Mechanism Of S1PR1 In Trigeminocervical Complex Involved In The Central Sensitization Of Chronic Migraine

PART Ⅰ S1PR1 IS INVOLVED IN CENTRAL SENSITIZATION IN CHRONIC MIGRAINEObjectiveChronic migraine(CM)is defined as a headache that lasts for at least15 days per month for more than three months,with migraine-like attacks for at least 8 days.Due to the poor response and high disability of CM treatment,it often brings a serious burden to individuals and society.The in-depth study of the pathophysiological mechanism of CM is of great significance for clinical work,among which central sensitization is considered to be the main pathological mechanism of CM.In recent years,various pain model studies have found that sphingosine 1 phosphate receptor 1(S1PR1)is involved in the occurrence and maintenance of pain.However,whether S1PR1 is involved in the development of central sensitization in chronic migraine has not been investigated.Therefore,this study will explore the expression of S1PR1 in the trigeminocervical complex(TCC)and its effect on central sensitization in an animal model of CM.Methods1.This study used a mouse model of repeated intraperitoneal injection(i.p)nitroglycerin(NTG,10mg/kg).The same dose of saline was administered as a control group.Before each administration,the pain threshold in the periorbital and hindpaw of mice were detected.2.After the model was constructed,we first detected the changes of S1PR1 at the m RNA and protein levels in the TCC site by Western blot(WB)and real-time quantitative polymerase chain reaction(q PCR).Then,immunofluorescence staining was used to observe protein expression and cell localization of S1PR1 in the TCC area.3.To further explore the role of S1PR1 in the central sensitization of CM,we administered the specific inhibitor of S1PR1,W146(1 mg/kg,i.p),the functional antagonist FTY720(0.01mg/kg,0.1mg/kg and 1 mg/kg,i.p)and S1PR1 agonist(SEW2871,20mg/kg,i.p)before intraperitoneal injection of NTG in mice.The changes of periorbital and hindpaw pain thresholds of mice were detected before administration and 2 hours after NTG administration.In addition,we also analyzed the expression changes of calcitonin gene related peptide(CGRP)and c-fos in TCC by WB and immunofluorescence staining.Results(1)Expression and localization of S1PR1 in TCC in NTG model1.Repeated NTG stimulation induced a significant decrease in periorbital mechanical pain threshold,hindpaw mechanical pain threshold and thermal pain threshold in mice.Compared with the Saline group,the expression of S1PR1 in the TCC site was up-regulated at both m RNA(p<0.01)and protein(p<0.05)levels after repeated NTG administration.2.The results of immunofluorescence co-staining showed that S1PR1 could co-localize with microglia,astrocytes and neurons in the TCC site.Compared with the Saline group,the S1PR1 positive cells in the NTG group increased significantly(p<0.001).(2)Effects of S1PR1 intervention on NTG-induced central sensitization1.The results of behavioral analysis showed that compared with the Saline group,intraperitoneal injection of W146 and FTY720 in mice could significantly alleviate the periorbital and hindpaw hyperalgesia induced by repeated NTG stimulation.However,intraperitoneal injection of S1PR1 agonists before NTG administration did not alleviate hyperalgesia in mice.2.CGRP,as an important biological marker of CM,increased in the TCC after repeated NTG injection by WB and immunofluorescence staining(p<0.01).W146 and FTY720 significantly reduced NTG-induced CGRP release(p<0.001).3.p-ERK and c-fos are important indicators of central sensitization.W146 and FTY720 significantly reduced the up-regulation of p-ERK and c-fos in the TCC of mice induced by repeated NTG injections(p<0.05).ConclusionS1PR1 was up-regulated at both protein and m RNA levels in a CM mouse model with repeated NTG injections,suggesting that S1PR1 may be involved in the development of chronic migraine.Antagonizing the S1PR1 receptor could significantly relieve hyperalgesia in mice,and also reduce the expression of CGRP,p-ERK and c-fos in the TCC,which further confirms that S1PR1 is involved in the central sensitization of CM.PART Ⅱ S1PR1 MEDIATES MICROGLIA ACTIVATION IN CENTRAL SENSITIZATION IN CHRONIC MIGRAINEObjectiveNumerous studies have shown that microglia activate could promote the development of central sensitization.Our previous study also found that activation of microglia in the TCC could promote the development of CM.S1PR1 is widely expressed in the central nervous system,and a large number of studies have shown that antagonizing S1PR1 signaling can inhibit the activation of microglia and alleviate the development of various central nervous system diseases.Furthermore,targeting S1PR1 reduced central sensitization-induced neuroinflammation in the spinal dorsal horn in neuropathic pain models.However,in chronic migraine,whether antagonizing S1PR1 can regulate microglia to alleviate central sensitization remains unclear.Therefore,in this section we will explore the cellular and molecular mechanisms underlying the role of S1PR1 in chronic migraine,focusing on neuroinflammation.Methods1.First,we cultured BV-2 microglia in vitro,and established an inflammation model by LPS stimulation.The expression changes of microglial M1 marker i NOS after administration of S1PR1 antagonist were observed by q PCR and immunofluorescence staining techniques.At the same time,the expression changes of inflammatory factors released by microglia were detected by q PCR technology.2.In animal model,the effect of S1PR1 antagonists on NTG-induced microglial activation at TCC sites was explored by immunofluorescence staining and immunoblotting staining.At the same time,the expression of S1PR1 antagonists on inflammatory factors in the TCC site was analyzed.Results1.First,in vitro study,we found that S1PR1 antagonists can significantly inhibit the expression of i NOS in LPS-induced microglia,and can inhibit the inflammatory factor produced by LPS-stimulated BV-2microglia at the m RNA level.2.In animal models,the results of immunofluorescence staining showed that antagonizing S1PR1 could reduce the number of Iba1-positive cells in the TCC area(p<0.001),and could reduce the expression level of microglial M1 marker i NOS protein(p<0.05).In addition,immunoblotting results showed that antagonizing S1PR1 inhibited the expression of TNF-αin the TCC area.ConclusionThis part of the study found that antagonizing S1PR1 could significantly inhibit the activation of microglia and the release of related inflammatory mediators,suggesting that S1PR1 may participate in the central sensitization of chronic migraine by mediating the activation of microglia.PART Ⅲ S1PR1 IS INVOLVED IN CENTRAL SENSITIZATION OF CHRONIC MIGRAINE VIA STAT3 SIGNALING PATHWAYObjectiveSignal transducers and activators of transcription 3(STAT3)are important transcription factors involved in cell growth,proliferation,differentiation and immune regulation.In CNS disease models,the STAT3 signaling pathway is involved in the regulation of microglial activation and neuroinflammation.S1PR1 is a G protein-coupled receptor that activates the STAT3 signaling pathway by activating tyrosine kinases and serine/threonine kinases.Therefore,in this part of the study,we explored whether the STAT3 signaling pathway mediates microglial activation as a downstream pathway of S1PR1 and participates in central sensitization in chronic migraine.Methods1.BV-2 microglia were cultured in vitro,and BV-2 cells were stimulated with LPS(0.1ug/ul)to observe the activation of STAT3 signaling pathway after LPS stimulation at different time points in the inflammation model.BV-2 microglia were pretreated with S1PR1 receptor antagonist to observe the expression changes of STAT3 pathway.2.To establish a chronic migraine mouse model stimulated by repeated NTG,to observe the changes of STAT3 expression in the mouse TCC by immunoblotting,and to further observe the co-localization of phosphorylated STAT3 and microglia by immunofluorescence staining.The effect of S1PR1 receptor antagonist on STAT3 in mouse TCC was observed by western blotting.3.Mice were given AG490(20mg/kg,i.p),a STAT3 signaling pathway inhibitor,before NTG injection to further explore whether the STAT3 signaling pathway was involved in the central sensitization of chronic migraine.An equal volume of solvent was used as a control(Vehicle,VEH).Before administration and 2 hours after NTG injection,the changes of mechanical pain threshold and thermal pain threshold in the periorbital and hindpaw of mice were detected to reflect the acute and preventive treatment effects of AG490 on chronic migraine.At the same time,immunoblotting and immunofluorescence staining were used to further observe the effect of AG490 on the expression of CGRP and c-fos in TCC.Finally,the effect of AG490 on the microglial M1 activation marker i NOS was observed by immunoblotting and immunofluorescence staining.Results(1)Regulation of S1PR1 on STAT3 signaling pathway in microglia1.The results of the in vitro study showed that with the increase of LPS stimulation time,the expression of p-STAT3 protein in BV-2 microglia gradually increased,and the most obvious after 12 hours of LPS stimulation(p<0.05).After pretreatment with W146 and FTY720,LPS-induced p-STAT3 was significantly reduced,and the total amount of STAT3 was not changed significantly.2.In the NTG-induced CM mouse model,the results of immunoblotting showed that the expression of p STAT3 was significantly up-regulated in the TCC site(p<0.001),and the expression of p STAT3 in microglia was found by immunofluorescence staining.The trend of up-regulation of p-STAT3 expression at TCC sites decreased after administration of S1PR1 antagonist in mice.(2)STAT3 signaling pathway is involved in the central sensitization of chronic migraine(1)Behavioral results showed that AG490 could significantly alleviate the basic mechanical and thermal hyperalgesia induced by repeated NTG administration.At the same time,the acute hyperalgesia produced after each NTG injection was significantly relieved(p<0.05).(2)The results of western blotting and immunofluorescence staining showed that AG490 could reduce the up-regulation of CGRP and c-fos in the TCC of mice(p<0.001).In addition,AG490 also reduced NTG-induced expression of the microglial M1-type activation marker i NOS.ConclusionThe results of this part of the study indicate that S1PR1 regulates the activation of STAT3 signaling pathway.Inhibition of STAT3 signaling pathway can significantly alleviate the NTG-induced decrease in acute and basal pain thresholds in mice,and can reduce the release of CGRP and the activation of c-fos at the TCC site after NTG administration.The STAT3 signaling pathway is also expected to become a clinical therapeutic target for chronic migraine.