ABSTRACT
Neonatal hyperbilirubinemia is a common clinical condition caused mainly by the increased production and decreased excretion of bilirubin. Current treatment is aimed at reducing the serum levels of bilirubin. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme that generates bilirubin. In this study we intended to suppress HO-1 using the RNA interference technique. Small interfering RNA (siRNA)-A, -B, and -C were designed based on human HO-1 (hHO-1) mRNA sequences. siRNA was transfected into a human hepatic cell line (HL-7702). hHO-1 transcription and protein levels were then determined. In addition, the inhibitory effect of siRNA on hHO-1 was assessed in cells treated with hemin or transfected with an hHO-1 plasmid. siRNA-C showed the most potent suppressive effect on hHO-1. This inhibition is dose and time dependent. Compared with control, both hemin and hHO-1 plasmids up-regulated hHO-1 expression in HL-7702 cells. However, the up-regulation was significantly attenuated by siRNA-C. Furthermore, the decrease in hHO-1 activity was coincident with the suppression of its transcription. Finally, siRNA-C was shown to reduce hHO-1 enzymatic activity and bilirubin levels. Thus, this study provides a novel therapeutic rationale by blocking bilirubin formation via siRNA for preventing and treating neonatal hyperbilirubinemia and bilirubin encephalopathy at an early clinical stage.
Subject(s)
Bilirubin/metabolism , Gene Expression Regulation, Enzymologic , Heme Oxygenase-1/metabolism , Hyperbilirubinemia, Neonatal/enzymology , Kernicterus/enzymology , RNA, Small Interfering , Cell Line , Heme Oxygenase-1/genetics , Hemin/pharmacology , Humans , Hyperbilirubinemia, Neonatal/drug therapy , Hyperbilirubinemia, Neonatal/genetics , Kernicterus/drug therapy , Kernicterus/genetics , Liver/enzymology , Oxidation-Reduction , RNA, Small Interfering/genetics , RNA, Small Interfering/therapeutic use , Up-RegulationABSTRACT
Unconjugated bilirubin (UCB) induces both apoptosis and necrosis in neurons. To investigate the role of caspases and excitotoxicity in UCB-induced cell death, we exposed NT2-N neurons to 5 microM UCB (a concentration known to induce apoptosis) or 2 microM staurosporine (positive apoptosis control) and investigated the effects of treatments with the specific caspase-3 inhibitor, zDEVD.FMK (20 and 100 microM), or the general caspase inhibitor, zVAD.FMK (20 and 100 microM), and/or the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (10 microM) during a 24- or 48-h exposure. UCB increased caspase-3 activity 2.3-fold after 6 h. Despite this, treatment with zDEVD.FMK did not prevent cell death. zVAD.FMK enhanced neuronal survival by reducing apoptotic nuclear fragmentation, while MK-801 enhanced survival by reducing apoptotic nuclear condensation; both without affecting the MTT assays. Combined treatment reduced both apoptotic morphologies (without affecting necrosis), and this effect was also reflected in the MTT assays [corrected] We conclude that NMDA receptor-mediated pathways and caspase-mediated pathways are involved in UCB-induced cell death in human NT2-N neurons. Concomitant inhibition of both pathways results in synergistic protection.
Subject(s)
Apoptosis/drug effects , Bilirubin/toxicity , Caspase Inhibitors , Dizocilpine Maleate/pharmacology , Neurons/drug effects , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Amino Acid Chloromethyl Ketones/pharmacology , Caspase 3 , Cysteine Proteinase Inhibitors/pharmacology , DNA Fragmentation/drug effects , Drug Synergism , Enzyme Activation/drug effects , Humans , Kernicterus/enzymology , Kernicterus/metabolism , Neurons/physiology , Tumor Cells, CulturedABSTRACT
We studied the relationship between changes in auditory brainstem responses (ABR) and serum and cerebrospinal fluid levels of neuron-specific enolase (NSE) in hyperbilirubinemic 2- to 8-d-old piglets. Infusion of a stabilized solution of bilirubin resulted in serum bilirubin levels of 571.1 +/- 48.8 mumol/L (mean +/- SEM) after 6 h. ABR were obtained at baseline and then hourly until the piglets were killed. We measured peak amplitudes and latencies for waves I-V, as well as latency for the post-V trough. Changes in amplitudes and latencies were analyzed as slopes because of heterogeneous variances. Over time, a significant reduction was observed in peak II-V amplitudes of bilirubin-infused piglets, but not in those of corresponding controls. No change was observed in latencies. NSE was analyzed by RIA. Serum NSE remained stable throughout the experiment (means 5.1-6.6 micrograms/L) and did not differ between the groups. Cerebrospinal fluid NSE values also remained stable, and no differences that could be ascribed to hyperbilirubinemia were detected. We conclude that hyperbilirubinemia induced significant changes in piglet ABR amplitudes without concomitant evidence of severe neuronal compromise, as might have been indicated by significant increases in serum and/or cerebrospinal fluid NSE levels. This provides further support to the clinical impression that early ABR changes during hyperbilirubinemia may be reversible.
Subject(s)
Evoked Potentials, Auditory, Brain Stem/physiology , Jaundice, Neonatal/physiopathology , Animals , Animals, Newborn , Cell Membrane Permeability , Humans , Infant, Newborn , Jaundice, Neonatal/enzymology , Kernicterus/enzymology , Kernicterus/physiopathology , Phosphopyruvate Hydratase/blood , Phosphopyruvate Hydratase/cerebrospinal fluid , SwineABSTRACT
The activities of choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD), markers of cholinergic and GABAergic terminals, were measured in growing and adult Gunn rats, which possess an autosomal recessive gene for jaundice or kernicterus. In the olfactory tubercle, hippocampus, neostriatum, thalamus, and hypothalamus of the animals with kernicterus, the development of the cholinergic pathways was delayed, but by the adult stage it was normal, while there was practically no action on the innervation by GABAergic neurons, at least as indicated by the chemically measured parameters.
