Analgesic Actions And Mechanisms Of Zhengtian Pill Via Modulating Nociceptive Hypersensitivity In A Rat Animal Model Of Migraine

Objective:To explore the effects and mechanisms of Zhengtian Pill?ZTP?on migraine in rat model induced by nitroglycerin via assessing the analgesic actions and influence on nociceptive sensitization.Methods:1 Analgesic effects of ZTP.The hot plate test and acetic acid-induced writhing test were performed to assess the effects of ZTP on nociceptive response to thermal and chemical stimuli.Formalin test were used to assess the effects of ZTP on the biphasic nociceptive responses.After ZTP administration for 15 days,the nitroglycerin-induced rat model of migraine was utilized to observe the behavioral effects of ZTP.2 Influence of ZTP on nociceptive mediators.After the formalin test,all mice were sacrificed and serum and brain samples were collected.The levels of serum prostaglandin E2?PGE2?and nitric oxide?NO?,cerebral glutamate?Glu?and calcitonin gene-related peptide?CGRP?in mice were measured by Ultraviolet spectrophotometry,Nitrate reductase approach and ELISA kits,respectively.3 Influence of ZTP on neurotransmitters in brain of migraine rats.The brain tissues were dissected at150min after nitroglycerin injection and homogenized.The contents of glutamate?Glu?,?-aminobutyric acid?GABA?,norepinephrine?NE?,serotonin?5-HT?,and acetylcholine?Ach?in rat brain were measured by ultra-performance liquid chromatography–tandem mass spectrometry?UPLC-MS?.4 Influence of ZTP on the spectrum of amino acids in brain of migraine rats.The levels of various amino acids in rat brain,including aspartate?Asp?,serine?Ser?,glycine?Gly?,histidine?His?,arginine?Arg?,threonine?Thr?,alanine?Ala?,cysteine?Cys?,tyrosine?Tyr?,valine?Val?,and methionine?Met?,were assayed by high-performance liquid chromatography with fluorescence detection?HPLC-FLD?5 Influence of ZTP on the c-fos protein expression in brain of migraine rats.The expression of c-fos protein in brainstem and brain of migraine rats was analysed by immunohistochemistry and western blotting,respectively.Results:1 Analgesic effects of ZTP.When high-and middle-dose groups were compared with CMC control group,there were significant increases in the latency of nociceptive response to thermal stimuli at 60,90,and 120min after ZTP administration?P<0.05?.A significant increase of latency time to writhing reaction and a reduction of numbers of writhing were seen in these groups compared with that in CMC group in acetic acid test in mice?P<0.05?.Furthermore,The total time of licking,biting the injected paw in high-and middle-dose groups was?P<0.05?significantly less than that in CMC group in the both phases in formalin test.when compared with the nitroglycerin-treated rats,pre-treatment with high-and middle-dose of ZTP showed a significant increase in the latency of red-ear occurrence and significant decrease in the frequency of head-scratching and climbing the cage?P<0.05?.2 Influence of ZTP on nociceptive mediators.The levels of peripheral serum PGE₂ as well as of cerebral Glu in three doses of ZTP group were makedly lower than that in CMC group in formalin-evoked mouse model?P<0.05?.CGRP contents in middle-and low-dose of ZTP groups were also significantly lower than that in CMC-treated mice?P<0.05?.Whereas,a significant increase of NO concentrations in peripheral serum of mice in these two groups was observed as compared to that in the CMC group.3 Influence of ZTP on neurotransmitters in brain of migraine rats.The levels of NE in high-and low-dose of ZTP group and 5-HT in high-dose group were significantly higher than that in model group?P<0.05?.However,pre-treatment with three doses of ZTP did not produce significant differences in Ach contents compared to the model group?P>0.05?.4 Influence of ZTP on the spectrum of amino acids in brain of migraine rats.Pre-treatment with middle-dose of ZTP produced obvious reduction in brain Glu and Tyr contents when compared with the model rats?P<0.05?.There were marked decreases in Arg contents in brain of rats in three ZTP group compared with that in the model group?P<0.01?.However,the GABA contents in three ZTP group were significantly greater than that in model group?P<0.05?.The Ser contents in high-and low-dose of ZTP group were also greater than that in model group?P<0.05?.NO significant differences were detected in the contents of other amino acids in the ZTP groups as compared to the model group,such as Asp?Gly?His?Ala?Cys?Tyr?Val and Met.5 Influence of ZTP on the c-fos protein expression in brain of migraine rats.The results of immunohistochemistry showed the localization of c-fos immunoreactivity was in the cytoplasm in brainstem neuron cells.The c-fos protein expression level in model group was significantly higher than that in CMC control group?P<0.01?.The c-fos expression level in high and middle dose of ZTP group was significantly lower than that in model group?P<0.01?.Furthermore,the western blotting results showed when compared with the vehicle group,there was a significant increase in the c-fos protein expression in the nitroglycerin-treated rats?P<0.05?.Pre-treatment with three doses of ZTP showed a significant decrease in c-fos protein expression when compared with the migraine model rats?P<0.05?.Conclusions:The results of the present study show that ZTP may inhibit release of peripheral PGE₂ and promote synthesis of peripheral NO.Besides,our data show ZTP can decrease the levels of central Glu,CGRP,Arg,and Tyr.Meanwhile,ZTP may increase the levels of NE,5-HT,Ser,and GABA.All of these caused the downregulation of c-fos protein expression.These findings suggest that ZTP might exert antimigraine effects through analgesia and the reduction of nociceptive sensitization.