Nitrogen Dioxide Inhalation Contributes To The Development Of Migraine In SD Rat And Its Molecular Mechanism

Objective:By constructing a NO₂-exposed rat model and a nitroglycerin-type migraine rat model,we explored the correlation between NO₂ exposure and migraine,and further explored the molecular mechanism of action between NO₂ exposure and migraine occurrence by in vivo and in vitro experiments.Methods:To explore the relationship between NO₂ and migraine,50 rats were randomly divided into 5 groups of 10 rats each,which were the control group,NTG group（nitroglycerin-type migraine animal model group）and different doses of NO₂ group,in which NO₂ exposure was carried out by NO₂ whole-body dynamic dyeing cabinet for whole-body exposure,and the differences in behavioral and biological indexes of rats in each group were examined after different treatments respectively.To explore the effect of Transient Receptor Potential Vanilloid Receptor 1（TRPV1）on NO₂-induced migraine,40rats were randomly divided into 4 groups of 10 each and then intervened with daily intraperitoneal injections of TRPV1 receptor antagonist（capsazepine,CZP）starting on day 8 of NO₂ exposure,and differences in behavioral and biological indicators were examined in each group.To further explore the molecular mechanism of action between NO₂exposure and migraine onset,the levels of NO₂^-/NO₃^-,an in vivo derivative of NO₂,were measured by treating primary trigeminal neuron cells with NO₂^-/NO₃^-,CZP,TRPV1 receptor agonist（capsaicin,Cap）,VE and H₂O₂ for 24h,respectively,to detect changes in biological indicators associated with migraine.Behavioral indicators in rats were compared with mechanical pain thresholds,RGS and behavioral scores.ELISA was used to determine biological indicators of ROS,GSH,IL-1β,TNF-α,SP and CGRP in peripheral plasma and midbrain,SP and CGRP in trigeminal ganglion and IL-1β,TNF-α,SP and CGRP in cells.Western bolt was used to detect TRPV1 and c-Fos proteins in midbrain,trigeminal ganglion and cells expression.Cellular reactive oxygen levels reactive oxygen species were detected by the ROS assay kit.Results:Exploring the relationship between NO₂ and migraine,medium and high dose NO₂exposure resulted in lower mechanical pain thresholds and higher RGS and behavioral scores in rats compared with blank controls,with statistically significant differences（P<0.05）,higher ROS（P<0.05）and lower GSH（P<0.05）,higher IL-1βand TNF-α（P<0.05）,higher CGRP and SP（P<0.05）and increased TRPV1 and c-Fos protein expression（P<0.05）.To explore the effect of TRPV1 on NO₂-induced migraine,alleviate behavioral changes in migraine thresholds,RGS and behavioral scores induced by NO₂（P<0.05）and changes in oxidative,inflammatory and pain-related neuropeptide levels with statistically significant differences（P<0.05）.Treatment of primary trigeminal neuron cells with in vivo derivatives of NO₂ showed the same changes in oxidative,inflammatory and pain-related neuropeptide levels indicators as in NO₂-exposed rats,and the differences were statistically significant（P<0.05）Changes in oxidative,inflammatory and pain-related neuropeptide levels indicators caused by NO₂ in vivo derivative exposure were alleviated by the use of CZP and VE,respectively,with statistically significant differences（P<0.05）.Conclusions:（1）NO₂ exposure leads to migraine in rats.（2）NO₂ exposure positively regulates TRPV1 receptor and promotes the release of pain related neuropeptides,resulting in migraine;（3）NO₂can induce migraine by activating ROS-TRPV1 pathway.