Study On The Effects Of [D-Lys~3]-GHRP-6 Which Is Ghrelin’s Receptor Antagonist On Morphine Tolerance In Mice

Objectives:Morphine is a classical kind of exogenous opioid receptor agonist with good analgesic action.But long-term use can lead to drug tolerance,which has an impact on its clinical application.So,exploring the mechanisms of morphine tolerance contributes to apply morphine more effectively to relieve pain in clinical treatment.However,the mechanisms of morphine tolerance is unclear.The aim of this study was to explore the effects of morphine tolerance by using ghrelin receptor antagonist[D-Lys~3]-GHRP-6 and further investigate the related molecular mechanisms.The finish of the study will lay a foundation on exploring the mechanisms of morphine tolerance and provide references for finding medicines which can reduce the side-effects of morphine.Methods:The effects and mechanisms of[D-Lys~3]-GHRP-6 in morphine tolerance were explored through establishing chronic morphine tolerance model.The molecular biological methods were applied to assay the expressions of relevant genes,proteins and the changes of inflammatory factors’level.Double-labeling immunofluorescence were used to confirm the locations of the related proteins.Results:1.The data of tail flick test indicated that the mice injected morphine graduallyproduced tolerance from day 1 to day 6.[D-Lys~3]-GHRP-6 inhibited the morphinetolerance,(P<0.05).The above changes had significant differences and statisticalsignificances.2.The data of RT-PCR revealed that the transcriptions of Oprm1,Oprd and NOS1increased in morphine group,and decreased in morphine+[D-Lys~3]-GHRP-6group.The transcriptions of Grin1 decreased in morphine group,and increased inmorphine+[D-Lys~3]-GHRP-6 group.The transcriptions of ghrelin increased inmorphine group,and increased more in morphine+[D-Lys~3]-GHRP-6 group.Thetranscriptions of GHSR didn’t change obviously in morphine group,however,itincreased more in morphine+[D-Lys~3]-GHRP-6 group.The above changes hadsignificant differences and statistical significances.3.Based on the results of RT-PCR,further researches were detected usingWestern-blot.The results showed that the expressions of Grin1 decreased,andincreased in morphine+[D-Lys~3]-GHRP-6 group.The expressions of Oprm1increased in morphine group,and decreased in morphine+[D-Lys~3]-GHRP-6group.The above changes had significant statistically significant.4.The data of ELISA showed that the levels of TNFαraised and the level of IL-1βreduced in morphine group.Comparing to it,the levels of TNFαreduced andIL-1βraised in morphine+[D-Lys~3]-GHRP-6 group.The above changes hadsignificant statistically significant.5.The fluorescence microscope results demonstrated that the proteins of Oprm1,Grin1 and ghrelin were mainly distributed in LV,LSD,LSV,LSI,cg,cc and CPu.Conclusion:Ghrelin receptor antagonist[D-Lys~3]-GHRP-6 had obvious effects on inhibiting morphine tolerance.The mechanisms of[D-Lys~3]-GHRP-6 inhibiting morphine tolerance were through the variations of the Grin1,Oprm1 and NOS1’s expressions.Furthermore,these mechanisms were related to the levels of TNFαand IL-1β.Ghrelin,Grin1 and Oprm1 proteins were mainly distributed in the areas around ventricles.