Fourthly the distribution sites after i c

Fourthly, the distribution sites after i.c.v. injection of FAM-ghrelin(1–7)-NH2 were explored in mice using the technologies of fluorescence labeling. The green fluorescence of FAM-ghrelin(1–7)-NH2 was strongly distributed in these structures of lateral ventricle, lateral septal nucleus, dorsal part, oriens layer of the hippocampus, hippocampal fissure, corpus callosum, and dorsal 3rd ventricle in mice. Among these structures, the dorsal 3rd ventricle and hippocampus have the highest expressions of ghrelin, GHS-R1α and ORs (Bunzow et al., 1994; Diano et al., 2006; Ferrini et al., 2009; Hou et al., 2006). It showed that maybe the dorsal 3rd ventricle and hippocampus were the most important and the direct sites of antinociception after i.c.v. injection of ghrelin(1–7)-NH2. In our present study, the antinociceptive effects of ghrelin(1–7)-NH2 (i.c.v.) were completely antagonized by OR antagonist. Furthermore in our previous study, the antinociception induced by deltorphin II, which is the selective ligand of OPRD wasn’t blocked by the co-injection (i.c.v.) of [D-Lys3]-GHRP-6(Liu et al., 2016). The results suggested that i.c.v. injection of ghrelin(1–7)-NH2 initially directly activated the GHS-R1α then activated the opioid system to produce antinociception. In summary, our results indicated that ghrelin(1–7)-NH2 as the active fragment of ghrelin, could also produce dose- and time-related antinociceptive effects after i.c.v. injection in the acute pain in mice. The antinociceptive effects of ghrelin(1–7)-NH2 were mediated through GHS-R1α, OPRM (primary μ1-opioid receptor subtype) and OPRD. The distribution sites of ghrelin(1–7)-NH2 after i.c.v. injection included the dorsal 3rd ventricle and hippocampus which were the high CCK-8 sites of ghrelin, GHS-R1α and ORs. Moreover, the present study on the molecular levels also showed that the antinociceptive effects of ghrelin(1–7)-NH2 were connected with increasing the expressions of endogenous δ-opioid peptide PENK and OPRD. All these results indicated that ghrelin(1–7)-NH2 initially activated the GHS-R1α, then activated the OPRM, as well as increased the release of endogenous PENK to activate of OPRD to produce antinociception. Further investigation is required to explain the mechanisms of ghrelin(1–7)-NH2 in pain. These results suggested that ghrelin(1–7)-NH2 had a novel role of alleviating pain, and might be a new potential strategy for developing new analgesic drugs.
Conflicts of interest
Acknowledgements This work was supported by the grant from National Natural Science Foundation of China (No. 21302085, 81660246), by the grant from the Natural Science Foundation of Jiangxi Province (No. 20181BAB205027).
Introduction Ghrelin is a circulating growth hormone-releasing and appetite inducing brain-gut peptide (Cummings, 2006) that is predominantly synthesised by the endocrine X/A-like cells in the gastric mucosa (Korbonits et al., 2004). It is the endogenous ligand for the previously ‘orphan’ growth hormone secretagogue-receptor (GHS-R), a G protein-coupled receptor with seven transmembrane domains. There are two splicing variants of the GHS-R gene: the functioning full-length product GHS-R1a and a truncated variant GHS-R1b. One of the most important established roles of ghrelin is the regulation of appetite and energy homeostasis (Kojima et al., 2004, Korbonits et al., 2004). Both central and peripheral administrations of ghrelin cause an increase in appetite and body weight (Kamegai et al., 2001, Tschop et al., 2000, Wren et al., 2001a). Ghrelin acts mainly in the hypothalamus by binding to its receptors in areas that are important for appetite regulation, namely the arcuate nucleus, paraventricular nuclei, dorsomedial region, central nucleus of amygdale and the nucleus of solitary tract (Mano-Otagiri et al., 2006, Olszewski et al., 2003). Ghrelin stimulates the secretion of orexigenic neuropeptide Y, agouti-related protein and orexin, and inhibits the release of anorexigenic compounds such as pro-opiomelanocortin, cocaine- and amphetamine-regulated transcript and α-melanocyte-stimulating hormone, thus resulting in an increase in appetite (Gao and Horvath, 2007, Kohno et al., 2003).