Function Of Spexin On Pain Perception In Mice

BackgroundSpexin(SPX)is a newly discovered endogenous neuropeptide,which is also known as neuropeptide Q.The spexin precursor peptide of human is encoded by the C12orf29 gene on the 12 th chromosome of genomes.The amino acid sequence of spexin is highly conserved in the vertebrates,and the gene and protein of spexin are widely distributed in central nervous and periphery systems.The spexin is involved in the regulation of many physiological functions,includingfeeding behavior,gastric motility,energy metabolism and glucose and lipid metabolism.The immunohistochemistry result shows that the spexin immune positive cells were detected in the brainstem,trigeminal ganglion and cerebral cortex of the rodents,which are closely related to nociceptive transmission.These results indicate that Spexin may paly a role in regulating nociceptive behavior..In this study,the formalin inducedacute inflammatory pain model and the acetic acid induced visceral pain model are employed and then analyze the analgesic effects and mechanisms of Spexin in acute inflammatory pain model nociceptive by behavior assessment,qRT-PCR,Western blot,immunohistochemistry methods areemployed.The formalin induced inflammatory pain animal model can simulate some characteristics of human pain after injuries.This model has good repeatability and widely applications for investigating the inflammatorypain and mechanisms.The acetic ace-induced visceralpain is also a commonly adopted model to evaluatethe pains in central nervous and periphery systems.ObjectivesTo Study the effects of Spexin in the pain.Methods(1)In the formalin tests,the nociceptive behaviors of mice were measured after intracerebroventricularly(i.c.v.)injections with different dosages of spexin(0.081-2.43 mg/kg)and the various antagonists for Galr2,Galr3,KOR and MOR.(2)The qRT-PCR is adopted to analyze the expression levels of Galr2,Galr3,Adcy1-9,Pomc,Penk,Pdyn,Oprm1,Oprd1,Oprk1,c-Fos,Junand Egr1 mRNA.(3)The Western blot is employed to measure the protein expression level of KOR after spexin injection,brain tissue and protein extract.(4)The immunohistochemistry applied to measure the number of c-Fos–positive neurons in the three subsection of periaqueductal gray(PAG)after Spexin injection,perfusion,fixation,tissue embedding,sectioning.(5)The influence of spexin on the writhing times of mice was investigated in the acetic acid–induced writhing tests.The qRT-PCR is adopted to analyze the expression levels of Galr2,Galr3,Adcy1-9,Pomc,Penk,Pdyn,Oprm1,Oprd1,Oprk1,c-Fos,Junand Egr1 mRNA in the brain tissue.Results(1)In formalin tests,the licking/biting durations of mice inphase I(earlyphase,0-10 min)was significantly reduced by the spexin(0.81 and 2.43 mg/kg),while that in the phase II(late phase,10-20 min),the licking/biting time was obviously decreased by the spexin(2.43 mg/kg).The positive control GAL(0.32 mg/kg)can remarkably reduce the durations of biting/biting time in both the early phase and late phase.(2)The qRT-PCR results show that i.c.v.spexin can significantly increase the Galr3 and the Adcy4 mRNAs levels.The Galr3 receptor antagonist SNAP37889(0.018 mg/kg)could significantly block the analgesia effect of spexin in the formalin tests.This indicated that the analgesic effect of spexin is mediated by Galr3.(3)The analgesic responses of the spexin(2.43 mg/kg)were blocked by the nor-BNI(73.47 mg/kg),which is the specific antagonist of kappa opioid receptor(KOR).The qRT-PCR result showed that the spexin can remarkably increase the Pdyn and Oprk1 mRNA levels,while it has no influence on the Pomc?Penk?Oprm1 and Oprd1 mRNA levels.Western blot result showed that the i.c.v spexin significantly increased the expression of KOR protein.This indictes that the analgesic effect of spexin was mediated by Pdyn/KOR.(4)The spexin increased the c-Fos mRNA level in the brain.T-5224(the c-Fos inhibitor)antagonized the increased Pdyn and Oprk1 mRNA induced by the spexin.The immunohistochemical analyses showed that the spexin(2.43 mg/kg)can remarkably increase the number of c-Fos positive-cells in dorsomedial,dorsolateral and ventrolateral parts of PAG of caudal(frontal fontanel-4.9 mm).This indicated that c-Fos was involved in the analgesic response of spexin.(5)In the acetic acid-induced visceral pain model,the number of writhing response of mice was significantly reduced after i.c.v.injection with spexin.And the central spexin significantly increased the Galr2,Adcy1,Pomc,Oprm1 and c-Fos mRNAs levels.Conclusions1.The injection of i.c.v.spexin(0.81 and 2.43 mg/kg)produce danalgesic effect inthe formalin induced inflammatory pain modelin mice,and this effect is mediated by Galr3/Adcy4/FOS/Pdyn/KOR pathway.2.In the acetic acid-induced visceralpain model,the analgesic effect of spexin(0.81 and 2.43mg/kg)is correlated with the genes of Galr2,Adcy1,c-Fos,Pomc and Oprm1.