Gene knockdown with lentiviral vector-mediated intrathecal RNA interference of protein kinase C gamma reverses chronic morphine tolerance in rats

Although morphine is a widely used opioid analgesic, morphine tolerance (MT) has limited the use of the drug because it creates the necessity for high doses. Protein kinase C (PKC), especially the PKCγ isoform, is considered to play a key role in the development of MT. Because RNA interference provides a powerful method for the investigation of gene function, and lentiviral delivery systems have been approved for human use, this present study examined rats tolerant to morphine to determine whether an intrathecal injection of a lentiviral vector of PKCγ short hairpin RNA (LV-shPKCγ) down-regulated the expression of the PKCγ gene and reversed MT.MT was induced by intrathecal morphine (10 µg b.i.d.) for six consecutive days. A lentiviral-mediated short hairpin RNA (shRNA) system was synthesized to deliver the PKCγ shRNAs to the spinal cord of the rats with MT. Mechanical and thermal paw withdrawal threshold were assessed to determine the analgesic effects of morphine. Expression of PKCγ mRNA and protein was determined by reverse transcriptase-polymerase chain reaction and western blotting analysis, respectively.The chronic administration of morphine induced a stabilized analgesic tolerance. A single injection of LV-shPKCγ significantly reversed morphine antinociceptive tolerance. Compared to the control group, PKCγ mRNA and protein levels were dramatically down-regulated in the LV-shPKCγ group.A single injection of LV-shPKCγ reversed MT by reducing the expression of PKCγ in the spinal cord. These findings indicate that the use of LV-shPKCγ might be a potential strategy for therapy in MT.