The Mechanism Of Proteasome In The Development Of Chronic Morphine Tolerance

Opioids are commonly used in treatment of acute and chronic pain, but long-term application can lead to chronic opioid tolerance. Clarifying the mechanism of opioid tolerance is important for its prevention and treatment. There have been many articles that illustrate the mechanism of morphine tolerance.Recent reports indicate that glutamate transporters play a crucial role in neuropathic pain and neurotoxicity.At the mean time,the inhibition of proteasome activity attenuate neuropathic pain behaviors in rats. However,the mechanisms at cellular level are still unknown.The ubiquitin-proteasome system(UPS) is a major non-lysosomal proteolytic pathway that degrades cellular proteins including those have important roles in the regulation of cell growth and function.It is now widely appreciated that the UPS plays its role in a variety of cellular pathways, including cell growth and proliferation, apoptosis, ptotein quality control, DNA repair, transcription,and immune response.Ubiquitination is a process involving three enzymes:E1(ubiquitin-activating enzyme), E2(ubiquitin-conjugating enzyme),and E3(ubiquitin ligase).Interaction between an E3ligase and its target is considered as a key step in determining the selectivity of UPS for a target and its subsequent proteasomal degradation,a process that is subject to intracellular modulation by various upstream regulators.It is reported that glutamate transporter plays an important role in the signal transduction of morphine tolerance and hyperalgesia.It is also reported that chronic morphine exposure induces posttranscriptional downregulation of EAAC1and increases proteasome activity. Whether the downregulation of glutamatetransporter is regulated by proteasome after chronic morphine treatment has not been reported. In this dissertation, we researched the effect of the proteasome inhibitor in morphine tolerance, anticipating providing a new idear for future treatment and studies in the field of morphine tolerance.Objective: To study the effect of proteasome in the development of morphine tolerance and its mechanism in the intracellular signal transduction. Methods:42Sprague-Dawley rats weighed300～350g were randomly divided into7groups (n=6):saline group (S group),chronic morphine tolerance group (M group), morphine+MG-1320.5,1.25,2.5,5ug group (M/MG group) and MG-1322.5ug group (MG group). They were intrathecally (i.t.) administrated by saline10μl, morphine10μg, morphine lOμg+MG-1320.5,1.25,2.5,5ug, MG-1322.5ug alone respectively twice a day for6days. Tail flick test and hot plat test were given at8:30on day1,3,5,7. After that the rats are euthanasiaed.The segment of spinal L3～Ls were taken away for Western blotting (n=5) and RT-PCR (n=5) to examine the protein expression of glutamatetransporter, GLAST and EAAC1in the spinal cord dorsal horn. RT-PCR was used to test the mRNA expression of PTEN and Nedd4in the spinal cord dorsal horn (n=5).ResuIts:After the continuous injection of morphine, the latency of both tail flick and hot plat decline gradually. Morphine tolerance formed stably in group M after intrathecal infusion of morphine. Compared with group M, the TFL in group M/MG was still higher than the group M and group S on day7, MG132dose-dependently prevent the development of morphine tolerance. Western blot result showed that the expression of glutamate transporter GLAST and EAAC1decreased in group M compared with group S (P<0.05). Compared with group M, the expression of glutamate transporter GLAST and EAAC1increased in group M/MG (P <0.05).RT-PCR result showed that the mRNA expression of PTEN and Nedd4increased in group M compared with group S (P<0.05). Compared with group M, the mRNA expression of PTEN and Nedd4decreased in group M/MG (P<0.05).Conclusion:Spinal glutamatetransporter may play an important role in the development of morphine tolerance through the ubiquitin-proteasome signal pathway. Moreover, PTEN contributes to morphine tolerance by inducing the ubiquitin ligase Nedd4mRNA expression in spinal cord dorsal horn.In summary, chronic morphine tolerance was established by intrathecal administration of morphine for7d. In our studies, we found that glutamatetransporter plays an important role in the development of morphine tolerance through the ubiquitin-proteasome signal pathway, indicating that we could change the proteasome activity to prevent and treat morphine tolerance.