Yuji Omiya

Epithelial transient receptor potential ankyrin 1 (TRPA1)-dependent adrenomedullin upregulates blood flow in rat small intestine

The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells (Kono T et al. J Crohns Colitis 4: 161-170, 2010). TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.

Daikenchuto (TU-100) ameliorates colon microvascular dysfunction via endogenous adrenomedullin in Crohn’s disease rat model

BackgroundDaikenchuto (TU-100), a traditional Japanese medicine, has been reported to up-regulate the adrenomedullin (ADM)/calcitonin gene-related peptide (CGRP) system, which is involved in intestinal vasodilatation. The microvascular dysfunction of the intestine in Crohn’s disease (CD), due to down-regulation of the ADM/CGRP system, is etiologically related to the recurrence of CD. Therefore, we investigated the vasodilatory effect of TU-100 in a CD rat model.MethodsColitis was induced by the rectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in rats. Laser Doppler blood flowmetry was used to measure colonic blood flow. ADM, CGRP, and their receptors in the ischemic colon were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme immunoassays. Additionally, we determined whether the intestinal epithelial cell line IEC-6 released ADM in response to TU-100.ResultsTU-100 increased blood flow in ischemic segments of the colon but not in hyperemic segments. Pretreatment with an antibody to ADM abolished the vasodilatory effect of TU-100. CGRP levels and βCGRP mRNA expression were decreased in the ischemic colon, while protein and mRNA levels of ADM were unchanged. Hydroxy α-sanshool, the main constituent of TU-100, was the most active component in improving blood flow. Additionally, both TU-100 and hydroxy α-sanshool enhanced the release of ADM from IEC-6 cells.ConclusionsIn the ischemic colon, endogenous βCGRP, but not ADM, was decreased. Thus, it was concluded that TU-100 ameliorated microvascular dysfunction by the up-regulation of endogenous ADM in the CD rat model. TU-100 may be a possible therapeutic agent for gastrointestinal ischemia-related diseases including CD.

The analgesic mechanism of processed aconiti tuber: the involvement of descending inhibitory system

Tsumura-shuchi-bushi-matsu (TJ-3021) is a processed Aconiti tuber which has a potent antinociceptive action. The present study was undertaken to study the analgesic mechanism produced by TJ-3021. RCS (repeated cold stress) rats in hyperalgesia were markedly suppressed by oral administration of TJ-3021. Intrathecal and intraperitoneal administration of a selective alpha 2-adrenoreceptor antagonist, idazoxan (IDA), reduced significantly the analgesic effect of TJ-3021 in RCS rats. Methysergide (METH), a 5-HT receptor antagonist, demonstrated a similar effect, while intraperitoneal administration of opioid receptor antagonist, naloxone, did not produce the effect. Both oral and intracisternal administration of mesaconitine (MA) which is one of the main potent alkaloids contained in TJ-3021 produced analgesic effect in non-RCS rats.

Role of α2-adrenoceptors in enhancement of antinociceptive effect in diabetic mice

The present studies investigated behavioral and neurochemical aspects of the noradrenergic and serotonergic nervous systems in streptozotocin-induced diabetic mice. We previously reported that intrathecal (i.t.) injection of norepinephrine significantly potentiated antinociception in diabetic mice compared to that in non-diabetic mice, and that antinociception due to norepinephrine injection was completely abolished by pretreatment with yohimbine, an alpha2-adrenoceptor antagonist. The present studies demonstrated that i.t. injection of clonidine also showed more-potent antinociceptive activity in diabetic mice than in non-diabetic mice, but that i.t. methoxamine injection did not affect diabetic or non-diabetic mice. The antinociceptive potency due to i.t. injection of 5-HT was significantly lower in diabetic than in non-diabetic mice. In a neurochemical study, we found that the density of [3H]-rauwolscine binding sites in spinal alpha2-adrenoceptors was significantly higher in diabetic than in non-diabetic mice, but that the binding affinity was unchanged. Spinal norepinephrine turnover was determined by measuring the decline in tissue norepinephrine concentration at 3 h after injection of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine. The spinal norepinephrine concentration decreased to 43.7% from the baseline in non-diabetic mice, while it was 21.0% in diabetic mice. These results suggest that, based on the decrease of norepinephrine release in the spinal cord, up-regulation of spinal alpha2-adrenoceptors caused the increase of antinociception due to i.t. injection of an alpha2-adrenoceptor agonist in streptozotocin-induced diabetic mice, and it seemed that the stimulation of alpha2-adrenoceptors potentiated the antinociceptive effect. Thus, the spinal noradrenergic systems play an important moderating role in diabetes-induced neuropathic pain.

Changes in analgesia-producing mechanism of repeated cold stress loading in mice

Functional changes in opioid receptors involved in analgesia of repeated cold stress (RCS)-loaded mice were investigated. The antinociceptive potency of morphine (4 mg/kg, PO) was not affected in normal mice by norbinaltorphimine (10 mg/kg, SC), but treatment with this agent resulted in a lower level of morphine-induced antinociception in RCS-loaded animals. The antinociceptive activity of U-50488H (3 mg/kg, SC) was increased in RCS-loaded mice. In contrast to hypersensitivity to U-50488H (1 and 10 microg, IT) noted in RCS-loaded mice, the antinociception induced by DAMGO (0.1 and 1 microg, ICV) was reduced compared to that of normal animals. Diazepam (1 mg/kg/day SC) was given during RCS loading, and this agent prevented the development of hyperalgesia and the decrease in the antinociceptive activity of DAMGO (1 microg, ICV) in RCS-loaded mice, but there was no effect on the enhancement of the antinociceptive potency of U-50488H (10 microg, IT). These results indicate that the RCS-loaded mice were hyposensitive to supraspinal mu-opioid receptor-mediated antinociception, whereas their antinociceptive activities through kappa-opioid receptor in the spinal cord were increased. Hypofunction of the supraspinal mu-opioid receptor due to anxiety may explain the mechanism involved in the lowering of the nociceptive threshold in RCS-loaded animals.

Antinociceptive effect of U-50488H, a κ-opioid agonist, in streptozotocin-induced diabetic mice

We compared the antinociceptive activity of a κ‐opioid agonist, U‐50488H, in streptozotocin‐induced diabetic mice with that in non‐diabetic mice. Subcutaneously administered U‐50488H (3 and 10 mg kg−1) showed a more potent antinociceptive effect, as evaluated by the tail‐pressure method, in diabetic mice than in non‐diabetic mice. Increased antinociceptive activity of U‐50488H observed in diabetic mice was also observed in mice given U‐50488H intrathecally (3 and 10 μg). However, there were no differences observed between diabetic and non‐diabetic mice given U‐50488H intracerebroventricularly (3 and 10 μg). Although the antinociceptive effect of U‐50488H (3 mg kg−1, s.c.) in non‐diabetic mice was increased by treatment with PD135158 (100 ng, i.c.v.), a cholecystokininB (CCKB) antagonist, the antinociceptive activity of U‐50488H which was enhanced in diabetic mice was not influenced by PD135158. Moreover, the increased antinociceptive activity of U‐50488H (3 mg kg−1, s.c.) in diabetic mice diminished when desulfated octapeptide of cholecystokinin (3–100 ng, i.c.v.), a CCKB agonist, was administered. These results suggested that diabetic mice were selectively hyper‐responsive to spinal κ‐opioid receptor‐mediated antinociception. The function of the analgesia inhibitory system in which cholecystokinin is used as a transmitter might be diminished in diabetic mice.

Preventive effect of oral goshajinkigan on chronic oxaliplatin-induced hypoesthesia in rats.

Oxaliplatin, a widely used chemotherapeutic agent, induces peripheral neuropathy that manifests itself as two distinct phases: acute cold hyperesthesia and chronic peripheral hypoesthesia/dysesthesia. The latter is a serious dose-limiting side effect that can often lead to withdrawal of treatment. We have developed a rat model expressing both phases and used the model to investigate the action of goshajinkigan (GJG), a traditional Japanese herbal medicine, which was reported to ameliorate oxaliplatin-induced neuropathy in a placebo-controlled double-blind randomized phase II study. In this study, neuropathy was induced by injection of oxaliplatin twice weekly for 8 wks. The maximum level of cold hyperesthesia was observed at 4 wks with heat hypoesthesia developing later. Microscopy studies revealed atrophy of axons of myelinated sciatic nerve fibers in oxaliplatin-treated rats at 8 wks. Co-administration of GJG ameliorated both abnormal sensations as well as histological damage to the sciatic nerve. A pharmacokinetic study revealed numerous neuroprotective components of GJG that are rapidly absorbed into the blood. GJG and some of its components attenuated the generation of oxaliplatin-induced reactive oxygen species, which is a possible mechanism of oxaliplatin-induced neurotoxicity. The present study provides a useful animal model for oxaliplatin-induced neurotoxicity as well as a promising prophylactic agent.

Analysis of the antioxidative function of the radioprotective Japanese traditional (Kampo) medicine, hangeshashinto, in an aqueous phase

Oral mucositis (OM) is a common and painful complication of radiotherapy for head and neck cancer. Hangeshashinto (HST), a Japanese traditional medicine, is known to alleviate radiotherapy- and/or chemotherapy-induced OM; however, the detailed mechanism has not yet been clarified. The aim of the present study was to clarify the details of the antioxidative functions of HST against reactive oxygen species (ROS) produced by radiation. The hydroxyl radical (•OH)–scavenging ability and the reduction ability was simultaneously measured using a modified electron paramagnetic resonance (EPR) spin-trapping method. The superoxide (O2•−)–scavenging ability was estimated by an EPR redox probing method. Water suspensions of powdered HST and of its seven constitutive crude drugs were tested. In addition, some of the main water-soluble ingredients of the crude drugs were also tested. HST was found to scavenge both •OH and O2•−. Furthermore, HST was observed to reduce relatively stable nitroxyl radicals. Glycyrrhizae Radix (kanzo), Ginseng Radix (ninjin), Zizyphi Fructus (taiso) and glycyrrhizin (an ingredient of kanzo) were all found to be relatively good •OH scavengers. Scutellariae Radix (ogon) and Coptidis Rhizoma (oren) demonstrated reducing ability. In addition, acteoside and berberine chloride, which are water-soluble ingredients of ogon and oren, respectively, also demonstrated reducing ability. Oren exhibited oxidative ability at higher concentrations, which may have a function in maintaining catalytic redox action. The antioxidative function of HST probably worked via a balance of scavenging ROS, reducing stable free radicals, and some minor oxidizing activities.

Ignavine: a novel allosteric modulator of the μ opioid receptor

Processed Aconiti tuber (PAT) is used to treat pain associated with various disorders. Although it has been demonstrated that the κ opioid receptor (KOR) signaling pathway is a mediator of the analgesic effect of PAT, active components affecting opioid signaling have not yet been identified. In this study, we explored candidate components of PAT by pharmacokinetic analysis and identified ignavine, which is a different structure from aconitine alkaloids. A receptor binding assay of opioid receptors showed that ignavine specifically binds the μ opioid receptor (MOR), not the KOR. Receptor internalization assay in MOR-expressing cell lines revealed that ignavine augmented the responses produced by D-Ala(2)-N-Me-Phe(4)-Gly-ol(5)-enkephalin (DAMGO), a representative MOR agonist, at a low concentration and inhibited it at a higher concentration. Ignavine also exerted positive modulatory activity for DAMGO, endomorphin-1 and morphine in cAMP assay. Additionally, ignavine alone showed an analgesic effect in vivo. In silico simulation analysis suggested that ignavine would induce a unique structural change distinguished from those induced by a representative MOR agonist and antagonist. These data collectively suggest the possibility that ignavine could be a novel allosteric modulator of the MOR. The present results may open the way for the development of a novel pain management strategy.

Effects of Hangeshashinto on Growth of Oral Microorganisms.

Oral mucositis (OM) in cancer patients induced by chemotherapy or radiotherapy has a significant impact on quality of life, and causes considerable morbidity. Oral microorganisms are likely to intensify the inflammatory process and aggravate the formation of ulcers. Hangeshashinto (HST), a Japanese kampo medicine, has been reported to be effective when used as a gargle for the treatment of OM. To clarify the effects of HST on oral microorganisms, we assessed its antimicrobial activity against 27 microbial species, including 19 oral bacteria and one fungus. HST extract inhibited the growth of Gram-negative bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella melaninogenica, Tannerella forsythia, Treponema denticola, and Porphyromonas asaccharolytica, though inhibitory effects were less pronounced for Gram-positive bacteria and the fungal strain. We then investigated the effects of antibacterial activities on 15 purified ingredients of HST and determined that baicalein, berberine, coptisine, [6]-shogaol, and homogentisic acid actively inhibited the growth of these bacteria. These findings showed that HST inhibits the growth of specific Gram-negative periodontopathogenic bacteria, which are significant pathogens in OM, without disturbing the normal oral flora. Our data suggest that HST may be a useful treatment for OM in patients undergoing anticancer treatment.