Hepatitis C infection is a global health problem. Spontaneous viral clearance was observed in approximately 30% of individuals with acute infection. In the therapy using a combination of pegylated interferon-α and ribavirin, approximately 50% of chronic hepatitis C patients infected with high viremia of hepatitis C virus infection (HCV) genotype 1 reached a sustained viral response. These findings were strongly expected to reflect variations of the host genome. To reveal genetic effects against viral clearance or treatment response, four independent groups applied a genome-wide association study (GWAS) to HCV infection. These groups almost simultaneously reported a strong association of interleukin (IL)-28B polymorphisms with viral clearance or final decision of HCV therapy. The discovered single nucleotide polymorphisms (SNPs) also revealed the enigma that the viral clearance rate was dependent on ethnic type. The significant SNPs are useful for prediction prior to treatment because of the strong association with clinical outcome. In addition, the unexpected results revealed by GWAS could promote the development of a novel drug related to IL-28B. Herein, we present current understanding in regard to the relationship between host variations and clinical outcome of hepatitis C.
The factors involved in the progression of non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH) are not fully understood and thus it is urgently needed to elucidate these factors. Steatosis is not causal in the development of NASH, but rather it sensitizes the liver to the damaging effects of second hits such that stressors innocuous to a healthy liver lead to the development of NASH in the steatotic liver. In the previous study, most of the hepatic lipid metabolite profiles were similar in the NAFL and NASH groups. However, very-low-density lipoprotein (VLDL) synthesis, especially hepatic microsomal triglyceride transfer protein (MTP) mRNA expression, was impaired in the NASH group. Moreover, NASH showed significantly higher incidence of minor alley appearance compared with NAFL, indicating the possibility of association between NASH pathogenesis and decreased congenital MTP activity. MTP is one of the enzymes that transfer triglycerides to nascent apolipoprotein B, producing VLDL and removing lipid from the hepatocyte. A growing body of literature suggests that the measurement of hepatic MTP expression may be helpful for diagnosis; and moreover, hepatic MTP activator may be a possible therapeutic agent for the treatment of NASH.
We have developed an in vivo mouse model [the green fluorescent protein (GFP) / carbon tetrachloride (CCl4) model] and reported that infused GFP-positive bone marrow cells administered via a tail vein efficiently repopulated cirrhotic liver tissue under conditions of persistent liver damage induced by CCl4. Moreover, bone marrow cells infused into the liver improved liver function and ameliorated liver fibrosis with higher expression of matrix metalloproteinase 9 (MMP-9), consistent with improved survival rate. Based on these findings, we started a multicenter clinical trial of autologous bone marrow cell infusion (ABMi) therapy for decompensated liver cirrhosis patients and demonstrated the efficacy of this approach without unexpected complications. However, this therapy involves bone marrow aspiration under general anesthesia and is not indicated for patients for whom general anesthesia is difficult. We therefore aimed to develop a new liver regeneration therapy in which cells having a curative effect on liver cirrhosis are isolated and cultured from a small amount of autologous bone marrow aspirated under local anesthesia and infused back into the same subject. Herein, we present results for the GFP/CCl4 model and ABMi therapy and future prospects for a new liver regeneration therapy.
Selective agonists of the sigma-1 (σ1) ligand–operated chaperone protein, like igmesine or PRE-084, are antidepressants in preclinical depression models. σ1-Protein activation may contribute to the antidepressant efficacy of drugs known to act as selective serotonin-reuptake inhibitors (SSRI) or noradrenaline reuptake inhibitors through direct or indirect involvement of the σ1-receptor in the drug effect. We here compared antidepressant effects in two behavioral procedures, the forced swimming test (FST) and conditioned fear stress (CFS). The involvement of the σ1-receptor was examined using a co-treatment with the σ1-antagonist BD1047 or using σ1-knockout (KO) mice. Igmesine but not PRE-084 decreased FST immobility. The SSRI fluoxetine and sertraline, but not fluvoxamine, and the tricyclic antidepressants imipramine, desipramine, and amitriptyline were also effective. Only the effect of igmesine was blocked by BD1047 or in σ1-KO mice. Igmesine, PRE-084, fluvoxamine, and sertraline decreased the CFS immobility in a BD1047- and σ1-KO–sensitive manner. Among tricyclics, only amitriptyline was effective and its effect was unaffected by BD1047 or in σ1-KO mice. The behavioral effects induced by mixed σ1-receptor/SSRI antidepressants, like fluvoxamine or sertraline, may therefore involve a non-selective action at both targets. Moreover, the CFS appears to more reliably uncover a σ1 pharmacological component in antidepressant screening.
We investigated actions of uridine 5′-triphosphate (UTP) in rat aorta, cerebral and mesenteric arteries, and their single myocytes. UTP (≥10 μM) elicited an inward-rectifying current strongly reminiscent of activation of P2X1 receptor, and a similar current was also induced by α,β-methylene adenosine 5′-triphosphate (ATP) (≥100 nM). UTP desensitized α,β-methylene ATP–evoked current, and vice versa. The UTP-activated current was insensitive to G-protein modulators, TRPC3 inhibitors, or TRPC3 antibody, but was sensitive to P2-receptor inhibitors or P2X1-receptor antibody. Both UTP (1 mM) and α,β-methylene ATP (10 μM) elicited similar conductance single channel activities. UTP (≥10 μM) provoked a dose-dependent contraction of de-endothelialized aortic ring preparation consisting of phasic and tonic components. Removal of extracellular Ca2+ or bath-applied 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) (30 μM) or nifedipine (10 μM) completely inhibited the phasic contraction while only partially reducing the tonic one. The tonic contraction was almost completely abolished by additional application of thapsigargin (2 μM). Similar biphasic rises in [Ca2+]i were also evoked by UTP in rat aortic myocytes. In contrast to the low expression of TRPC3, significant expression of P2X1 receptor was detected in all arteries by RT-PCR and immunoblotting, and its localization was limited to plasma membrane of myocytes as indicated by immunohistochemistry. These results suggest that UTP dually activates P2X1-like and P2Y receptors, but not TRPC3. [Supplementary materials: available only at http://dx.doi.org/10.1254/jphs.10281FP]
We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the bidirectional cystine/Glu antiporter in undifferentiated osteoblastic MC3T3-E1 cells. In this study, we investigated the expression profile of the xCT subunit of the antiporter as well as the master regulator of osteoblastogenesis runt-related transcription factor-2 (Runx2) in ovariectomized mouse bone. In spinal columns isolated 28 days after ovariectomy, a marked reduction was seen with the intensity of Von Kossa staining used as an index of ossification. In femurs of these ovariectomized mice, a significant decrease was seen in mRNA and protein levels of Runx2 along with increased expression of both mRNA and the corresponding protein for the xCT subunit. To evaluate the possible role of the antiporter in osteoblastogenesis, stable transfectants were established with the xCT subunit toward the culture with osteoblastic differentiation inducers in MC3T3-E1 cells. In stable xCT transfectants cultured under differentiation conditions, marked decreases were seen in nodule formation, Ca2+ accumulation, and osteoblastic marker gene expression, in addition to downregulation of both mRNA and the corresponding protein for Runx2. Runx2 promoter activity was markedly stimulated in MC3T3-E1 cells transfected with a responsive promoter plasmid after the culture under differentiation conditions, while transient and stable transfection with xCT expression vector invariably prevented the stimulation through an activator protein-1 site. These results suggest that Runx2 expression would be negatively regulated by the cystine/glutamate antiporter expressed by osteoblastic cells at the level of gene transactivation.
Oxidative stress plays pivotal roles in aging, neurodegenerative disease, and pathological conditions such as ischemia. We investigated the effect of sulforaphane and 6-(methysulfinyl) hexyl isothiocyanate (6-HITC), a naturally occurring isothiocyanate, on oxidative stress–induced cytotoxicity using primary neuronal cultures of rat striatum. Pretreatment with sulforaphane and 6-HITC significantly protected against H2O2- and paraquat-induced cytotoxicity in a concentration-dependent manner. Sulforaphane and 6-HITC induced the translocation of nuclear factor E2–related factor 2 (Nrf2) into the nucleus and increased the expression of γ-glutamylcysteine synthetase (γ-GCS), a rate-limiting enzyme in glutathione synthesis, and the intracellular glutathione content. Treatment with reduced glutathione (GSH) and N-acetyl-L-cysteine, a substance for glutathione synthesis, significantly prevented the cytotoxicity induced by H2O2 and paraquat. Moreover, exposure to L-buthionine-sulfoximine, an irreversible inhibitor of γ-GCS, suppressed the protective effects of sulforaphane and 6-HITC. In contrast, sulforaphane and 6-HITC increased heme oxygenase-1 (HO-1) expression in neurons. However, zinc-protophorphyrin IX, a competitive inhibitor of HO-1, did not influence the protective effects of sulforaphane and 6-HITC. These results suggest that sulforaphane and 6-HITC prevent oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular glutathione content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant response element pathway.
Contributions of gluconeogenesis suppression in liver, kidney, and intestine as major gluconeogenic organs to the glucose-lowering effect of CS-917, a fructose 1,6-bisphosphatase inhibitor, was evaluated in overnight-fasted Goto-Kakizaki (GK) rats. CS-917 decreased plasma glucose by suppressing glucose release and lactate uptake from liver but not from kidney and intestine. These results suggest that hepatic gluconeogenesis suppression predominantly contributes to the glucose-lowering effect of CS-917 in GK rats. Moreover, the mechanism by which CS-917 decreased plasma glucose more in overnight-fasted GK rats than in non-fasted ones was investigated. Lactate uptake from liver was suppressed by 15 mg/kg of CS-917 in both states, but glucose release from liver and plasma glucose were decreased only in the overnight-fasted state. CS-917 at 30 mg/kg decreased hepatic glycogen content in both states and depleted it in the overnight-fasted state. In the non-fasted GK rats, co-administration of CS-917 with CP-91149, a glycogen phosphorylase inhibitor, suppressed hepatic glycogen reduction by CS-917 and decreased plasma glucose more than single administration of CS-917. These results suggest that gluconeogenesis suppression by CS-917 was counteracted by hepatic glycogenolysis especially in the non-fasted state and that combination therapy with CS-917 and CP-91149 is efficacious to decrease plasma glucose in GK rats.
We revealed that pre-treatment with docetaxel (DOC) 12 h before adriamycin (ADR) administration significantly reduced ADR-induced toxic death compared with the simultaneous dosing schedule that was commonly used in previous studies. We considered that pre-treatment with DOC relieves ADR-induced cardiotoxicity. In this study, we investigated the influence of DOC on the pharmacokinetics and pharmacodynamics of ADR in order to clarify the mechanism by which DOC pre-treatment relieves ADR-induced cardiotoxicity. When ADR and/or DOC was intravenously administered, the DOC pre-treatment (DOC-ADR) group showed significantly less toxic death than the ADR-alone group. We examined hepatopathy, nephropathy, leukopenia, and cardiotoxicity, all of which can cause toxic death. Of these toxicities, ADR-induced cardiotoxicity was significantly relieved in the DOC-ADR group. To elucidate the mechanism by which DOC pre-treatment relieved ADR-induced cardiotoxicity, lipid peroxidation as a proxy for the free radical level and the pharmacokinetics of ADR were measured. There was no difference in the pharmacokinetics of ADR between the ADR and DOC-ADR groups. On the other hand, the DOC-ADR group showed significantly inhibited lipid peroxidation in the heart compared with the ADR group. It was considered that DOC pre-administration inhibited ADR-induced free radicals and decreased cardiotoxicity.
Mineralocorticoid receptor (MR) antagonists, such as spironolactone (SPI) and eplerenone (EPL), are useful for the treatment of hypertension and heart failure. However, the use of these two agents has been limited due to endocrine disturbance (SPI) and poor drug action (EPL). In our search for safer and more effective MR antagonists, we identified SM-368229 as a novel non-steroidal MR antagonist. SM-368229 showed strong MR inhibitory activity with IC50 values of 0.021 and 0.13 μM in the binding assay and reporter-gene assay, respectively. The selectivity of SM-368229 for MR was 18-fold higher than that for other steroid receptors, such as androgen, progesterone, and glucocorticoid receptors. SM-368229 dose-dependently increased urinary Na+/K+ ratio with an ED50 value of 5.6 mg/kg in adrenalectomized rats treated with deoxycorticosterone acetate, and its efficacy was superior to that of SPI (ED50 = 14 mg/kg) or EPL (ED50 = 147 mg/kg). Moreover, even at high doses of 100 and 300 mg/kg, SM-368229 showed very weak anti-androgenic effect in methyltestosterone-treated male rats and no progestagenic effect in estrus cycle synchronized female rats. These findings indicate that SM-368229 may offer a new promising therapeutic option for the treatment of hypertension and heart failure.
Gene regulation during in vitro differentiation into adipocytes was examined in rat dental pulp–derived cells. Insulin, 3-isobutyl-1-methylxanthine, and dexamethasone were added to induce adipogenesis. Cells containing lipid droplets were observed after induction as in 3T3 L1 cells. Rat dental pulp–derived cells showed their potential to differentiate into adipocytes in vitro. In both types of cells, the pluripotent markers Oct-3/4 and Sox2 were downregulated during differentiation, whereas the expression of Nanog was not significantly changed during differentiation. Interestingly, in the dental pulp–derived cells, the level of Oct-3/4 was transiently induced at 1 week after induction and then significantly decreased during differentiation. Based on the expression profiles determined using GeneChip Arrays, 3418 probes across 10 clusters showed a difference in expression at 1, 2, and 3 weeks after induction versus before induction. Notably, genes in the PPAR signaling pathway including Pparγ, Fabp4, and the C/EBP family were upregulated by more than 3-fold. Upregulation of the PPAR pathways seems to be a critical signal transduction pathway in this differentiation system. These findings indicate that dental pulp–derived cells are a potential source of adipogenic cells, and their gene expression profile could be useful in future regenerative medicine applications.
In the disease states of urinary frequency and urgency, atropine-resistant contractions are known to be involved, in addition to contractions mediated by cholinergic nerves. This study was undertaken to investigate the mechanism underlying the development of atropine-resistant contractions using the representative antimuscarinic drugs solifenacin and tolterodine and also propiverine that has Ca2+ channel–antagonizing activity in addition to antimuscarinic activity. Rat models of urinary frequency were established by intravesical infusion of acetylcholine (ACh) (cholinergic nerve–mediated urinary frequency model), acetic acid (AcOH) [non-adrenergic non-cholinergic nerve (NANC)-mediated urinary frequency model], or CaCl2 (atropine-resistant contractions-mediated urinary frequency model). Cystometrograms were obtained to measure the micturition parameters following oral administration of the aforementioned drugs. Propiverine increased the micturition weight in all the urinary frequency models. Solifenacin and tolterodine increased the micturition weight in the ACh-induced urinary frequency model but neither had any effect in the AcOH- or CaCl2-induced urinary frequency models. While antimuscarinic drugs are, in general, effective for the control of urinary frequency and incontinence, use of drugs possessing inhibitory effects on contractions mediated by cholinergic as well as NANC nerve transmission or Ca2+ influx into smooth muscles is recommended for management of the symptoms in disease states in which atropine-resistant contractions, such as Ca2+- and capsaicin-sensitive sensory nerves, are involved.
The aim of the current study was to characterize comparatively the binding of muscarinic receptor in the lung of rats intratracheally administered anticholinergic agents (tiotropium, ipratropium, glycopyrrolate) used clinically to treat chronic obstructive pulmonary disease (COPD) and asthma. Binding parameters of [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) were determined in tissues (lung, bladder, submaxillary gland) of rats intratracheally administered tiotropium, ipratropium, and glycopyrrolate. The in vitro binding affinity of tiotropium for the receptors was 10 – 11-fold higher than those of ipratropium and glycopyrrolate. Intratracheal administration of tiotropium (0.6 – 6.4 nmol/kg) caused sustained (lasting at least 24 h) increase in the apparent dissociation constant (Kd) for [3H]NMS binding in rat lung compared with the control value. Concomitantly, there was a long-lasting decrease in the maximal number of binding sites (Bmax) for [3H]NMS. Similary, ipratropium and glycopyrrolate at 7.3 and 7.5 nmol/kg, respectively, brought about a significant increase in Kd for [3H]NMS binding. The effect by ipratropium was observed at 2 h but not 12 h, and that by glycopyrrolate lasted for 24 h. Both agents had little influence on the muscarinic receptors in the bladder and submaxillary gland. The present study provides the first evidence that tiotropium, ipratropium, and glycopyrrolate administered intratracheally in rats selectively bound muscarinic receptors of the lung, and tiotropium and glycopyrrolate had a much longer-lasting effect than ipratropium.
Uromodulin storage diseases are characterized by hyperuricemia of underexcretion type and renal insufficiency. Although these diseases are caused by mutations in the UMOD gene that encodes the kidney-specific glycoprotein uromodulin, the effect of uromodulin mutation on the kidney has not been clearly established. In this study, we investigated the effect by comparing transgenic mice expressing human uromodulin with and without mutation. Change in the intracellular localization of human uromodulin protein was shown in the kidney of transgenic mice expressing mutant human uromodulin by a deglycosylation experiment. Then, we determined by microarray technology and quantitative real-time PCR that the strongly induced gene in the kidney of these mice was 5-α-reductase 2, an enzyme that converts testosterone into the more potent androgen. Moreover, the expressions of androgen-induced genes β-glucuronidase, ornithine decarboxylase structural 1, and cytochrome P450 4a12a were increased. The increase in mRNA levels of urate reabsorptive transport system urate transporter 1 could be investigated, but the changes in its protein level and renal urate handling could not be demonstrated. Therefore, it is suggested that a uromodulin mutation may be responsible for the enhancement of renal androgen action. [Supplementary Table: available only at http://dx.doi.org/10.1254/jphs.10240FP]
We investigated the effects of dexamethasone on hepatocyte growth factor (HGF)-induced DNA synthesis and proliferation in serum-free primary cultures of adult rat hepatocytes. Isolated hepatocytes were cultured at a density of 3.3 × 104 cells/cm2 in Williams’ medium E containing 5% newborn bovine serum and various concentrations of dexamethasone for 1, 2, and 3 h. After a 3-h attachment period, the medium was then changed, and cells were cultured in serum-free dexamethasone (10−10 M)-containing Williams’ medium E with or without glucocorticoid receptor antagonists. After addition of dexamethasone to the culture medium, the growth-stimulating effects of HGF (5 ng/mL) on the primary cultured hepatocytes were time- and dose-dependently inhibited. The mineralcorticoid aldosterone (10−7 M) did not produce the same growth-inhibitory effects as dexamethasone (10−8 M). The inhibitory effects of dexamethasone were reversed by treatment with the glucocorticoid-receptor antagonist mifepristone (RU486, 10−6 M) or a monoclonal antibody against glucocorticoid receptor (100 ng/mL). In addition, the growth-inhibitory dose of dexamethasone did not affect HGF-induced receptor tyrosine kinase and extracellular signal-regulated kinase 2 phosphorylation. These results indicate that dexamethasone dose-dependently delays and inhibits HGF-induced DNA synthesis and proliferation through its own intracellular receptor in primary cultures of adult rat hepatocytes.
Cytochrome P450 (CYP)-mediated drug interactions caused by Kampo medicine have not been investigated sufficiently. The current study was conducted to reveal the effect of anchusan, a commonly used Kampo formula for gastrointestinal disease, on CYP3A-mediated drug metabolism in rats. The pharmacokinetics of midazolam (MDZ) was investigated after the single or one-week administration of anchusan (500 mg/kg) to evaluate its inhibitory and inducible effect on CYP3A, respectively. MDZ was administrated 16 h after the last anchsan treatment in the multiple dose study, while their intervals were 2 or 16 h in the single dose study. Unexpectedly, the multiple-pretreatment of anchusan increased the AUC of MDZ by 2.4-fold rather than decreasing it, and the CYP3A contents and activities were unchanged in hepatic and intestinal microsomes of these rats. In contrast, no significant inhibitory effects on MDZ metabolism were observed by the single anchusan pretreatment. In vitro study showed that the preincubation of anchusan and some of its component extracts with rat liver microsomes reduced CYP3A activity in a time- and NADPH-dependent manner. These results suggested that anchusan increased the serum MDZ concentration in rats, at least in part, by the time-dependent inhibition of CYP3A.
Sesamin is a major lignan in sesame seed. We confirmed that ingestion of sesamin and α-tocopherol synergistically reduced the concentration of blood cholesterol in rats given a high-cholesterol diet. To elucidate the molecular mechanism behind this effect, we analyzed the gene-expression profiles in rat liver after co-ingestion of sesamin and α-tocopherol. Six-week-old male Sprague-Dawley rats were fed a 1% cholesterol diet (HC) or HC containing 0.2% sesamin, 1% α-tocopherol or sesamin + α-tocopherol for 10 days. Blood samples were collected on days 1, 3, 7, and 10 and livers were excised on day 10. The gene expressions of ATP-binding cassette, sub-family G (WHITE), members 5 (ABCG5) and 8 (ABCG8) were significantly increased, while the gene expression of apolipoprotein (Apo) A4 was significantly decreased. ABCG5 and ABCG8 form a functional heterodimer that acts as a cholesterol efflux transporter, which contributes to the excretion of cholesterol from the liver. ApoA4 controls the secretion of ApoB, which is a component of low-density-lipoprotein cholesterol. These studies indicate that the cholesterol-lowering mechanism underlying the effects of co-ingestion of sesamin and α-tocopherol might be attributable to increased biliary excretion of cholesterol and reduced ApoB secretion into the bloodstream.
We investigated whether the descending noradrenergic system regulates allergic itch. Mosquito allergy of the hind paw elicited biting, an itch-related response, in sensitized mice. The biting was inhibited by intrathecal clonidine and reversed by yohimbine, an α2-adrenoceptor antagonist. The biting was increased by intrathecal pretreatment with the catecholaminergic neurotoxin 6-hydroxydopamine and the α-adrenoceptor antagonist phentolamine but not the serotonergic neurotoxin 5,7-dihydroxytryptamine. We propose that α2-adrenoceptors are involved in the inhibition of allergic itch in the spinal cord and that the descending noradrenergic system exerts a tonic inhibition on the itch signaling. The serotonergic system may not be involved.
Tramadol has been widely used as analgesic. O-Desmethyl tramadol (ODT) is one of the main metabolites of tramadol, having much greater analgesic potency than tramadol itself. Substance P receptors (SPR) are well known to modulate nociceptive transmission within the spinal cord. In this study, we investigated the effects of ODT on SPR expressed in Xenopus oocytes by examining SP-induced Ca2+-activated Cl− currents. ODT inhibited the SPR-induced Cl− currents at pharmacologically relevant concentrations. The protein kinase C (PKC) inhibitor bisindolylmaleimide I did not abolish the inhibitory effects of ODT on SP-induced Ca2+-activated Cl− currents. The results suggest that the tramadol metabolite ODT inhibits the SPR functions, which may be independent of activation of PKC-mediated pathways.