Study On The Molecular Mechanisms Of Antinociception Induced By Ghrelin And Ghrelin?1-7?-NH₂ In Mice

Objectives:Ghrelin,a peptide hormone produced principally from the stomach,has been reported to act a variety of biological functions.Recent studies highlight that ghrelin has important roles in pain.Our preliminary results also suggested that ghrelin could induce analgesia to mice acute pain by i.c.v.administration.However,the mechanism of ghrelin-induced analgesia is still unclear.In addition,ghrelin(1-7)-NH2,an active fragment of ghrelin,was screened to research for its simple structure.The aim of this study was to explore the analgesic mechanisms of ghrelin as well as the analgesic effects and mechanisms of ghrelin(1-7)-NH2.Thus these findings may be a new strategy to clarify the mechanisms of their analgesia and lay the foundation for the further study.Methods:The tail withdrawal test was used to study the relationships between ghrelin and the opioid receptors,the analgesic effects of ghrelin(1-7)-NH2 as well as its relationships with the opioid system;Then we further explored the central distributions of ghrelin and ghrelin(1-7)-NH2 respectively after i.c.v.administration using fluorescence labeling method;Additionally,RT-PCR and Western blot were applied to assay the expressions of relevant genes and proteins.Results:1.The i.c.v.administration of ?-FNA,NTI and nor-NBI could not induce analgesia.And the results showed that NTI,but not ?-FNA or nor-NBI,could significantly antagonize the antinociception of ghrelin(P<0.001).2.The immunofluorescent microscope data indicated that FAM-ghrelin were mainly distributed in LV,LSD,3V,DG,Hippocampus etc.after i.c.v.administration.3.According to the results of RT-PCR,ghrelin significantly up-regulated the expressions of PENK mRNA and the level of OPRD mRNA.However,ghrelin had no effects on the expressions of other relevant genes.4.The results of Western-blot showed that ghrelin had no effect on the level of GHSR protein,while the expressions of PENK protein and OPRD protein were significantly increased compared to the control group.5.The[D-Lys3]-GHRP-6 could not reverse the antinociception of Deltorphin ?treatment(P>0.05).6.The i.c.v.injection of ghrelin(1-7)-NH2 induced a time and concentration-dependently increasing of tail withdrawal latency,but this antinociception could be antagonized by[D-Lys3]-GHRP-6 or naloxone.7.The fluorescence microscope results demonstrated that FAM-ghrelin(1-7)-NH2 were mainly distributed in LV,LSD,cc,3 V,Hippocampus,hf,Or etc.8.According to the results of RT-PCR,the expressions of PENK mRNA and OPRD mRNA were significantly up-regulated compared to control group(P<0.01)after administration of ghrelin(1-7)-NH2.However,ghrelin(1-7)-NH2 could not modify other related gene expressions.9.The Western-blot results showed that the levels of PENK protein and OPRD protein were significantly higher than control group,whereas GHSR protein expression was no significant difference compared to control group.Conclusion:1.Intracerebroventricular injection of ghrelin(1-7)-NH2 produced a obviously analgesic effect of time and concentration-dependently.2.After i.c.v.injection,ghrelin and ghrelin(1-7)-NH2 mainly distributed around the ventricles.The analgesia of them was via a mechanism of activating the GHS-Rla initially,subsequently up-regulating the expression of PENK and OPRD,and then PENK combined with OPRD to produce an antinociceptive effect.3.The analgesia mechanisms of ghrelin and ghrelin(1-7)-NH2 are similar as well as the central distribution.Furthermore,ghrelin(1-7)-NH2 has potential to be an analgesic drug for its simple structure.