The Mechanism And Effect Of The Spexin On The Inflammatory Pain Induced By Formalin In Mice

Background Spexin,also known as NPQ,its amino acid sequence(NWTPQAMLYLKGAQ-NH2)is highly conserved in the evolution of different vertebrates.Spexin m RNA and protein are widely expressed in the central nervous system and peripheral tissues of human,rodents and fish.So far,the physiological and pathological functions of spexin in energy metabolism,cardiovascular,appetite and defecation have been confirmed.In the central nervous system of rodents,spexin m RNA and protein have been detected in various brain regions,such as brainstem,trigeminal ganglia,brain cortex ect,which is known to be closely associated with nociceptive transmission.This suggests that spexin may be involved in the regulation of pain response.Formalin inflammatory pain model,which is produced by injecting formalin to cause sustained not a transient nociceptive stimulus and response,and the pain response is reproducible and quantifiable.It is widely used to evaluate the analgesic effect of different drugs,and it is one of the best models for preclinical pain research.However,there are few reports about the effects of spexin on acute inflammatory pain.Therefore,it is necessary to study the effect of spexin on acute inflammatory pain.This text is aimed to study the mechanism and effects of i.c.v.spexin and its antagonist on pain response in the mice formalin pain model by using PCR,RT-q PCR,ELISA,Western blot and PCR Array methods.Objectives To investigate the mechanism and effect of supraspinal spexin on inflammatory pain.Methods1.The mouse formalin model and groups After adaptation for 30 min,the mouse was taken out of the chamber and 20 ?l of 1.0% formalin solution was injected subcutaneously(s.c.)into the dorsal surface of the right hindpaw,using a microsyringe with a 26-gauge needle,each animal was immediately returned to the observation chamber and time(s)spent licking or biting the formalin-injected paw was measured with a hand-held stopwatch for each 5-min block for 30 min.Dosage regimen: The mice were divided into experimental group and control group.Spexin,non-amidated spexin,naloxone,bicuculline,normal saline(NS)were administrated in a total volume of 4?l at a constant rate of 10 ?l/min attached to a 10-?l Hamilton microsyringe.After 5 min,the mouse was taken out of the chamber and 20 ?l of 1.0% formalin solution was injected subcutaneously(s.c.)into the dorsal surface of the right hindpaw,using a microsyringe with a 26-gauge needle.And then the behavioral assay was started.2.Real-time quantitative PCR(RT-q PCR)was performed to detect the expression levels of target genes m RNA Total RNA was extracted from brain tissue of each mouse,then the m RNA was reverse transcribed and the c DNA was synthesized.After10 or 15 min i.c.v.injection of spexin or saline,gene expression of MOR,KOR,DOR,POMC,PDYN,PENK in brain will be detected by using the real-time quantitative PCR.3.ELISA was performed to detect the protein expression of PDYN After 15 min of i.c.v.administration with spexin or saline,the total proteins were extracted from mouse brain tissues and the content of PDYN by using ELISA.4.Western blot was performed to detect the protein expression of KOR After i.c.v.of spexin or saline 15 min,total proteins were extracted from the mouse brain,the protein expression of KOR was detected by using Western blot.5 PCR Array was performed to detect the expression levels of related genes Total RNA from brain tissue of each mouse was reversed transcribed to synthesize c DNA,after i.c.v.of spexin or saline 10 or 15 min,to examine m RNA levels of related 84 genes by using the RT2 PCR Array 96 wells.Results1.Spexin significantly reduced the licking/biting time in the mouse formalin test.Compared with the control group,spexin significantly reduced the licking/biting time at the dose of 10 nmol and 30 nmol/mouse(p<0.01,p<0.001,respectively)in early phase.And the licking/biting time significantly decreased at the dose of 30 nmol/mouse in the late phase(p<0.05).2.I.c.v.non-amidated spexin had no influence on paw-licking/biting time in the mouse formalin test.Compared with control group,i.c.v.non-amidated spexin at the dose of 30 nmol/mouse have no influence on paw-licking/biting time in both the early pahse and late phase(p=0.30,p=0.12,respectively).3.The opioid receptor antagonist naloxone significantly antagonized the antinociceptive effect of spexin.However,the GABAA receptor antagonist bicuculine failed to block the antinociceptive response induced by spexin in the mouse formalin test.Compared to control group,i.c.v.naloxone(10 nmol/mouse)alone did not significantly alter pain response induced by formalin in either early or late phase(p=0.94,p=0.79,respectively).However,co-administration(i.c.v.)of naloxone significantly antagonized the antinociceptive effect of spexin in early phase(p<0.01 as compared with spexin,p=0.40 as compared with control)and late phase(p<0.05 as compared with spexin,p=1.00 as compared with control).In comparison with the control group,the paw-licking/biting time of the two phases not changed after i.c.v.administration of bicuculine(early phase,p=1.00;late phase,p=0.82).Unfortunately,the dose of10 nmol/mouse bicuculine failed to block the antinociceptive response induced by 30 nmol/mouse spexin in the early and late phase in mouse formalin test(p=0.87,p=0.96 as compared with spexin;p<0.001,p<0.05 as compared with control,respectively).4.Spexin significant increased the PDYN and KOR m RNAs level after i.c.v.spexin in mice.Spexin did not influence the POMC or PENK m RNA level compared with control group(p=0.54,p=0.55,respectively).However,spexin significant increased the PDYN m RNA level compared with the control group(p<0.05).Compared with the control group,the expression level of MOR or DOR m RNA not changed(p=0.50,p=0.84,respectively).However,the KOR m RNA level significantly increased(p<0.05).5.The ELISA showed that spexin significant increased the PDYN total proteins expression level after i.c.v.spexin in mice compared to control group(p<0.05).6.The Western blot showed that spexin significantly increased the KOR total proteins expression level after i.c.v.spexin in mice as compared to control group(p<0.05).7.PCR Array and RT-q PCR analysis showed that fos gene expression increased after i.c.v.spexin in mice brain.The PCR Array analysis showed that fos gene expression was increased with about 2.3-fold in brain after spexin treatment.In addition,RT-q PCR showed that spexin significantly upregulated the fos gene expression(p<0.05).Conclusions1.I.c.v.spexin produced antinociceptive effect in formalin-induced inflammatory pain mouse model.2.The C-terminal non-amidation spexin had no influence on paw-licking/biting time in the formalin test.3.The antinociceptive effect induced by i.c.v.spexin was mediated through ?-opioid receptor but not GABAA receptor.4.The antinociceptive effect induced by i.c.v.spexin was mediated through activating dynorphin/?-opioid receptor system by fos,but not ?-opioid receptor or ?-opioid receptor.