The Analgesic Effects And Mechanisms Of Ghrelin(1-5)-EM2,a Hybrid Peptide Based On μ-Opioid/Ghrelin Receptor,in CFA-induced Chronic Inflammatory Pain

Objectives:Ghrelin,as an endogenous polypeptide with analgesic effects,has attracted the attention of researchers in recent years.Our experimental group had comfirmed that ghrelin and its active fragments had better analgesic effects in acute pain in previous studies.Ghrelin active fragments have octanoyl structure and also have the abilities to cross the blood-brain barrier.Endomorphins,as endogenous ligands of μ opioid receptor,have good analgesic effects.The side effects of endomorphins are reduced compared to morphine,but they still cannot penetrate the blood-brain barrier.In our laboratory,ghrelin(1-5)-EM2,EM2-ghrelin(1-5),ghrelin(1-9)-EM2 and EM2ghrelin(1-9)based on μ-opioid/ghrelin receptor were formed by combining ghrelin active fragments with EM-2.The results of our experimental group showed that ghrelin(1-5)-EM2 had good analgesic effects on acute pain and neuropathic pain,and the tolerance was low in previous studies.Therefore,our experiments will first study the blood-brain barrier permeabilities of four hybrid peptides.Secondly,our experiment will study the analgesic effect of ghrelin(1-5)-EM2 in carrageenan、acetic acid writhing、formalin inflammatory pain and the analgesic effect and mechanism in CFA chronic inflammatory pain.Our experiments will lay the foundation for subsequent experiments.Methods:The hybrid peptides were synthesized using solid-phase synthesis.These hybrid peptides were connected with Cy7 and were explored whether they had blood-brain barrier permeabilities.Four inflammatory pain models(carrageenan,formalin,acetic acid writhing and CFA chronic inflammatory pain)were established to study the analgesic effects of ghrelin(1-5)-EM2 through behavioral tests.In the CFA chronic inflammatory pain model,the tolerance of the hybrid peptide ghrelin(1-5)-EM2 was studied through continuous administration.HE staining was used to detect its improvements in the pathological state of the inflammatory site.Western blot and immunofluorescence staining were used to study its analgesic mechanisms in CFA chronic inflammatory pain.Results:1.The hybrid peptide ghrelin(1-5)-EM2 could pass through the blood-brain barrier.2.Intrathecal injection of ghrelin(1-5)-EM2 had significant analgesic effects in four inflammatory pain models.3.Ghrelin(1-5)-EM2 produced good analgesic effects in CFA chronic inflammatory pain,and did not produce tolerance after continuous intrathecal injection.Ghrelin(1-5)-EM2 had certain degree of improvements on the skin of the inflammatory site in mice.4.Growth hormone secretagogue receptor 1α(GHS-R1α)antagonist and opioid receptor antagonist could antagonize the analgesic effects of ghrelin(1-5)-EM2.5.In CFA chronic inflammatory pain,after continuous injection of ghrelin(1-5)-EM2,there was no significant change in serum levels of tumor necrosis factor-α(TNFα),interleukin-1β(IL-1β)and interleukin-6(IL-6).6.Western blot and immunofluorescence staining experiments showed that ghrelin(1-5)-EM2 exerted its analgesic effects by inhibiting the mitogen-activated protein kinase(MAPK)pathway and inhibiting the activation of non-selective cation channel TRPV1 in CFA chronic inflammatory pain.Conclusion:Ghrelin(1-5)-EM2 could produce significant analgesic effects in carrageenan,formalin,acetic acid writhing and CFA chronic inflammatory pain.Ghrelin(1-5)-EM2 could penetrate the blood-brain barrier.Ghrelin(1-5)-EM2 did not produce tolerance in CFA chronic inflammatory pain,and exerted its analgesic effects by inhibiting the mitogen-activated protein kinase(MAPK)pathway and inhibiting the activation of non-selective cation channel TRPV 1.Ghrelin(1-5)-EM2 could improve the skin of the inflammatory site.