158 Morphine Enhances Hepatitis C Virus Replicon Expression Y. Li*, T. Zhang, S. D. Douglas, W. Z. Ho Children's Hosp of Philadelphia, Univ of Pennsylvania Sch of Med, PA
Background: Although injection drug users (IDUs) are the single largest risk factor for Hepatitis C Virus (HCV) transmission, there is little information available regarding whether substance abuse enhances HCV replication and promotes HCV disease progression. Therefore, we investigated whether morphine alters HCV mRNA expression in a HCV replicon-containing cell clone (Huh.8) that derived from human hepatoma cells (Huh7).
Methods: RT-PCR and Western blotting were used to determine the expression of mu-opioid receptor at mRNA and protein levels, respectively. HCV RNA copy numbers in the Huh.8 cells were quantified by real-time RT-PCR. pNF-kappaB-Luc, a plasmid containing NF-kappaB promoter linked with a luciferase gene, was transfected into Huh7 cells to study the effect of morphine on the NF-kappaB activity. CD8+ T lymphocytes were purified from PBMC isolated from whole blood of HCV-infected adult subjects using MACS CD8 Microbeads.
Results: Both Huh.8 and Huh7 cells express mu-opioid receptor. The addition of morphine to the Huh.8 cell cultures resulted in significant increase of HCV mRNA expression. Morphine diminished the anti-HCV effect of IFN-alpha in Huh.8 cells. This stimulatory effect of morphine on HCV mRNA expression was abolished by naltrexone (an opioid receptor antagonist) or beta-funaltrexamine (a specific mu-opioid receptor antagonist). Investigation of the mechanisms responsible for morphine action revealed that morphine activated NF-kappaB promoter and caffeic acid phenethyl ester (CAPE), a potent and specific inhibitor of activation of NF-kappaB, abolished morphine-activated HCV RNA expression in Huh.8 cells. In addition, culture supernatants from the activated CD8+ T-lymphocytes isolated from HCV-infected adult subjects, when added to Huh.8 cell cultures, significantly inhibited HCV RNA expression. This CD8+ T-cell-mediated anti-HCV effect was partially reversed by the addition of morphine to Huh.8 cell cultures.
Conclusions: Our data indicate that morphine may play an important role as a positive regulator of HCV replication in human liver cells in vivo. This enhancing effect of morphine on HCV RNA expression may compromise IFN-alpha therapy as well as CD8+ T-cell-mediated immunity against HCV infection.
