Inhibition of the activation of hepatic stellate cells by arundic acid via the induction of cytoglobin.

BACKGROUND: The activation of hepatic stellate cells plays a central role in the development of liver fibrosis during chronic liver trauma. The aim of the present study was to identify a compound that inhibits the activation process of stellate cells. METHODS: Rat primary cultured stellate cells and a human stellate cell line (LX-2) were used. The effects of arundic acid on the expression of α-smooth muscle actin, collagen 1α1, and cytoglobin were evaluated. RESULTS: Arundic acid (300 µM) inhibited the activation of primary rat stellate cells, as determined by morphological transformation and α-smooth muscle actin expression, after both prophylactic and therapeutic treatment. The level of α-smooth muscle actin mRNA showed a dose-dependent decrease in response to arundic acid, and 50 µM arundic acid exhibited the maximum inhibition of collagen 1α1 mRNA expression. In contrast, arundic acid triggered an unexpected increase in mRNA and protein levels of cytoglobin, the fourth globin in mammals expressed exclusively in hepatic stellate cells. The effect of arundic acid on the level of α-smooth muscle actin mRNA was abrogated in HSCs treated with cytoglobin siRNA. Arundic acid decreased the expression of collagen 1α1 mRNA in LX-2 cells. CONCLUSION: Arundic acid affects the activation process of hepatic stellate cells via the unexpected induction of cytoglobin.

Neuroprotective effect of arundic acid, an astrocyte-modulating agent, in mouse brain against MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes the damage of dopaminergic neurons as seen in Parkinson's disease. Oxidative stress has been as one of several pathogenic hypotheses for Parkinson's disease. Here we investigated whether arundic acid, an astrocyte-modulating agent, can protect against alterations of nitric oxide synthase (NOS) and superoxide dismutase (SOD) expression on MPTP neurotoxicity in mice, utilizing an immunohistochemistry. For this purpose, anti-tyrosine hydroxylase (TH) antibody, anti-dopamine transporter (DAT) antibody, anti-Cu/Zn-SOD antibody, anti-Mn-SOD antibody, anti-nNOS antibody, anti-eNOS antibody and anti-iNOS antibody were used. The present study showed that the arundic acid had a protective effect against MPTP-induced neuronal damage in the striatum and substantia nigra of mice. The protective effect may be, at least in part, caused by the reductions of the levels of reactive nitrogen (RNS) and oxygen species (ROS) against MPTP neurotoxicity. These results suggest that the pharmacological modulation of astrocyte may offer a novel therapeutic strategy for the treatment of Parkinson's disease. Furthermore, our results provide further evidence that a combination of nNOS inhibitors, iNOS inhibitors and free radical scavengers may be effective in the treatment of neurodegenerative diseases. Thus our present results provide valuable information for the pathogenesis of degeneration of the nigrostriatal dopaminergic neuronal pathway.

Effects of OP-1206 alpha-CD on walking dysfunction in the rat neuropathic intermittent claudication model: comparison with nifedipine, ticlopidine and cilostazol.

The systemic treatment effects of OP-1206 alpha-CD (17S-20-dimethyl-trans-delta 2-PGE1 alpha-cyclodextrin clathrate), a prostaglandin E1 (PGE1) analogue, on walking dysfunction, spinal cord blood flow (SCBF) and skin blood flow (SKBF) were assessed in the rat neuropathic intermittent claudication (IC) model in comparison with nifedipine (dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate), ticlopidine (5-[(2-chlorophenyl)methyl]-4,5,6,7-tetrahydrothieno[3,2-C]pyridine hydrochloride) and cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)-butoxy]-3,4-dihydro-2(1H)-quinolinone). Two pieces of silicone rubber strips were placed in the lumbar (L4 and L6) epidural space in rats. After surgery, walking function was measured using a treadmill apparatus. SCBF and SKBF were measured using a laser-Doppler flow meter. Drugs were administered orally twice a day for 11 days from day 3 post-surgery. Treatment with OP-1206 alpha-CD significantly improved walking dysfunction on days 5, 7 and 14, and improved SCBF on day 14 post-surgery. SKBF remained unaffected. Treatment with nifedipine, ticlopidine or cilostazol had no significant effects on any of the parameters measured in this model. These data suggest that the therapeutic effect of OP-1206 alpha-CD is primarily mediated by the improved local SCBF at the territory of spinal stenosis and not due to improvement of peripheral perfusion and/or antiplatelet activity.

Effects of orally administered OP-1206 alpha-CD with loxoprofen-Na on walking dysfunction in the rat neuropathic intermittent claudication model.

An orally active prostaglandin E1 analogue, OP-1206 alpha-CD improves walking dysfunction in the rat spinal stenosis model. Loxoprofen-Na, a non-steroidal anti-inflammatory drug, is used to relieve chronic pain in patients with lumbar spinal canal stenosis. To determine whether the OP-1206 alpha-CD in combination with loxoprofen-Na could induce a greater therapeutical effect on walking dysfunction and spinal cord blood flow (SCBF) than OP-1206 alpha-CD treatment alone after chronic spinal stenosis in the rat. Spinal stenosis was induced by placing two pieces of silicon rubber strips in the lumbar (L4 and L6) epidural space of rats. After surgery, walking function was measured using a treadmill apparatus and SCBF was measured using a laser-Doppler flow meter. Drugs were administered orally twice a day for 11 days from the day 3 post-surgery. OP-1206 alpha-CD elicited a significant improvement of walking dysfunction on days 7 and 14 post-surgery and significantly increased spinal cord blood flow on day 15, whereas walking dysfunction and SCBF of rats treated with loxoprofen-Na alone remained unchanged. Combined treatment of OP-1206 alpha-CD with loxoprofen-Na did not provide additive therapeutical effect. These results suggest that a significant improvement seen after OP-1206 alpha-CD treatment is primarily mediated by improvement of the local spinal cord blood flow. This effect is not ameliorated or potentiated by a combined treatment with loxoprofen-Na.

Structure-activity study of L-amino acid-based N-type calcium channel blockers.

Synthesis and structure-activity relationship (SAR) study of L-amino acid-based N-type calcium channel blockers are described. The compounds synthesized were evaluated for inhibitory activity against both N-type and L-type calcium channels focusing on selectivity to reduce cardiovascular side effects due to blocking of L-type calcium channels. In the course of screening of our compound library, N-(t-butoxycarbonyl)-L-aspartic acid derivative 1a was identified as an initial lead compound for a new series of N-type calcium channel blockers, which inhibited calcium influx into IMR-32 human neuroblastoma cells with an IC(50) of 3.4 microM. Compound 1a also exhibited blockade of N-type calcium channel current in electrophysiological experiment using IMR-32 cells (34% inhibition at 10 microM, n=3). As a consequence of conversion of amino acid residue of 1a, compound 12a, that include N-(t-butoxycarbonyl)-L-cysteine, was found to be a potent N-type calcium channel blocker with an IC(50) of 0.61 microM. Thus, L-cysteine was selected as a potential structural motif for further modification. Optimization of C- and N-terminals of L-cysteine using S-cyclohexylmethyl-L-cysteine as a central scaffold led to potent and selective N-type calcium channel blocker 21f, which showed improved inhibitory potency (IC(50) 0.12 microM) and 12-fold selectivity for N-type calcium channels over L-type channels.

L-Cysteine based N-type calcium channel blockers: structure-activity relationships of the C-terminal lipophilic moiety, and oral analgesic efficacy in rat pain models.

This study was performed to determine the structure-activity relationships (SAR) of L-cysteine based N-type calcium channel blockers. Basic nitrogen was introduced into the C-terminal lipophilic moiety of L-cysteine with a view toward improvement of its physicochemical properties. L-Cysteine derivative 9 was found to be a potent and selective N-type calcium channel blocker with IC(50) of 0.33 microM in calcium influx assay using IMR-32 cells and was 15-fold selective for N-type calcium channels over L-type channels. Compound 9 showed improved oral analgesic efficacy in the rat formalin induced pain model and the rat chronic constriction injury (CCI) model, which is one of the most reliable models of chronic neuropathic pain, without any significant effect on blood pressure or neurological behavior.

The effects of OP-1206 alpha-CD on walking dysfunction in the rat neuropathic intermittent claudication model.

UNLABELLED: IV prostaglandin E1 improves clinical symptoms in patients with spinal canal stenosis. In the present study, we assessed the effects of OP-1206 alpha-CD, an orally active prostaglandin E1 analog, on walking dysfunction in the rat neuropathic intermittent claudication model. To induce spinal stenosis, two pieces of silicon rubber were placed in the lumbar (L4-6) epidural space in rats. Postsurgical walking function was measured using a treadmill apparatus. Spinal cord blood flow (SCBF) and skin blood flow (SKBF) were measured using a laser-Doppler flowmeter. OP-1206 alpha-CD was administered orally bid for 11 days from postoperative Day 3. In Control nontreated rats, a significant walking dysfunction was observed from Day 1 after the induction of spinal stenosis and persisted for 14 days when compared with the Sham-Operated group. On postoperative Day 15, SCBF revealed a significant reduction in the territory of spinal stenosis, although SKBF was not affected. OP-1206 alpha-CD significantly improved walking dysfunction on postoperative Days 5 (300 microg/kg), 7 (150 and 300 microg/kg), and 14 (150 and 300 microg/kg) when compared with the Vehicle-Treated group. On postoperative Day 15, the decrease in SCBF was significantly (150 and 300 microg/kg) improved by OP-1206 alpha-CD treatment, albeit SKBF remained unaffected. These data show that oral treatment with OP-1206 alpha-CD is effective in improving walking dysfunction induced by spinal canal stenosis, and this therapeutic effect is likely mediated by improved SCBF at the territory of spinal stenosis. IMPLICATIONS: Intermittent motor dysfunction is a clinical symptom associated with partial spinal compression. The present study provides evidence that oral treatment with the prostaglandin E1 analog (OP-1206 alpha-CD) is effective in improving motor dysfunction and spinal cord blood flow in rats with spinal compression.

Structure-activity study of L-cysteine-based N-type calcium channel blockers: optimization of N- and C-terminal substituents.

Synthesis and structure-activity relationship (SAR) studies of L-cysteine-based N-type calcium channel blockers are described. In the course of exploring SAR of the N- and C-terminal substituents, the L-cysteine derivative was found to be a potent N-type calcium channel blocker with an IC(50) value of 0.14 microM on IMR-32 assay. Compound showed 12-fold selectivity for N-type over L-type calcium channels on AtT-20 assay.

Involvement of cathepsin E in exogenous antigen processing in primary cultured murine microglia.

We have attempted to elucidate an involvement of cathepsin E (CE) in major histocompatibility complex class II-mediated antigen presentation by microglia. In primary cultured murine microglia, CE was localized mainly in early endosomes and its expression level was markedly increased upon stimulation with interferon-gamma. Pepstatin A, a specific inhibitor of aspartic proteases, significantly inhibited interleukin-2 production from an OVA-(266-281)-specific T helper cell hybridomas upon stimulation with native OVA presented by interferon-gamma-treated microglia. However, pepstatin A failed to inhibit the presentation of OVA-(266-281) peptide. The possible involvement of CE in the processing of native OVA into antigenic peptide was further substantiated by that digested fragments of native OVA by CE could be recognized by OVA-specific Th cells. Cathepsin D also degraded native OVA into antigenic peptide, whereas microglia prepared from cathepsin D-deficient mice retained an ability for antigen presentation. On the other hand, the requirement for cysteine proteases such as cathepsins S and B in the processing of invariant chain (Ii) was confirmed by immunoblot analyses in the presence of their specific inhibitors. In conclusion, CE is required for the generation of an antigenic epitope from OVA but not for the processing of Ii in microglia.