Inhibition Of S1PR3 In Central Amygdala Alleviated Pain And Anxiety-Like And Depression-Like Behavior In CCI Mice

BackgroundNeuropathic pain is a common chronic pain.Typical symptoms include hyperalgesia,abnormal pain and spontaneous pain.In addition to sensory abnor malities,patients are often accompanied by negative emotions,such as anxiety and depression.Chronic pain and anxiety and depression promote each other.Up to now,the pathogenesis is still unclear and the therapeutic effect of drugs is limited.Sphingosine 1-phosphate(S1P)binds to sphingosine 1-phosphate receptors(S1PRs)on the cell surface and participates in the regulation of pain in the central and peripheral nervous systems.Recent studies have shown that sphingosine 1-phosphate receptor subtype 3(S1PR3)mediates pain,anxiety and depression like behavior.After the central nervous system was stimulated by S1 P,the activity of cerebral cortex and amygdala was the strongest.N-methyl-D-aspartate receptor subtype 2B(NR2B)of the central nucleus of the amygdala(CeA)is involved in the regulation of pain,anxiety and depression like behavior.S1 PRs can phosphorylate NR2 B by regulating cyclic adenosine monophosphate,change synaptic plasticity and lead to neuropathic pain.Whether S1PR3 of CeA can change neuropathic pain and anxiety depression like behavior by regulating NR2 B is unclear.Therefore,in this study,the chronic sciatic nerve compression injury(CCI)model was used.Firstly,a new target S1PR3 involved in the comorbidity of pain and depression was screened,and then the agonists and antagonists of S1PR3 or si S1PR3 were used respectively.The behavioral effects of interfering with S1PR3 on pain sensation and pain emotion and the expression of NR2 B and its phosphorylation were confirmed by in vivo experiments.ObjectiveTo study whether S1PR3 in the central amygdala is involved in neuropathic pain,anxiety and depression like behavior and its mechanism.Methods1.Establishment of chronic sciatic nerve compression injury model(CCI).2.Use 0.07 g and 0.4g von ferry hair and thermal radiometer to detect mechanical pain and thermal pain respectively.Anxiety like behavior was tested by open field and the elevated plus maze elevated,and depression like behavior was tested by forced swimming and tail suspension.3.The changes of S1PR1-S1PR5 in different brain regions were detected by RT-PCR.4.Detect the distribution of S1PR3 in neurons,astrocytes,and microglia in CeA under pain by immunofluorescence.5.Brain stereotactic injection of S1PR3 agonist,antagonist,and siRNA3 to detect the changes of pain feeling and painful emotion.6.The protein expression changes of S1PR3,NR2 B,and p NR2 B in CeA were detected by Western blot.Results1.CCI model can induce mechanical pain,thermal pain and anxiety depression like behavior in mice.Compared with sham group,the frequency of mechanical foot contraction and the latency of thermal foot contraction in CCI group were significantly increased(P <0.05).The results showed that the number of times of mice entering the maze and the open arm in the elevated group was significantly reduced compared with that in the open arm group and the open arm group;The central area residence time,open arm residence time and times of CCI mice in the easy anxiety group were significantly lower than those in the sham group,and there was no difference between the difficult anxiety group and the sham group(P < 0.05).The results of forced swimming and tail suspension test showed that the resting time of mice in CCI group was significantly prolonged;The resting time of CCI mice in easy depression group was more than that in sham group,and there was no significant difference between CCI mice in difficult depression group and sham group(P < 0.05).2.The expression of S1PR3 gene and protein in the contralateral CeA region of CCI mice increased.q PCR detected different subtypes of S1 P in different brain regions(ACC,NAC,CeA,VTA,Hip)on the 7th day after CCI.Compared with sham group,the S1PR3 m RNA level of CeA in CCI group was significantly higher(P < 0.05).Immunofluorescence showed that S1PR3 was expressed in Ce C,Ce L and Ce M of the three subregions of CeA in naive mice,and the S1PR3 protein level of CeA increased on days 3,7,14 and 21 after CCI(P < 0.05).Immunofluorescence double labeling results showed that compared with sham group,the expression of S1PR3 of CeA in CCI group was significantly increased,mainly in neurons,and had no co localization with astrocytes and microglia.The S1PR3 protein level of CeA increased on days 3,7,14 and 21 after CCI(P < 0.05).3.Injection of S1PR3 agonist CYM5541 into the CeA region of naive mice could induce pain behavior.Compared with DMSO group,the frequency of mechanical foot contraction and the latency of thermal foot contraction increased on the 1st,2nd and 3rd day after administration of selective S1PR3 receptor allosteric agonist CYM5541(P < 0.05).4.Inhibition of S1PR3 in CeA region can alleviate pain,anxiety and depression like behavior in CCI mice.Compared with siRNA1 and siRNA2,only siRNA3 could significantly reduce the protein expression of S1PR3(P < 0.05).The behavioral results showed that compared with the control group,the frequency of mechanical foot contraction and the latency of thermal foot contraction were reduced after injection of specific antagonists CAY10444 and siRNA3 respectively.The analgesic effect of CAY10444 group lasted for two days and that of siRNA3 group lasted for at least five days(P <0.05).Compared with the control group,CAY10444 and siRNA3 significantly increased the time of CCI mice in the central area of the open field and the residence time and times of entering the open arm of the elevated plus maze.The proportion of anxiety prone mice after DMSO \ si NC intervention was not significantly different from that before intervention,while the proportion of anxiety prone mice after CAY10444 \ siRNA3 intervention was significantly lower than that before intervention(P < 0.05);In the forced swimming and tail suspension test,the resting time decreased.The proportion of prone to depression mice after DMSO \ si NC intervention was not significantly different from that before intervention,while the proportion of prone to depression mice after CAY10444 \ siRNA3 intervention was significantly lower than that before intervention(P < 0.05)。5.The expression of NR2 B and p NR2 B in CeA region of CCI mice increased.Western blot showed that NR2 B and its phosphorylation in the contralateral CeA region of CCI group were significantly higher than those in sham group(P < 0.05).Immunofluorescence double labeling showed that NR2 B and S1PR3 had obvious co-labeling.6.Regulating S1PR3 in CeA region can change the protein expression of NR2 B and p NR2 B.Western blot showed that injection of CYM5541 into the CeA region of naive mice significantly increased the expression of NR2 B and p NR2B(P < 0.05).Compared with the control group,injection of CAY10444 and S1PR3 siRNA3 after CCI could significantly reduce the expression of NR2 B and p NR2B(P < 0.05).ConclusionS1PR3,which inhibits CeA in central amygdala,alleviates pain,anxiety and depression-like behavior in CCI mice through NR2 B.