| BackgroundMigraine is one of the common types of primary headache,it shows recurrent episodes,unilateral or bilateral,moderately severe pulse sampleheadache,often accompanied by nausea,vomiting,sound and light stimulation and daily activities can make the headache aggravated,a small number of patients with visual,feel or motility aura before the onset of headache."Guidelines for the diagnosis and treatment of migraine in China"(2011)reported in migraine prevalence in China women 3.3%?32.6%,0.7%?16.1%for men.Epidemiological survey in 2012 migraine prevalence was 9.3%in our country.High rates of the attack and the healing rate is low,the lack of safe and effective treatment measures,which seriously affect the patient's quality of life.The 2010 global burden of disease investigation will be listed as the world seventh migraine high disabling diseases.Trigeminal neurovascular reflex theory is the study of the pathogenesis of migraine prevailing doctrine,it combined nerves,blood vessels,and neurotransmitters,mainly involves three mechanisms:supply intracranial meningeal blood vessels to dilate,perivascular nerves release of vasoactive substances inhibition caused by neurogenic inflammation and central pain transmission is reduced.Activation and sensitization of the trigeminovascular system is the key to a migraine headache.Expression of immediate early gene c-fos positive is the neuronal activation marker,which is rich in expression in the spinal trigeminal nucleus caudal portion,when the cells are stimulated c-fos nucleus transferred to the cytoplasm and translated into fos protein,to test the fos protein can be indirect response the distribution of the function related cells in the brain.Recent studies have found that extracellular signal-regulated kinase(extracellular signal-egulatedkinase,ERK)regulating the treatment of neuropathic pain a new target,migraine is a chronic neuropathic pain.ERK/MAPK participate in a variety of neural plasticity,such as pain in the generation and maintenance,ERK/MAPK activation for neuropathic pain development and maintenance is necessary.Transient receptor potential vanilloid subtype 1(transient receptor potential vanilloid 1,TRPV1 receptor)and purinergic receptors P2X3 receptors,are non-selective cationchannel,and they highly expressed in both associated with pain,the small diameter sensory neurons in the trigeminal sensory neurons.In recent years,P2X3 receptors and TRPV1 receptors in the pathogenesis of migraine attacks more people's attention,when migraine,trigeminal sensory neurons of P2X3 receptors and TRPV1 receptors expression significantly increased.Western medicine for the treatment of migraine has not been radical curative treatment,only control of acute attacks and prophylaxis.Drug treatment of migraine include acute onset of treatment and prophylactic treatment,including acute exacerbation of therapeutic agents into specific drugs[ergot amine derivatives,triptans,calcitonin gene-related peptide(CGRP)receptor antagonist]and non-specific drugs[nonsteroidal anti-inflammatory drug,Bobby duly class sedatives,opiates,etc.]categories.However,studies show that the triptans and ergot alkaloids adverse drug reactions were more,it can not be used in children and adolescents.Nonsteroidal anti-inflammatory drug adverse reactions and the presence of gastrointestinal bleeding risk.Currently used in the prophylactic treatment of migraine drugs include:?-blockers,calcium channel blockers,antiepileptic agents,antidepressants,nonsteroidal anti-inflammatory drug,also has many adverse effects and contraindications.Migraine belongs to traditional Chinese medicine "headache","head wind","migraine","migraine," etc.According to ancient medical migraine record my supervisor with his clinical experience,and summarized the etiology and pathogenesis of migraine as wind,wet,stasis and asthenia,and in 1985 created the Zhengtian Pills.The prescriptions contains of the ChuanXiong Cha Tiao San,Mahuangfuzixixin soup,Taohongsiwu decoction,Sitengxiaozhen soup,include Ligusticum chuanxiong hort,Uncaria,white peony root,rehmannia,Chinese angelica,peach,red flower,Angelica,ephedra,aconite,Asarum,wind,Rhizoma,Angelica,Millettia and other components of Chinese medicine,for Chuanxiong and Uncaria King as jun medicine,with Shufeng blood,nourishing Pinggan,Tongluozhitong and other effects,can be used in the treatment of exogenous pathogenic wind,blood stasis,deficiency dystrophy,hyperactivity caused by migraine,tension headache,nervous headache,cervical spondylosis headache,premenstrual headache.In the Clinical application of nearly 30 years,the Zhengtian Pills is the National Essential Drug treatment of migraine and has been exported overseas,with annual sales of about 500 million yuan for the country to bring a higher social and economic benefits.Clinical randomized controlled double-blind multi-center study showed Zhengtian Pills can significantly reduce the intensity of migraine attack,extent,reduce attack frequency,shorten the duration of clinical efficacy,patient were well tolerated,and no significant adverse reactions.But the current study of the Zhentian Pills treatment of migraine is still at the clinical efficacy and Simple pharmacodynamic evaluation,and lack of microscopic understanding of biological regulatory networks,especially for migraine pathogenesis of newly discovered lack of research on cytokines.The Zhentian Pills is lack of research in stage,making it difficult to obtain recognition of the international medical community,restricted the internationalization of the Zhentian Pills treatment of migraine.Therefore,we believe that in recent years played an important role in pain transmission in the process of P2X3 receptors,TRPV1 receptor and ERK signaling pathway research to clarify whether positive Pill by affecting their expression and thus exert inhibitory effect on migraine.Tertramethylpyrazine(TMP)is a main alkaloid monomers extracted from Chuanxiong:4-methyl pyrazine which is a typical Calcium channel blocker,through inhibit overexpression of P2X3 receptor and ERK pathway of primary sensory neurons to suppressed a variety of acute or chronic pain of rat plantar acute nociceptive responses,chronic constriction injury and burns.TMP clinical reports about the treatment of migraine is a lot,but experimental studies is so few,and the TMP's specific mechanism of action is unclear.In order to make clear of TMP migraine whether inhibition affects their expression,we detect migraine rat trigeminal sensory neurons of P2X3 receptors,TRPV1 receptor,ERK signaling pathway and c-fos.ObjectiveBy creating a classic representative of migraine animal models,using a variety of experimental techniques,from behavioral,genetic and protein levels observed Zhengtian Pills and Tetramethylpyrazine on experimental migraine rats trigeminal vascular system for c-fos,ERK,TRPV1 P2X3 expression and to provide experimental data for elucidating the analgesic mechanism of Zhengtian Pills and TMP treatment of migraine,and lay a solid scientific theoretical basis for the clinical application of traditional Chinese medicine and modern promote Chinese medicine theory and international,so Zhengtian Pills produces better social and economic benefits.Methods1 Nitroglycerin migraine rat model preparation and conduct observationsSD male rats were randomly divided into:control group,model group,Zhengtian Pills group,TMP group.Zhengtian Pills group were givenl.62 g·kg-1·d-1,Zhengtian Pills group,the control group and model group were given 1 ml·d-1 0.9%sodium chloride solution orally,TMP group were given Ligustrazin Hydrochloride injection diluted with saline by intraperitoneal injection at a dose of 100 mg kg-1·d-1,the rats continuous administration of 7 d.Model group,Zhengtian Pills group,TMP rats after last administration 30 min,subcutaneous injection of 10 mg·kg-1 Nitroglycerin injection,preparation of migraine animal model,the control rats injected subcutaneously 0.5 ml 0.9%sodium chloride solution over the same period.In rats had red ears,forelegs frequent scratching head,climbing cage,irritability and other behavior deemed successful model.OUTCOME MEASURES:(1)after subcutaneous injection of glycerol nitrate ears red emergence time and disappearance time;(2)the start time and end time of scratching after subcutaneous injection of nitroglycerin.More than scratching for 5 consecutive times as a symbol of scratching start time,rats scratching to less than 5 times within 30 min and performance burnout or fatigue as a symbol of the scratching ends.(3)the number of rats climbing cage scratching after subcutaneous injection of nitroglycerin of each period.Duration segmented using a method of counting,as a period of time to observe every 30 minutes,continuing to observe the time after 3h of modeling,in a single period of less than five times of scratching their heads as a symbol of scratching stop time.2 Specimen collection and index detection2.1 ImmunofluorescenceAfter each group of rats modeling 4 h 10%chloral hydrate anesthesia with 4%paraformaldehyde perfusion-fixed,paraffin-embedded sections.Sections were dewaxed and hydrated,heat antigen retrieval,endogenous peroxidase after the fire,5%solution of bovine serum albumin(BSA)blocking solution 15 min to block nonspecific antigen,followed by addition of an anti-4 overnight at ? environment.Dropping fluorescent secondary antibodies,incubated in the dark 1 h,between each of the links are with phosphate buffered saline(PBS)rinsing 5 min × 3 times,plus anti-quencher,neutral gum cementing inverted fluorescence microscope observation,red fluorescent protein particles,the control group with PBS was used as a labeled antibody.2.2 Western blottingThe rats underwent modeling 4 h 10%chloral hydrate anesthesia,rapid ice coverage,-80 ? stored in liquid nitrogen.50 mg of the sample were ground in liquid nitrogen,was added 150?l RIPA lysis buffer and 1.5 ?l phenyl methyl sulfonyl fluoride(as PMSF)were incubated on ice for 30 min,4 ? 1500 r · mim-1 centrifuged 15 min(centrifugal radius 13.5 cm).Supernatant,protein concentration was determined by BCA method.50 ?g of each sample from each by SDS-PAGE electrophoresis,transferred PDVF membrane,blocking with 5%skim milk at room temperature 1 h,TBST washed 3 times,primary antibody was added,incubated overnight at 4 ? incubated with secondary antibody at room temperature 1 h,ECL Chemistry in the IS 2000 MM luminescence imaging system(Kodak)was exposed photograph,Molecular imaging software Version 4.0(Kodak)software analysis strip the gamma value to ?-actin as an internal control,each in triplicate.2.3 RT-PCRThe rats underwent modeling 4 h 10%chloral hydrate anesthesia,rapid ice coverage.In accordance with the kit instructions extraction and cDNA transcription of total RNA of each sample to the housekeeping gene GAPDH as an internal reference.Primers were synthesized by Shanghai Invitrogen Biotechnology Co.,primer sequences are as follows:P2X3:upstream primer:5'-TCTTGAGGGTAGGGGATGTG-3',downstream primer 5'-CACACCCAGCCGATCTTAAT-3';TRPV1:upstream primer:5'-TGACTACCGGTGGTGTTTCA-3 ' upstream and downstream primers:5'-GCTGGGTGGCATGTCTATCT-3';GAPDH:upstream primer:5'-ATTCTCAGCAATGCATC-3',downstream primer 5'-ATGGACTGTGGtcATGAGCC-3'.A total of 25 ?l reaction system:12.5ul SYBR core mix,10.5ul DEPC water,lul cDNA template,0.5ul P2X3/TRPV1 upstream primer,0.5ul P2X3/TRPV1 downstream primer.PCR reaction conditions:95 ?10min,95 ? 10s,60 ? 30s,72 ? reaction 30s,a total of 40 cycles.PCR instrument reading of GAPDH and P2X3 threshold cycle using the relative quantification method(2-??CT)is calculated for each sample P2X3mRNA ACT value.3 Statistical AnalysisUsing SPSS 13.0 software for statistical analysis,measurement data to x ± s,F test first,using the t-test or one-way ANOVA between groups were compared when equal variance assumed,when heterogeneity of variance using Welch to compared between groups.When difference between whole group,using Bonferroni or Dunnett's T 3 for multiple comparison,when P<0.05 as the difference was statistically significant.Results1.After a control group injected with normal saline in addition to start within 30 min scratching and climbing cage slightly conspicuous phenomenon,the rest period showed no abnormal activity,earless red performance.After 4 min of injected subcutaneously nitroglycerin,Model group,Zhengtian Pills group and TMP group were appears red ears,frequent scratching head,climbing cage and other phenomena,this phenomenon in 30?90min after the model reached the peak for about 2 to 3 hours.The start time of scratching and ear red appearance between model group,Zhengtian Pills group and TMP group have no significant difference(P = 0.495,P =0.830).The duration of scratching of model group were longer than Zhengtian Pills group and TMP group(P<0.001),between Zhengtian Pills group and TMP group showed no significant difference(P<0.001).Red ear duration of model group was significantly longer than Zhengtian Pills group and TMP group(P<0.001).The frequency of climbing cage and scratching of Zhengtian Pills group and TMP group in each period of time after administration of 30min was significantly reduced compared with the model group(P<0.001).2.Immunofluorescence results showed that:The rats trigeminal ganglion(TG)nerve to varying degrees C-fos,P2X3,TRPV1 expression element has,in the cytoplasm showed a uniform expression status,no expression in the nucleus,mainly in small diameter neurons.Under normal conditions the control group also a small amount of expression,in model group expression was significantly higher than the control group,the expression intensity of Zhengtian Pills group and TMP group was reduced compared with the model group.3.Immunofluorescence results showed that:The rats spinal tract of trigeminal nerve and spinal trigeminal nucleus caudalis have different levels of C-fos,P2X3,TRPV1 expression,expression intersity of the model group was higher than other groups.4.Western blotting results showed that:The protein expression of ERK,P2X3 and TRPV1 in rats TG and Trige.minal nerve cervical spinal complex(TCC)showed that the model group was significantly higher than in the control group(P<0.05),Zhengtian Pills group and TMP group protein expression was significantly lower than model group(P<0.05).5.RT-PCR results showed that:The TRPV1 mRNA and P2X3 mRNA expression levels in TG and TCC of the model group was significantly increased than the control group(P<0.05),The mRNA expression of Zhengtian Pills group and TMP group was lower than in model group(P<0.05).Conclusion1.Zhengtian Pills and TMP can inhibit the nitroglycerin-induced rat model of migraine behavioral changes can reduce migraine rats climbing cage,scratching their heads,ears red duration,reducing climbing cage per unit time,scratching episodes frequency,can be seen and TMP Zhengtian Pills can shorten the duration of a migraine headache onset,reducing the degree of attack.2.c-Fos,ERK,P2X3,TRPV1 in nitroglycerin-induced migraine rat TCC and TG were abundantly expressed.3.Zhengtian Pills and TMP can inhibit over-expression of c-Fos,ERK,p-ERK,P2X3 and TRPV1 in TCC and TG of nitroglycerin migraine rat.4.The inhibition of Zhengtian Pills and TMP on migraine may simultaneous act on P2X3 receptors,TRPV1 receptor,ERK and c-fos,but also perhaps by inhibiting P2X3 receptors and TRPV1 receptor expression,thereby affecting the downstream activation of ERK signaling pathway. |
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