The Role And Mechanism Of SIRT1in The Morphine Tolerance

Background:Morphine is a powerful analgesic for treating moderate to severe pain in clinical. However, long-term morphine administration induces tolerance, and it require larger doses to elicit the same amount of analgesia. High doses of morphine result in many side-effects, such as respiratory depression, urinary retention, nausea, vomiting and dizziness. Thus antinociceptive tolerance and the high doses required to achieve effects have limited the use of morphine. Many factors have been reported to relate to morphine tolerance, but the mechanism behind morphine tolerance is still unclear.It is domenstrated that morphine tolerance and neuropathic pain share many common mechanism,and the discovery of epigenetic mechanisms of neuropathic pain provided us important information to study the mechanisms of morphine tolerance.Epigenetic regulation including DNA methylation, histone modifications (phosphorylation, methylation and acetylation) and small RNA regulation. Histone acetylation modification can regulate gene transcription through the acetylation or deacetylation modification of specific lysine (Lys) residues ofcore histone protein. Little research has been conducted regarding the epigenetic mechanismsin in morphine tolerance.So the successful completion of this study will provide new direction in morphine tolerance research. Objectives:To observe the mechanical and thermal nociception changes before and after induction of morphine antinociceptive tolerance; to the global Ac-H3and SIRT1expression changes in the lumbar spinal cord;to observe the effect of IT resveratrol on the Onset and development of morphine tolerance and choose the the minimum effective dose; to observe the effect of IT Resveratrol on morphine tolerance induced variantions of Ac-H3and SIRT1in the Lumbar Spinal Cord.Methods:1. Adult male prague-Dawley (SD) rats (Weight280-320g) were divided randomly into3groups. The latencies of hindlimb withdrawal response to radiant heat and mechanical stimulation were measured before implanted with intrathecal catheter following the methods of Yaksh. In group normal saline, morphine injection (10μg b.i.d. for13days) was replaced with normal saline injection In group morphine tolerance I (MTI), morphine tolerance was induced by an intrathecal treatment regimen (10μg of morphine b.i.d. for6days consecutive days),then the morphine treatment was changed to10μg everyday. In group morphine tolerance Ⅱ (MTⅡ),morphine tolerance was induced by an intrathecal treatment regimen (10μg of morphine b.i.d. for13days consecutive days).After the test of paw withdrawa mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) every two days after surgery,choose the appropriate morphine tolerance group as the morphine tolerance model in the next study.2. Adult male prague-Dawley (SD) rats implanted with intrathecal catheter were divided randomly into2groups. The latencies of hindlimb withdrawal response to radiant heat and mechanical stimulation were measured before morphine injection, morphine tolerance group (MT) was the morphine tolerance model chosen from above study.In group normal saline, morphine injection was replaced with normal saline injection. After the test of paw withdrawa mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) every two days after surgery,the rats were sacrified for qRT-PCR detection of SIRT1mRNA,western blot detection of Ac-H3,Ac-H4and SIRT1expressions.3. Male SD rats fitted with an intrathecal catheter successfully, randomly assigned to seven groups.They are NS group,MT group,Resveratrol (RES)15μg group,Resveratrol (Res)30μg group,Resveratrol (Res)60μg group,Resveratrol (Res)300μg group,dimethyl sulfoxide (DMSO) group.PWMT and PWTL were tested in all rats。10μg of morphine b.i.d. for13days in MT group,Res (15μg,30μg,60μg,300μg) groups and DMSO group. While normal saline injection instead of morphine in NS group.From day7to13different doses of resveratrol and DMSO was injected in the corresponding groups. PWMT and PWTL of all the rats were tested every two days during the experiment. Then Select the minimum effective dose of resveratrol for subsequent tests.4. Adult male SD rats weighting280-320g were implanted with intrathecal catheter were randomly divided into4groups:mormal saline group (NS group),morphine group (MT group),resveratrol group (Res group) and DMSO group.After measuring the base thermal pain threshold,10μg of morphine b.i.d. for13days in MT group,RESgroup and DMSO group,normal saline instead morphine in NS group. From day7to13, resveratrol (30μg/10μl) and DMSO (10μl) was injected in RES group and DMSO group everyday. PWTL of all rats were measured every two days.On the day13, the rats were sacrified under deep anesthesia and their lumbarspinal cords were moved quickly for immunohistochemical, qRT-PCR and WB analysis,to observe the expression changes of Ac-H3, SIRT1and NR2B in different groups.Results:1. There was no significant difference in PWMT and PWTL among all the rats before implation (P>0.05).During the experitment period,the thermal and mechanical pain threshold had no statistical significance (P>0.05). In groups MTⅠ and MTⅡ,injection of morphine made the pain threshold higher than the basic pain threshold on the day1and day3(P<0.05), but fail to analgesia on day7(P>0.0.5).during the day7to day13, Morphine tolerance state persist in the group MTⅡ,while Morphine tolerance reversed automatically in group MTⅠ. So the MTⅡ was chosen to be the morphine tolerance model in the next study.2. There was no significant difference in PWMT and PWTL among all the rats before implation (P>0.05).During the experitment period,the thermal and mechanical pain threshold had no statistical significance (P>0.05). In group MT, morphine fail to analgesia on day?(P>0.0.5).qRT-PCR and Western blot analysis revealed a decrease in the level of SIRT1mRNA and protein and a increase in the level of Ac-H3protein in the lumbar spinal cord of morphine tolerance rats (P<0.05).there was no significant defference in Ac-H4expression during MT group rats and NS group rats (P>0.0.5)3. There was no significant difference in base pain threshold among7groups rats (P>0.05). Chronic morphine exposure (10μg, i.t, b.i.d) in rats lead to a stabilized antinociceptive tolerance in MT group,Res (15μg,30μg,60μg,300μg) groups and DMSO group.30uμg,60μg and300μg resveratrol injection can significant reversed morphine tolerance start from the day9,and lasted to day13. the thermal pain threshold are significantly different from the base pain threshold (P<0.05).The15μg resveratrol had no effect on the morphine tolerance.there was no significant difference in reversal effection among Res30μg,Res60μg and Res300μg groups (P>0.05)4. There was no significant difference in base pain threshold among4groups rats (P>0.05).On the day13, immunohistochemistry revealed a significant decrease in the number of Sirt1cells in the dorsal horn of MT rats compared with those in NS rats. The activation of Sirtl by resveratrol resulted in an increased number of Sirtl cells in Res rats compared with the DMSO rats (P<0.05)qRT-PCR results suggested that NR2B mRNA increased but SIRT1mRNA decreased significantly in RES groups rats compared with MT groups (P<0.05). Results of western blot analysis during the MT groups and RES group consistented with qRT-PCR results, the difference is statistically significant (P<0.05)Conclusions:1. Chronic morphine exposure (10μg of morphine b.i.d.) in rats led to a antinociceptive tolerance. But the maintaince of morphine tolerance need the continuation of original dose of morphine injection.2. The down-regulation of SIRT1expression and up-regulation of Ac-H3accompanying with the disappearance of morphine analgesia effect indicates that the Ac-H3and SIRT1may play an important role in the development of morphine tolerance. 3. IT injection of resveratrol7days after morphine tolerance can reversed the morphine analgesia tolerance without a dose dependence.4. IT injection of resveratrol (30μg/d) for7days reversed the chronic morphine analgesia tolerance,meanwhile the morphine tolerance-induced down-regulation of SIRTl expression and up-regulation of Ac-H3and NR2B also were reversed.It indicates that the morphine tolerance reversal of resveratrol was completed by the SIRT1-depended way through changing the Ac-H3level of NR2B genes and affecting the gene transcription and expression.