Study Of The Mechanism Of5-HT_1B/1D Agonist On Migraine Therapeutic Effect

Background and purpose5-HT_1B/1Dagonist （Triptans） is based on theneurovascular theory of migraine and represent a specific medicine for migrainetreatment.5-HT_1B/1Dagonist can inhibits vasodilation of intracranial vessels byactivate the5-HT1Band5-HT1Dserotonin receptors,which are present on meningealvessels. Rizatriptan as positive control in an anti-migraine drugs research,in view of5-HT_1B/1Dagonist play the satisfactory therapeutic effect on migraine.The resultsshowed that rizatriptan significantly reduced expression of the mRNAs forproenkephalin and substance P, which are important mediators in the painmodulatory system, and they participate in central analgesia. That stimulate ourresearch interest on the mechanism of5-HT_1B/1Dagonist on migraine. Results fromsome studies have also demonstrated that5-HT_1B/1Dagonist block neurogenicinflammation and central transmission of nociceptive stimuli.However, the effects of5-HT_1B/1Dagonist on the cytokines in peripheral blood and trigeminal gangliafunction remain little studied,and its on the endogenous pain modulatory systemremain poorly understood.The present study utilized in vivo and in vitro experimentsto assess the influence of rizatriptan on the cytokines in peripheral blood,pain-relatedneuropeptide in trigeminal ganglia and functions of endogenous pain modulatorysystem to investigate the possible mechanism by which5-HT_1B/1Dagonist treatmigraine.Methods1. In vivo（1） Experimental animals,Migraine model establishment and interventions,Behavioral observation:A total of48rats were randomly divided into four groups （n=12）:normal control groups（A）,migraine model groups（B）,Rizatriptan control groups（C） and Rizatriptan treatment groups（D）.Rizatriptan control and treatment groups wereintragastrically perfused with rizatriptan,1mg/kg per day （according to the adultdaily dose）, and normal control and migraine model groups were perfused withnormal saline1mL per day. After7days, nitroglycerin （10mg/kg） wassubcutaneously injected into the buttocks of the rizatriptan treatment and migrainemodel groups to induce migraine. Normal saline （2mL/kg） was injected into thenormal control and rizatriptan control groups.At60–90minutes followingnitroglycerin injection, the total number of behavioral symptoms was measured （eachsymptom was scored with1point）.Behavioral symptoms include: frequent scratchingof the head with forepaws, frequent cage-climbing, tail biting, and to-and-fro motion.（2） Plasma CGRP、5-HT、 IL-1β and TNF-α content: Two hours afternitroglycerin injection, Approximately5mL of venous blood was harvested fromeach group. Plasma CGRP、5-HT、IL-1β and TNF-α content were determined usingELISA according to the manufacturer’s instructions.（3） Detection of PENK、SP、CGRP and CCK mRNA expression in trigeminalganglia of rats in each group: Two hours after nitroglycerin injection, rats wereanesthetized and then sacrificed. The trigeminal ganglia were isolated. PENK、SP、CGRP and CCK mRNA expression in trigeminal ganglia were determined usingSYBR Green I real-time quantitative PCR.（4） Detection of ENK、SP、CGRP and CCK expression in midbrain tissues of ratsin each group: Two hours after nitroglycerin injection, rats were anesthetized and thensacrificed. The midbrains were isolated. PENK、SP、CGRP and CCK mRNAexpression in midbrain tissues were determined using SYBR Green I real-timequantitative PCR. M-ENK、L-ENK、SP、CGRP and CCK-8expression in PAG weredetermined using Immunohistochemistry.2. In vitro（1） Trigeminal ganglion neurons culture:Ganglia isolated from3-to5-d-oldSprague Dawley rats。（2） Heat stress:Trigeminal ganglion neurons were cultured in standard incubatorfor7d, cultured for30min at38.5℃.Rizatriptan interventions:Trigeminal ganglion neurons were cultured in standardincubator for7d,20μL rizatriptan（1mmol/L） were added,the culture time was1h. （3） A total of24culture wells were randomly divided into four groups （n=6）:Normal control groups（A）,Heat stress groups（B）,Rizatriptan control groups（C） andRizatriptan+Heat stress groups（D）. Normal control groups（A）:Trigeminal ganglionneurons were cultured in standard incubator for7d. Heat stress groups（B）:Trigeminalganglion neurons were cultured in standard incubator for7d, cultured for30min at38.5℃.Rizatriptan control groups（C）:Trigeminal ganglion neurons were cultured instandard incubator for7d,20μL rizatriptan（1mmol/L） were added,the culture timewas1h. Rizatriptan+Heat stress groups（D）:Trigeminal ganglion neurons were culturedin standard incubator for7d,20μL rizatriptan（1mmol/L） were added,the culture timewas1h, cultured for30min at38.5℃.（4） PENK、SP、CGRP and CCK mRNA expression in Trigeminal ganglionneurons were determined using SYBR Green I real-time quantitative PCR.Results1. In vivo（1） Behavioral changes in experimental rats:Nitroglycerin injection resulted insignificantly increased rat behavioral scores, characterized by frequent scratching ofthe head with forepaws, frequent cage climbing, tail biting, and a to-and-fro motion.When a symptom appeared once, it was scored with1point. Higher scoresrepresented more severe headaches. Results demonstrated significantly increasedbehavioral scores in the migraine model group （P <0.05）. Compared with themigraine model group, behavioral scores were significantly lower in the rizatriptantreatment group （P <0.05）.（2） Plasma CGRP、5-HT、IL-1β and TNF-α content:①CGRP content wassignificantly higher in plasma of rats in the migraine model group compared with thenormal control groups（P <0.05）. No significant difference in CGRP content in the ratplasma between the both rizatriptan groups and the normal control groups （P>0.05）.No significant difference in CGRP content in the rat plasma between the bothrizatriptan groups and migraine model groups （P>0.05）.②Compared with theRizatriptan control groups,5-HT content in the rat plasma were significantly lower inthe normal control groups and migraine model groups （P <0.05）. Compared with the Rizatriptan treatment groups,5-HT content in the rat plasma were significantly lowerin the migraine model groups （P <0.05）.③IL-1β content in the rat plasma weresimilar among groups （P>0.05）.④TNF-α content in the rat plasma were similaramong groups （P>0.05）.（3） PENK、SP、CGRP and CCK mRNA expression in trigeminal ganglia of ratsin each group:①Compared with the normal control groups and Rizatriptan controlgroups,PENK mRNA expression in trigeminal ganglia of rats were significantlylower in the migraine model groups and Rizatriptan treatment groups（P <0.05）.PENK mRNA expression was significantly higher in trigeminal ganglia of rats in theRizatriptan control groups compared with the normal control groups（P <0.05）. PENKmRNA expression was significantly higher in trigeminal ganglia of rats in theRizatriptan treatment groups compared with the migraine model groups（P <0.05）.②SP mRNA expression was significantly higher in trigeminal ganglia of rats in theRizatriptan control groups compared with the normal control groups and migrainemodel groups（P <0.05）. SP mRNA expression was significantly lower in trigeminalganglia of rats in the Rizatriptan treatment groups compared with the Rizatriptancontrol groups（P <0.05）.③CGRP mRNA expression was significantly higher intrigeminal ganglia of rats in the migraine model groups and Rizatriptan treatmentgroups compared with the normal control groups and Rizatriptan control groups（P <0.05）.④CCK mRNA expression in trigeminal ganglia of rats were similar amonggroups （P>0.05）.（4） ENK、SP、CGRP and CCK expression in midbrain tissues of rats in each group:①Real-time quantitative PCR results revealed PENK mRNA expression wassignificantly lower in midbrains of rats in the Rizatriptan control groups andRizatriptan treatment groups compared with the normal control groups and migrainemodel groups（P <0.05）. Immunohistochemistry results revealed significantly moreM-ENK-positive cells at the trochlear nucleus level in the rat periaqueductal graymatter in the migraine model groups compared with the normal control groups （P <0.05）. Significantly more M-ENK-positive cells at the trochlear nucleus level in therat periaqueductal gray matter in the Rizatriptan control groups compared with the normal control groups （P <0.05）.②Real-time quantitative PCR results revealed SPmRNA expression was significantly lower in midbrains of rats in the migraine modelgroups compared with the normal control groups（P <0.05）. SP mRNA expressionwas significantly lower in midbrains of rats in the Rizatriptan control groups andRizatriptan treatment groups compared with the normal control groups and migrainemodel groups（P <0.05）. Immunohistochemistry results revealed significantly moreSP-positive cells at the trochlear nucleus level in the rat periaqueductal gray matter inthe normal control groups compared with the Rizatriptan control groups（P <0.05）.Significantly more SP-positive cells at the trochlear nucleus level in the ratperiaqueductal gray matter in the migraine model groups compared with theRizatriptan treatment groups （P <0.05）.③Real-time quantitative PCR resultsrevealed CGRP mRNA expression was significantly lower in midbrains of rats in theRizatriptan treatment groups compared with the migraine model groups（P <0.05）.Immunohistochemistry results revealed significantly more CGRP-positive cells at thetrochlear nucleus level in the rat periaqueductal gray matter in the migraine modelgroups compared with the Rizatriptan control groups and Rizatriptan treatmentgroups（P <0.05）.④Real-time quantitative PCR results revealed CCK mRNAexpression was significantly lower in midbrains of rats in the Rizatriptan controlgroups and Rizatriptan treatment groups compared with the normal control groups andmigraine model groups（P <0.05）. Immunohistochemistry results revealedsignificantly more CCK-8-positive cells at the trochlear nucleus level in the ratperiaqueductal gray matter in the normal control groups compared with theRizatriptan control groups（P <0.05）. Significantly more CCK-8-positive cells at thetrochlear nucleus level in the rat periaqueductal gray matter in the migraine modelgroups compared with the Rizatriptan treatment groups（P <0.05）.2. In vitro（1） Compared with the Normal control groups, CGRP mRNA expression intrigeminal ganglion neurons cultured were significantly higher in the Heat stressgroups（P <0.05）. Compared with the Normal control groups, CGRP mRNAexpression in trigeminal ganglion neurons cultured were significantly lower in the Rizatriptan control groups（P <0.05）. Compared with the Heat stress groups, CGRPmRNA expression in trigeminal ganglion neurons cultured were significantly lower inthe Rizatriptan+Heat stress groups（P <0.05）.（2） Compared with the Normal control groups, PENK mRNA expression intrigeminal ganglion neurons cultured were significantly lower in the Heat stressgroups（P <0.05）. Compared with the Normal control groups and Heat stress groups,PENK mRNA expression in trigeminal ganglion neurons cultured were significantlylower in the Rizatriptan control groups and Rizatriptan+Heat stress groups（P <0.05）.（3） Compared with the Normal control groups, SP mRNA expression intrigeminal ganglion neurons cultured were significantly lower in the Heat stressgroups（P <0.05）. Compared with the Normal control groups and Heat stress groups,SP mRNA expression in trigeminal ganglion neurons cultured were significantlylower in the Rizatriptan control groups and Rizatriptan+Heat stress groups（P <0.05）.（4） CCK mRNA expression in trigeminal ganglia neurons cultured were similaramong groups （P>0.05）.Conclusions1.5-HT content decreases in the plasma of the nitroglycerin-induced migrainerats.5-HT_1B/1Dagonist can restore the5-HT content in migraine rats plasma andinfluence the metabolic state of the5-HT system,2. CGRP expression increases in the trigeminal ganglia of the migraine rats.5-HT_1B/1Dagonist can decrease the expression of CGRP in the trigeminal ganglia ofthe migraine rats and exhibits neurogenic inflammation triggered by CGRP.3. PENK expression decreases in the trigeminal ganglia of the migraine rats.5-HT_1B/1Dagonist can increase the expression of PENK in the trigeminal ganglia ofthe migraine rats and play analgesic effect.4.5-HT_1B/1Dagonist can increase the expression of M-ENK in theperiaqueductal gray matter of the migraine rats and play analgesic effect.5.5-HT_1B/1Dagonist can exhibit the expression of CGRP in the periaqueductalgray matter of the migraine rats. Mitigate the CGRP-induced migrainepathophysiological changs and weaken the CGRP-induced inhibition of the analgesic effects of opioid peptides.6.5-HT_1B/1Dagonist can exhibit the expression of CCK-8in the periaqueductalgray matter of the migraine rats. weaken the CCK-induced inhibition of the analgesiceffects of opioid peptides.