ABSTRACT
Helicobacter pylori (Hp) infection is related to the pathogenesis of chronic gastric disorders and extragastric diseases. Here, we examined the anorexigenic and anxiogenic effects of Hp vacuolating cytotoxin A (VacA) through activation of hypothalamic urocortin1 (Ucn1). VacA was detected in the hypothalamus after peripheral administration and increased Ucn1 mRNA expression and c-Fos-positive cells in the hypothalamus but not in the nucleus tractus solitarius. c-Fos and Ucn1-double positive cells were detected. CRF1 and CRF2 receptor antagonists suppressed VacA-induced anxiety and anorexia, respectively. VacA activated single paraventricular nucleus neurons and A7r5 cells; this activation was inhibited by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. VacA causes anorexia and anxiety through the intracellular PLC-PKC pathway, migrates across the blood-brain barrier, and activates the Ucn1-CRF receptor axis.
Subject(s)
Anorexia/chemically induced , Anxiety/chemically induced , Cytotoxins/toxicity , Helicobacter pylori/cytology , Hypothalamus/drug effects , Urocortins/metabolism , Vacuoles/metabolism , Animals , Anorexia/genetics , Anxiety/genetics , Body Weight/drug effects , Eating/drug effects , Gerbillinae , Helicobacter pylori/physiology , Hypothalamus/cytology , Hypothalamus/metabolism , Mice , RNA, Messenger/genetics , Signal Transduction/drug effects , Urocortins/geneticsABSTRACT
BACKGROUND: Helicobacter pylori induces gastric damage and may be involved in the pathogenesis of gastric cancer. H. pylori-vacuolating cytotoxin, VacA, is one of the important virulence factors, and is responsible for H. pylori-induced gastritis and ulceration. The aim of this study is to assess whether several naturally occurring polyphenols inhibit VacA activities in vitro and in vivo. MATERIALS AND METHODS: Effects of polyphenols on VacA were quantified by the inhibition of: 1, vacuolation; 2, VacA binding to AZ-521 or G401 cells or its receptors; 3, VacA internalization. Effects of hop bract extract (HBT) containing high molecular weight polymerized catechin on VacA in vivo were investigated by quantifying gastric damage after oral administration of toxins to mice. RESULTS: HBT had the strongest inhibitory activity among the polyphenols investigated. HBT inhibited, in a concentration-dependent manner: 1, VacA binding to its receptors, RPTP(alpha) and RPTP(beta); 2, VacA uptake; 3, VacA-induced vacuolation in susceptible cells. In addition, oral administration of HBT with VacA to mice reduced VacA-induced gastric damage at 48 hours. In vitro, VacA formed a complex with HBT. CONCLUSIONS: HBT may suppress the development of inflammation and ulceration caused by H. pylori VacA, suggesting that HBT may be useful as a new type of therapeutic agent for the prevention of gastric ulcer and inflammation caused by VacA.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Flavonoids/metabolism , Flavonoids/pharmacology , Gastric Mucosa/drug effects , Gastric Mucosa/pathology , Helicobacter Infections/drug therapy , Helicobacter pylori/pathogenicity , Phenols/metabolism , Phenols/pharmacology , Administration, Oral , Animals , Bacterial Proteins/administration & dosage , Bacterial Proteins/metabolism , Catechin/metabolism , Catechin/pharmacology , Cell Line, Tumor , Flavonoids/therapeutic use , Gastric Mucosa/microbiology , Gastritis/drug therapy , Gastritis/microbiology , Gastritis/pathology , Helicobacter Infections/microbiology , Helicobacter Infections/pathology , Humans , Humulus/chemistry , Immunohistochemistry , Mice , Mice, Inbred C57BL , Phenols/therapeutic use , Plant Extracts/chemistry , Plant Extracts/metabolism , Plant Extracts/pharmacology , PolyphenolsABSTRACT
Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused by VacA. To define the region of RPTPbeta involved in VacA binding, we made mutants of human cDNA RPTPbeta-B, a short receptor form of RPTPbeta. Immunoprecipitation experiments to assess VacA binding to RPTPbeta-B mutants indicated that five residues (QTTQP) at positions 747-751 of the extracellular domain of RPTPbeta-B (which is commonly retained in RPTPbeta-A, a long form of RPTPbeta) play a crucial role in its interaction with VacA, resulting in vacuolation as well as Git-1 phosphorylation. Transfected cells expressing deletion mutant Delta752, which lacks QTTQP, or the double point mutant Delta747 (T748A,T749A) had diminished vacuolation in response to VacA. Treatment of RPTPbeta-B and Delta747 (which have QTTQP at 747-751) with neuraminidase and O-glycosidase diminished their VacA binding, whereas chondroitinase ABC did not have an effect. No inhibitory effect of pleiotrophin, a natural RPTPbeta ligand, on VacA binding to RPTPbeta-B or Delta747 was observed, supporting the conclusion that the extracellular region of RPTPbeta-B responsible for VacA binding is different from that involved in binding pleiotrophin. These data define the region in the RPTPbeta extracellular domain critical for VacA binding, in particular the sequence QTTQP at positions 747-751 with crucial threonines at positions 748 and 749 and are consistent with a role for terminal sialic acids possibly because of threonine glycosylation.