Effects Of Estrogen On A Model Of Rat Migraine By Regulating Nrf2/ARE Pathway

Migraine is a chronic neurovascular disease that can be recurrent,and it's onset may be related to the hypersensitivity of nerve and vascular.Oxidative stress affects the hypersensitivity of the central nervous system,and estrogen may play an important role in migraine attacks.The effects of estrogen on the Nrf2/ARE pathway in migraine patients have not been reported.This study investigated the effects of estrogen on Nrf2/ARE pathway of periaqueductal gray in the chronic migraine model.Part I The metabolism of estrogen in ratsObjective:To study the serum estradiol level in physiological state in rats and after supplement of exogenous estradiol in ovariectomized rats.Methods:We used enzyme linked immunosorbent assay?ELISA?to determine the level of estradiol in the serum collected from different stages of female rats,and after exogenous estradiol supplement in ovariectomized rats.Results:The level of estradiol in rat serum fluctuated from 24.52±14.87pg/mL to 112.03±27.11 pg/mL in the physiological state.A dose of 5?g/kg,20?g/kg or 100?g/kg per body weight estradiol administered subcutaneously resulted in a estradiol peak level of 106.10±23.26 pg/mL,399.40±23.16pg/mL and 2231.72±106.25 pg/mL respectively.Conclusions:A dose of 5?g/kg per body weight estradiol administration resulted in a estradiol peak level close to the physiological level.While 20?g/kg or 100?g/kg estradiol administration resulted in a higher peak level.Part II Establishment and evaluation of chronic migraine model in ratsObjective:To evaluate the chronic migraine rat model.Methods:We established the model by using inflammatory material to repeatedly stimulate the dura mater of ovariectomized female rats.And evaluated the model by behavioral indicators and the expression of CGRP and c-fos in PAG and TG.Results:The periorbital pain threshold of model group was significantly lower than that in the control group?P<0.01?,and the immunohistochemistry showed a significant increase in c-fos and CGRP expressions in the PAG and TG regions,with statistically significant differences?P<0.05?.Conclusions:This model is an ideal choice of chronic migraine in rats for it has high success rate and good model effect.Part III The effects of estrogen on Nrf2/ARE pathway in migraine modelObjective:To explore the relationship among estrogen,oxidative stress and migraine by studing the Nrf2/ARE signaling pathway in PAG reagon.Methods:We randomly divided the ovariectomied female rats into four groups:Vehicle group,inflammatory soup?IS?group,low dose estradiol group?IS+LE₂,5?g/kg?,and high dose estrogen group?IS+HE₂,100?g/kg?.Both inflammatory soup and estradiol or their solvent were injected to the rats per day for 14 days.Chemistry method,immunohistochemistry,Western blot,real-time polymerase chain reaction?RT-PCR?methods were applied to study the effects of estradiol on the expression of Malondialdehyde?MDA?,Superoxide Dismutase?SOD?,Nrf2 and HO-1 in rat periaqueductal gray,and on the level of headache.Results:The peri-orbit pain threshold of rats in the IS group was significantly lower than that of the control group and the vehicle group?P<0.01?.And we found that the c-fos and calcitonin gene-related peptide?CGRP?positive cells increased obviously in the PAG and trigeminal ganglion?TG?areas by immunohistochemistry method,the difference was statistically significant?P<0.05?.After the rats were given different doses of estradiol,the periorbital pain threshold gradually increased than the IS group.The expression of SOD,Nrf2 and HO-1 were all on the rise in the PAG area,but the expression of MDA,c-fos and CGRP decreased compared with the model group.Conclusions:1.Chronic migraine is associated with the activation of Nrf2/ARE pathway;2.Estradiol can exert anti-oxidative stress by increasing the expression of antioxidant proteins such as SOD,Nrf2 and HO-1;3.After estradiol intervention,the rats had a different degree of headache;4.Estradiol may affect chronic migraine by activating the Nrf2/ARE pathway.