Effect Of Electroacupuncture At Fengchi On The Expression Of CGRP In TGVS Of Migraine Rat

OBJECTIVEMigraine is an episodic neurological disorder,affecting about 15%population worldwide.Its attack manifests as unilateral and throbbing headache,sometimes accompanied with gastrointestinal disorder and sensitive to light and voice,serious attack can disable the patient.However,the current interventions can not cure migraine,a lot of additional research are needed to explore the pathophysiology of migraine.According to the present theory,activation and sensitization of the trigeminal vascular system(TGVS)pathway is thought to be responsible for migraine pain.Based on the role of 5-HT involved in TGVS,triptans were developed.In recent years,the role of calcitonin gene related peptide(CGRP)in activation and sensitization of TGVS has been confirmed.It has become a new therapy target for migraine treatment and the focus of development of new anti-migraine drugs in recent years.Acupuncture is a commonly analgesic intervention for pain,which has shown the effect of anti-migraine.However,the mechanism of acupuncture treatment of migraine is unclear.This study was to investigate the effect of EA on the CGRP level in TGVS ascending pathway.In order to explore the mechanism of anti-migraine effect of EA at GB20,the levels of CGRP protein and CGRP positive cells in TGVS neurons were detected by western blot and immunofluorescence and the expression of CGRP receptor-related signaling protein(pPKA and pCREB)was detected by WB.METHODS1 Grouping:male SD rats were randomly divided into 4 groups:control group(C),model group(M),electroacupuncture at GB20 group(EA);and electroacupuncture at non-acupoint group(NA).2 Modeling:In this study,a conscious rat model of migraine is used by stimulating dura mater around the superior sagittal sinus(SSS)electrically.The rats were anesthetized with pentobarbitone sodiumin and fixed in the brain stereotaxic.After exposing the skull,two cranial holes were drilled in the middle line of the skull(the anterior one is located 4 mm anterior to the bregma,the other was 6 mm posterior to the bregma);a pair of electrodes were installed in the cranial holes and fixed by screws.After a recovery period of 7 days,the electrodes were connected by stimulator which outputs the stimuli.3 Intervention methods:Control group(C),which only received the electrode implantation surgery;Model group(M),which received electrical stimulation of the SSS;Electroacupuncture at GB20 group(EA),which received EA at GB20(Fengchi)after electrical stimulation of the SSS;and Electroacupuncture at non-acupoint group(NA),which obtained electroacupuncture at a non-acupuncture point after electrical stimulation of the SSS.4 The detection of indicators and methods:An electronic von-frey anesthesiometer was used to measure the withdrawal thresholds in face and hind paw.The CGRP level in the jugular vein was detected by radioimmunoassay.The expression of CGRP was detected by WB and immunofluorescence.The downstream signaling pathway protein,pPKA and pCREB,was detected by WB.Results1 Behaviour test:Facial withdrawal threshold:the withdrawal thresholds of M were significantly lower than the C group(p<0.001);the NA group and the M group had no significant difference in the withdrawal thresholds(p>0.05).The threshold of EA group was significantly higher than the M group(p<0.001).Hind paw withdrawal threshold:the withdrawal threshold of M group were significantly lower than the C group(p<0.001);the NA group and the M group had no significant difference in the withdrawal thresholds(p>0.05).The threshold of EA group was significantly higher than the M group(p<0.05).2 CGRP level in the jugular vein:the M group was significantly higher than the C group(p<0.001).Compared with the M group,the EA group was significantly lower than the M group(p<0.001).There was no significant difference between the NA group and the M group(p>0.05).3 CGRP level in TGVS neurons:WB:Compared with the C group,the levels of CGRP protein in TG/TNC/VPM of the M group was significantly higher(p<0.001).Compared with the M group,the EA group had a significant lower level of CGRP protein in the TG/TNC/VPM group(p<0.05).The M group and the NA group had no significant difference(p>0.05).Immunofluorescence:Compared with the C group,the number of CGRP-positive cells in TG of the M group was significantly higher(p<0.01).Compared with the M group,the electroacupuncture at GB20 significantly decreased the number of CGRP-positive cells in TG(p<0.01).4 pPKA and pCREB:the M group had significantly higher phosphorylation level of PKA and CREB in TG than the C group(p<0.05),and the electroacupuncture at GB20 significantly reduced the increased phosphorylation of TG CREB after electrical stimulation(p<0.05),the NA group and the M group had no significant difference(p>0.05).CONLUSION1 Electroacupuncture at GB20 can effectively reverse the reduction of withdrawal thresholds after electrical stimulation;2 Electroacupuncture at GB20 can significantly reduce the CGRP levels in TGVS neurons and inhibit the expression of CGRP in TG;3 Electroacupuncture at GB20 can inhibit the phosphorylation of CREB(TG)in the TGVS neurons.4 Electroacupuncture at GB20 reduces the level of CGRP in TGVS neurons and inhibits the phosphorylation of CREB(TG),which may be one of the mechanisms of action of electroacupuncture in the treatment of migraine.