Based On The TLR4-MAPK Pathway To Explore The Mechanism Of Compound Lishao Tablets In Improving Headaches In Chronic Migraine Rats

Objective To investigate the pharmacological mechanism of Fu-Fang-Li-Shao Pill in the treatment of chronic migraine with liver-yang hyperactivity by inducing a rat model of chronic migraine by nitroglycerin.Method 60 Male Sprague-Dawley rats were randomly divided into 6 groups: Control group(n=10);Migraine group(n=10);FFLSP-L group(420 ?/?,n=10);FFLSP-M group(840 ?/?,n=10);FFLSP-H group(1680 ?/?,n=10);Flunarizine Hydrochloride group(FH,1 ?/?,n=10).The FFLSP group and the flunarizine hydrochloride group were intragastrically administered according to the corresponding doses.The Control group and the Migraine group were given the same amount of normal saline.Each group were given intragastric administration for 30 days,once a day.Volume is 0.1 m L/kg.On the 18 th,21st,24 th,27th and 30 th day after intragastric administration,the nitroglycerin solution was injected subcutaneously into the neck of rats according to the dosage of 10 mg/kg.Migraine group,FFLSP-L group,FFLSP-M group,FFLSP-H group,and FH group were required to prepare chronic migraine model.Control group were injected with equal volume of normal saline.After the model was established,the number of scratching heads and the duration of the ear redness were recorded to detect the headache relief in rats.Detection of NO production in serum were analyzed by Griess reagent.Detection the expression of iNOS,IL-1?,IL-6,TNF-? mRNA in rat plasma and iNOS,IL-1?,TNF-? mRNA in rat dura mater by qRT-PCR.ELISA detection IL-1?,IL-6,TNF-? of serum and IL-1?,IL-6,TNF-? protein levels of dura mater.Western blot was used to detect the expression of iNOS,CGRP,TLR4 and My D88 in dura mater and the phosphorylation levels of ERK,JNK and p38 protein.Results(1)Behavioral experiments showed that compared with the Control group,the number of chronic migraine rats catches increased significantly(P<0.05),and the duration of the ear redness was significantly prolonged(P<0.05).Compared with the Chronic migraine group,the number of the FFLSP-M ? FFLSP-H rats catches were significantly reduced(P<0.05),and the duration of the ear redness was significantly shortened(P<0.05).(2)qRT-PCR results showed that the expression of plasma inflammatory cytokines iNOS,IL-1?,IL-6,TNF-? mRNA and dural inflammatory factors iNOS,IL-1?,TNF-? mRNA level in chronic migraine rats were significantly increased than the Control group(P<0.05);when compared with the Chronic migraine group,the results of FFLSP-Mand FFLSP-H shows a down-regulate(P<0.05).(3)ELISA results showed that :Compared with the Control group,the levels of serum pro-inflammatory factors IL-1?,IL-6,TNF-? and dural proinflammatory factors IL-1? and TNF-? were significantly increased in the chronic migraine group(P< 0.05);and the levels of serum proinflammatory factors IL-1?,IL-6,TNF-? and dural proinflammatory cytokines IL-1? and TNF-? in the FFLSP-M and FFLSP-H were significantly decreased when compared with chronic migraine rats(P < 0.05).(5)Western blot analysis showed that the iNOS?CGRP?TLR4?My D88?p-ERK1/2?p-JNK1/2 and p-p38 proteins of chronic migraine group were significantly higher than Control group(P<0.05);At the same time,the expression of the above proteins in the FFLSP-M and FFLSP-H were significantly lower than that in the Chronic migraine group(P<0.05).Conclusion FFLSP can inhibit TLR4-MAPK signaling pathway,down-regulate pro-inflammatory factor expression,lighten peripheral blood and dura mater inflammation,and relieve headache.