Study On The Mechanism Involving CB1 Receptor In Electroacupuncture Preconditioning For The Prophylaxis Of Migraine

Objective:To investigate the molecular biology basis on electroacupuncture pretreatment for migraine prophylaxis and to study the mechanism of electroacupuncture pre-disposal for migraine prophylaxis by assaying the concentrations of PGE2 and CGRP in serum, the expression of CB1 receptor, C-fos, COX-2, CGRP in the trigeminal ganglion or trigeminal nucleus caudalis, and dural plasma protein extravasation in animal models of migraine.Methods:Sprague-Dawley rats were randomly divided into four groups:sham operation group (A) (n=8), model group(B) (n=8), electroacupuncture pretreatment+model group(C) (n=8), rimonabant antagonist+electroacupuncture pretreatment+model group(D) (n=8). The animal model of rats with migrane was established by right electrical stimulation of the trigeminal ganglion. The electroacupuncture pretreatment at acupoints"fengchi" and "waiguan" was used as the test factor. Immunohistochemistry was conducted to determine the protein expression of CB1 receptor and C-fos in trigeminal nucleus caudalis and protein expression of CB1 receptor, COX-2 and CGRP in right trigeminal ganglion. Western-blot and Real-Time PCR were applied to demonstrate protein and mRNA expression of CB1 receptor, COX-2 and CGRP in right trigeminal ganglion. Concentrations of PGE2 and CGRP in serum were measured by enzyme-linked immunosorbent assay. Plasma protein extravasation in the dura matter on both sides was photographed using an inverted phase contrast microscope and the average optical density was determined by a software program, then dural plasma protein extravasation ratio (stimulated side/contralateral side) was calculated.Results:1.Effects of electroacupuncture pretreatment on protein expression of CB1 receptor and C-fos in trigeminal nucleus caudalis of migraine rats:CB1 receptor protein expression of model group, electroacupuncture pretreatment+model group and rimonabant antagonist+electroacupuncture pretreatment+model group were significantly upregulated compared with sham operation group(p<0.05); CB1 receptor protein expression of electroacupuncture pretreatment+model group and rimonabant antagonist+electroacupuncture pretreatment+model group were significantly upregulated compared with model group(p<0.05); electroacupuncture pretreatment+model group had no significant difference in protein expression of CB1 receptor compared with rimonabant antagonist+electroacupuncture pretreatment+model group (P>0.05). C-fos protein expression of model group, electroacupuncture pretreatment+model group and rimonabant antagonist +electroacupuncture pretreatment+model group were significantly upregulated compared with sham operation group(p<0.05); C-fos protein expression of electroacupuncture pretreatment+model group and rimonabant antagonist +electroacupuncture pretreatment+model group were significantly downregulated compared with model group(p<0.05); C-fos protein expression of electroacupuncture pretreatment+model group was significantly downregulated compared with rimonabant antagonist+electroacupuncture pretreatment+model group (p<0.05).2.Effects of electroacupuncture pretreatment on expression of CB1 receptor, COX-2 and CGRP in right trigeminal ganglion of migraine rats:CB1 receptor protein and mRNA expression of model group, electroacupuncture pretreatment +model group and rimonabant antagonist+electroacupuncture pretreatment+model group were significantly upregulated compared with sham operation group(p<0.05); CB1 receptor protein and mRNA expression of electroacupuncture pretreatment+model group and rimonabant antagonist +electroacupuncture pretreatment+model group were significantly upregulated compared with model group(p<0.05); electroacupuncture pretreatment+model group had no significant difference in protein and mRNA expression of CB1 receptor compared with rimonabant antagonist+electroacupuncture pretreatment+model group (P>0.05). COX-2 and CGRP protein and mRNA expression of model group, electroacupuncture pretreatment+model group and rimonabant antagonist +electroacupuncture pretreatment+model group were remarkably upregulated compared with sham operation group(p<0.05); COX-2 and CGRP protein and mRNA expression of electroacupuncture pretreatment +model group and rimonabant antagonist+electroacupuncture pretreatment+model group were remarkably downregulated compared with model group(p<0.05); COX-2 and CGRP protein and mRNA expression of electroacupuncture pretreatment+model group were significantly downregulated compared with rimonabant antagonist+electroacupuncture pretreatment+model group (p<0.05).3.Effects of electroacupuncture pretreatment on levels of PGE2 and CGRP in external jugular vein blood of migraine rats:The contents of PGE2 and CGRP of model group, electroacupuncture pretreatment+model group and rimonabant antagonist+electroacupuncture pretreatment+model group were remarkably augmented compared with sham operation group(p<0.05); The concentrations of PGE2 and CGRP of electroacupuncture pretreatment+model group and rimonabant antagonist+electroacupuncture pretreatment+model group were remarkably reduced compared with model group(p<0.05); The concentrations of PGE2 and CGRP of electroacupuncture pretreatment+model group were significantly reduced compared with rimonabant antagonist+electroacupuncture pretreatment+model group (p<0.05).4.Effect of electroacupuncture pretreatment on dural plasma protein extravasation of migraine rats:dural plasma protein extravasation ratio of model group, electroacupuncture pretreatment+model group and rimonabant antagonist +electroacupuncture pretreatment+model group were remarkably increaseded compared with sham operation group(p<0.05); dural plasma protein extravasation ratio of electroacupuncture pretreatment+model group and rimonabant antagonist +electroacupuncture pretreatment+model group were remarkably decreaseded compared with model group(p<0.05); dural plasma protein extravasation ratio of electroacupuncture pretreatment+model group was significantly decreaseded compared with rimonabant antagonist+electroacupuncture pretreatment+model group (p<0.05).Conclusions:1.Electroacupuncture pretreatment downregulates C-fos protein expression by upregulating CB1 receptor protein expression in trigeminal nucleus caudalis of migraine rats.2.Electroacupuncture pretreatment downregulates COX-2/CGRP expression reduces PGE2/CGRP concentrations and dural plasma protein extravasation ratio by inducing CB1 receptor expression.3.Electroacupuncture pretreatment downregulates C-fos expression, COX-2/CGRP expression, reduces PGE2/CGRP concentrations and dural plasma protein extravasation ratio via inducing CB1 receptor expression, which may be one of mechanism of migrane prophylaxis.