin. Cells were treated with 0, 0.5, 1, 2, or 3 mM FA for 24 and 48 hr, or further treated with 500 µM clofibrate (CLO) to induce FABP1 levels. Cytotoxicity was determined using the WST-1 assay. Intracellular lipid droplets were quantitated following staining with Nile Red. Dichlorofluorescein (DCF) was used to assess the extent of intracellular reactive oxygen species (ROS). RESULTS: Cell viability decreased (p < 0.01) with an increase in lipid concentration. Intracellular lipid droplets accumulated significantly (p < 0.001) with an increase in long-chain fatty acid load, which was associated with a statistical increase (p < 0.05) in ROS levels. Early clofibrate treatment showed significant increases in intracellular FABP1 levels with significant decreases in ROS levels (p < 0.05). Silencing FABP1 expression using siRNA revealed that FABP1 was the main contributor for the observed intracellular ROS clearance. CONCLUSIONS: Characteristic cellular damage resulted from released ROS following a high fat load to hepatoma cells. The damage was attenuated through early treatment with clofibrate, which may act as a hepatoprotectant by inducing FABP1 expression and in this manner, suppress intracellular ROS levels. 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.

The Role of Extracellular Binding Proteins in the Cellular Uptake of Drugs: Impact on Quantitative In Vitro-to-In Vivo Extrapolations of Toxicity and Efficacy in Physiologically Based Pharmacokinetic-Pharmacodynamic Research.

A critical component in the development of physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) models for estimating target organ dosimetry in pharmacology and toxicology studies is the understanding of the uptake kinetics and accumulation of drugs and chemicals at the cellular level. Therefore, predicting free drug concentrations in intracellular fluid will contribute to our understanding of concentrations at the site of action in cells in PBPK/PD research. Some investigators believe that uptake of drugs in cells is solely driven by the unbound fraction; conversely, others argue that the protein-bound fraction contributes a significant portion of the total amount delivered to cells. Accordingly, the current literature suggests the existence of a so-called albumin-mediated uptake mechanism(s) for the protein-bound fraction (i.e., extracellular protein-facilitated uptake mechanisms) at least in hepatocytes and cardiac myocytes; however, such mechanism(s) and cells from other organs deserve further exploration. Therefore, the main objective of this present study was to discuss further the implication of potential protein-facilitated uptake mechanism(s) on drug distribution in cells under in vivo conditions. The interplay between the protein-facilitated uptake mechanism(s) and the effects of a pH gradient, metabolism, transport, and permeation limitation potentially occurring in cells was also discussed, as this should violate the basic assumption on similar free drug concentration in cells and plasma. This was made because the published equations used to calculate drug concentrations in cells in a PBPK/PD model did not consider potential protein-facilitated uptake mechanism(s). Consequently, we corrected some published equations for calculating the free drug concentrations in cells compared with plasma in PBPK/PD modeling studies, and we proposed a refined strategy for potentially performing more accurate quantitative in vitro-to-in vivo extrapolations (IVIVEs) of toxicity (efficacy) at the cellular level from data generated in cell assays. Overall, this present study may help to optimize the human dose prediction in preclinical and clinical studies, while prescribing drugs with narrow therapeutic windows that are highly bound to extracellular proteins and/or highly ionized at the physiological pH. This may facilitate building a more accurate safety (efficacy) profile for such drugs.

Recent insights into the biological functions of liver fatty acid binding protein 1.

Over four decades have passed since liver fatty acid binding protein (FABP)1 was first isolated. There are few protein families for which most of the complete tertiary structures, binding properties, and tissue occurrences are described in such detail and yet new functions are being uncovered for this protein. FABP1 is known to be critical for fatty acid uptake and intracellular transport and also has an important role in regulating lipid metabolism and cellular signaling pathways. FABP1 is an important endogenous cytoprotectant, minimizing hepatocyte oxidative damage and interfering with ischemia-reperfusion and other hepatic injuries. The protein may be targeted for metabolic activation through the cross-talk among many transcriptional factors and their activating ligands. Deficiency or malfunction of FABP1 has been reported in several diseases. FABP1 also influences cell proliferation during liver regeneration and may be considered as a prognostic factor for hepatic surgery. FABP1 binds and modulates the action of many molecules such as fatty acids, heme, and other metalloporphyrins. The ability to bind heme is another cytoprotective property and one that deserves closer investigation. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation and the adverse effects of starvation (energy deficiency).

Immune System Induction of Nerve Growth Factor in an Animal Model of Multiple Sclerosis: Implications in Re-Myelination and Myelin Repair.

Nerve growth factor (NGF) expression is augmented during neuroinflammation. However, its function in the dorsal root ganglia (DRG) and spinal cord (SC) during experimental autoimmune encephalomyelitis (EAE), the inflammatory model of Multiple Sclerosis, is indistinct. Thus, the role of antigenically induced NGF in Lewis rats under a state of EAE was considered. NGF mRNA and protein expression were highly increased in DRG and SC tissues in animals with EAE. Between 18 and 24 days post induction (dpi), NGF mRNA and protein were elevated in the DRG, correlating with neurological recovery. In the SC, an increase in NGF protein at 12 dpi was, in contrast, preceded by neurological recovery. NGF mRNA expression became elevated in the SC at 15 dpi at the onset of neurological improvement and amelioration of EAE. This study revealed that antigenic induction of the 25 kDa pro-NGF isoform is associated with the disease course of EAE. Our findings suggest the induction of NGF represents an adaptive response against immune-mediated neuroinflammation in the DRG and SC that likely contributes to the EAE attenuation.

Assessing steatotic liver function after ischemia-reperfusion injury by in vivo multiphoton imaging of fluorescein disposition.

Ischemia-reperfusion injury, a common complication during liver surgery where steatotic livers are more prone to the injury, may become more prevalent in the growing obese population. This study characterizes liver morphology toward understanding changes in subcellular function in steatotic livers exposed to ischemia-reperfusion injury through quantitative description of fluorescein distribution obtained by minimally invasive in vivo multiphoton microscopy using a physiologic pharmacokinetic model. Rats were fed a high-fat diet for 7 days to induce liver steatosis. Partial ischemia was induced after reperfusion for 4 hours, when fluorescein (10 mg/kg) was injected intravenously. Liver images, bile, and blood were collected up to 180 minutes after injection. Ischemia-reperfusion injury was associated with an increase in alanine transaminase levels and apoptosis. In addition, steatosis featured lipid droplets and an increase in fluorescein-associated fluorescence observed in hepatocytes via multiphoton imaging. Analysis of the hepatic concentration-time profiles has suggested that the steatosis-induced increase in fluorescein-associated fluorescence mainly arises by inducing hepatic fluorescein metabolism. The combination of ischemia-reperfusion with steatosis exacerbates these effects further. This was confirmed by fluorescence lifetime imaging microscopy showing a decreased average fluorescence lifetime of the liver, which is indicative of an increased production of the metabolite. Our results show the potential of noninvasive dye imaging for improving our understanding of liver disease induced by subcellular changes in vivo, providing further quantitative measures of metabolic and biliary liver function, and hence extending the qualitative liver function tests now available.

Multiphoton microscopy in defining liver function.

Multiphoton microscopy is the preferred method when in vivo deep-tissue imaging is required. This review presents the application of multiphoton microscopy in defining liver function. In particular, multiphoton microscopy is useful in imaging intracellular events, such as mitochondrial depolarization and cellular metabolism in terms of NAD(P)H changes with fluorescence lifetime imaging microscopy. The morphology of hepatocytes can be visualized without exogenously administered fluorescent dyes by utilizing their autofluorescence and second harmonic generation signal of collagen, which is useful in diagnosing liver disease. More specific imaging, such as studying drug transport in normal and diseased livers are achievable, but require exogenously administered fluorescent dyes. If these techniques can be translated into clinical use to assess liver function, it would greatly improve early diagnosis of organ viability, fibrosis, and cancer.

Effects of long-term hepatic ischemia-reperfusion injury on the function of P-glycoprotein in vivo in rats.

PURPOSE: Ischemia-reperfusion injury is a common complication in liver surgery with oxidative stress related graft failure as a potential complication. The oxidative stress could affect hepatic drug transporters such as P-glycoprotein, which is crucial in the hepatic clearance of certain immunosuppressant drugs. Thus,, it is important to study its function after ischemia-reperfusion injury in vivo. Rhodamine 123 is a fluorescent substrate of P-glycoprotein and its hepatic disposition can be visualized using multiphoton microscopy in vivo using anaesthetized animals. The aim of this study was to investigate the effect of long-term ischemia-reperfusion injury on P-glycoprotein function in hepatocytes using in vivo multiphoton microscopy. METHODS: Localized ischemia was induced for 1 hour in rats. The liver was reperfused for 4, 24, 48 hours or 1 week, where-after rhodamine 123 was injected intravenously. Multiphoton microscopy imaged the liver and bile was collected continuously up to 6 hours following drug administration. The liver was harvested for histology and protein expression of P-glycoprotein. RESULTS: Ischemia-reperfusion injury resulted in extensive liver damage, inflammatory cell infiltration and apoptosis in the midzonal and centrilobular regions of the liver acinus. P-glycoprotein protein expression decreased. Cellular concentration of rhodamine 123 increased as visualized by multiphoton microscopy, which was confirmed with decreased excretion of rhodamine 123 in collected bile. CONCLUSIONS: This study showed reduced function of P-glycoprotein in ischemia-reperfusion injury as reflected by decreased biliary excretion of Rhodamine 123, as well as reduced protein expression of the transporter. Multiphoton microscopy could be used to visualize and quantitate the intracellular levels of rhodamine 123. These findings stipulate the importance of using multiphoton microscopy to understand transmembrane drug flux and reflect on careful drug dosing after hepatic surgery.

Hepatoprotective role of liver fatty acid binding protein in acetaminophen induced toxicity.

BACKGROUND: FABP1 has been reported to possess strong antioxidant properties. Upon successful transfection of the Chang cell line, which has undetectable FABP1 mRNA levels, with human FABP1 cDNA, the Chang cells were shown to express FABP1. Using the transfected and control (normal) Chang cells and subjecting them to oxidative stress, transfected cells were reported to be associated with enhanced cell viability. This study extends those observations by investigating the effect of FABP1 on acetaminophen (AAP)-induced hepatotoxicity. We hypothesized that presence of FABP1 would enhance cell viability compared to control cells (vector transfected cells). METHODS: Following AAP treatment of Chang FABP1 transfected and control cells, cell viability, oxidative stress, and apoptosis were evaluated using lactate dehydrogenase (LDH) release, the fluorescent probe DCF, and Bax expression, respectively. RESULTS: FABP1 cDNA transfected cells showed greater resistance against AAP toxicity than vector transfected cells. Significantly lower LDH levels (p < 0.05) were observed as were lower DCF fluorescence intensity (p < 0.05) in FABP1 cDNA transfected cells compared to vector transfected cells. FABP1 expression also attenuated the expression of Bax following AAP induced toxicity. CONCLUSION: FABP1 attenuated AAP-induced toxicity and may be considered a cytoprotective agent in this in vitro model of drug induced oxidative stress.

Membrane potential differences and GABAA receptor expression in hepatic tumor and non-tumor stem cells.

The ability to differentiate tumor initiating stem cells (TISCs) from healthy, normal stem cells (NSCs) could have important diagnostic and therapeutic implications for patients with hepatocellular carcinoma (HCC). The aim of this study was to document and compare cell membrane potentials (PDs) and GABAA receptor subunit expression in hepatic TISCs and NSCs. PD values were determined in CD133(+) Huh-7 TISCs and CD133(+) WBF344 NSCs by single channel microelectrode impalement. GABAA receptor subunit expression was documented using immunohistochemistry (IH) in both cell lines as well as surgically resected HCC and healthy liver tissues. TISCs were significantly depolarized compared with NSCs (-4.0 ± 1.8 versus -11.0 ± 2.4 mV, respectively; p < 0.05). GABAA α6 subunit expression was either absent or markedly attenuated, while γ3 subunit expression was abundant in TISCs and HCC compared with NSCs and healthy liver tissues. Exposure to the GABAA receptor agonist muscimol caused hyperpolarization of TISCs (Δ -4.4 ± 1.1) but depolarization of NSCs (Δ + 5.2 ± 2.3) and attenuation of TISC proliferative activity. We conclude that TISCs and NSCs have significantly different cell membrane potentials and these differences are associated with differences in GABAA receptor subunit expression.

Evaluation of percutaneous permeation of repellent DEET and sunscreen oxybenzone from emulsion-based formulations in artificial membrane and human skin.

Insect repellent DEET and sunscreen ingredient oxybenzone play an essential role in minimizing vector-borne diseases and skin cancers. The purpose of this study was to investigate the effects of emulsion type, addition of thickening agent and droplet size in three emulsion-based lotions on percutaneous permeation of DEET and oxybenzone using in vitro diffusion experiments, in order to minimize overall systemic permeation of the substances. Formulation C (water-in-oil emulsion) significantly increased overall permeation of DEET through human skin (56%) compared to Formulation A (oil-in-water emulsion). Formulation B (oil-in-water emulsion with thickening agent xanthan gum) significantly decreased the size of oil droplet containing DEET (16%), but no effect on oil droplets containing oxybenzone. Adding xanthan gum also increased overall permeation of DEET and oxybenzone (21% and 150%) when compared to Formulation A; presence of both ingredients in Formulation B further increased their permeation (36% and 23%) in comparison to its single counterparts. Overall permeation of oxybenzone through LDPE was significantly higher by 26%-628% than that through human skin; overall permeation of DEET through human skin was significantly higher by 64%-338% than that through LDPE.

Saikosaponin A of Bupleurum chinense (Chaihu) elevates bone morphogenetic protein 4 (BMP-4) during hepatic stellate cell activation.

BACKGROUND: Saikosaponin a (SSa) is a compound extracted from a Chinese herb which has been widely used in treating liver diseases such as liver fibrosis. However, the mechanism of SSa in treatment of liver fibrosis still remain unclear. Our previous study demonstrated that BMP4 stimulated the expression of smooth muscle alpha actin (α-SMA) in the liver. Therefore, the current study investigates the effect of SSa on BMP4 expression during hepatic stellate cell activation in a human hepatic stellate cell line. METHODS: LX-2 cells were cultured in DMEM/F12 with fetal bovine serum and treated with SSa in different times and concentrations. The expression of BMP4 was examined by both RT-PCR and western blot analysis. WST-1 proliferation reagent was used to evaluate cell proliferation. α-SMA and Bax protein expression was determined by western blot analysis. RESULTS: Both mRNA and protein levels of BMP-4 were significantly inhibited in LX-2 cells after 5 µM SSa treatment. SSa significantly inhibited LX-2 proliferation at the concentration of 5µM while BMP-4 had no effect on LX-2 proliferation. BMP-4 increased α-SMA expression in LX-2 while SSa reduced α-SMA expression. In addition SSa could neutralize the effect of BMP-4 on α-SMA expression. SSd also inhibited BMP4 expression but not NG. Bax protein expression was induced in these cells by 5 µM SSa. CONCLUSION: SSa could down-regulate BMP-4 expression and inhibit hepatic stellate cell activation. Therefore, SSa could be used for treatment of liver disease with elevated BMP-4 expression.

Intravital multiphoton microscopy can model uptake and excretion of fluorescein in hepatic ischemia-reperfusion injury.

The liver is important in the biotransformation of various drugs, where hepatic transporters facilitate uptake and excretion. Ischemia-reperfusion (I/R) injury is a common occurrence in liver surgery, and the developing oxidative stress can lead to graft failure. We used intravital multiphoton tomography, with fluorescence lifetime imaging, to characterize metabolic damage associated with hepatic I/R injury and to model the distribution of fluorescein as a measure of liver function. In addition to measuring a significant increase in serum alanine transaminase levels, characteristic of hepatic I/R injury, a decrease in the averaged weighted lifetime of reduced nicotinamide adenine dinucleotide phosphate was observed, which can be attributed to a changed metabolic redox state of the hepatocytes. I/R injury was associated with delayed uptake and excretion of fluorescein and elevated area-under-the-curve within the hepatocytes compared to sham (i.e., untreated control) as visualized and modeled using images recorded by intravital multiphoton tomography. High-performance liquid chromatography analysis showed no differences in plasma or bile concentrations of fluorescein. Finally, altered fluorescein distribution was associated with acute changes in the expression of liver transport proteins. In summary, multiphoton intravital imaging is an effective approach to measure liver function and is more sensitive in contrasting the impact of I/R injury than measuring plasma and bile concentrations of fluorescein.

Regulation of transforming growth factor beta 1 gene expression by dihydropteridine reductase in kidney 293T cells.

Quinoid dihydropteridine reductase (QDPR) is an enzyme involved in the metabolic pathway of tetrahydrobiopterin (BH4). BH4 is an essential cofactor of nitric oxide synthase (NOS) and can catalyze arginine to citrulline to release nitric oxide. Point mutations of QDPR have been found in the renal cortex of spontaneous Otsuka Long Evans Tokushima Fatty (OLETF) diabetic rats. However, the role of QDPR in DN is not clear. This study investigates the effects of QDPR overexpression and knockdown on gene expression in the kidney. Rat QDPR cDNA was cloned into pcDNA3.1 vector and transfected in human kidney cells (293T). The expression of NOS, transforming growth factor beta 1 (TGF-ß1), Smad3, and NADPH oxidase were examined by RT-PCR and Western blot analyses. BH4 was assayed by using ELISA. Expression of QDPR was significantly decreased and TGF-ß1 and Smad3 were increased in the renal cortex of diabetic rats. Transfection of QDPR into 293T cells increased the abundance of QDPR in cytoplasm and significantly reduced the expression of TGF-ß1, Smad3, and the NADPH oxidases NOX1 and NOX4. Moreover, abundance of neuronal NOS (nNOS) mRNA and BH4 content were significantly increased. Furthermore, inhibition of QDPR resulted in a significant increase in TGF-ß1 expression. In conclusion, QDPR might be an important factor mediating diabetic nephropathy through its regulation of TGF-ß1/Smad3 signaling and NADPH oxidase.