| General IntroductionBronchial asthma,a chronic inflammation of respiratory passage,is caused by a lot of multi-cytes and cell excitatory transmitters,such as mast cell,eosinophile granulocyte,T lymphocyte,macrophage,neutrophi and epithelial cell,etc.There are about 150,000,000 to 200,000,000 people who suffer from asthma all over the world.In our country,there are about 1500 to 20,000,000 asthma patients,the incidence of child is higher than adult's.Especially in the recent 20 years,the incidence and mortality of asthma increase contineously,with 20%～50%ratio per ten years,and has become the second disease besides cancer which cause death and disability.EOS(full name,so as the followings),the main effector cell in asthma pathogenesis,plays an important role in mediating inflammation of respiratory passage and participating asthma occurrence.TNF-αis the important promoter in asthma inflammation process,also stimulates the secretion of ET-1,and results in the contraction and multiplication of smooth muscle.ET-1 is the proinflammatory medium,which can increase the quantity of TNF-αand other substance.Moreover,the increasing of its synthesis and secretion is possibly the key relating the airway inflammation and remodeling in asthma.All these three factors are closely related to the airway inflammation,and TNF-αand ET-1 can promote and increase secretion mutually,and both of them can impact airway hyperreactivity.Transdermal theraputic system(TTS) or transdermal drug system(TDDS),a kind of dermal patch,is the controlled-release dosage to treat the general diseases through the skin administration,with the characters of high bioavailability,scheduled and longer acting time low drug toxicity and side effect,stable and long-lasting blood drug level,less and drug delivery times based on its advantages of without meeting the first-pass effect of hepatic,being destroyed by the gastrointestine,and avoiding the effect of many factors,such as gastrointestinal tract enzyme,digestive juice,PH etc.Crude herb moxibustion,also called drug moxibustion or blistering moxibustion,a kind of therapy to coat the acupoints or lesion with some skin irritating hebs,to make a local hyperemia and even blistering,like the moxibustion sore.Traditional crude herb moxibustion displays its therapeutic action by stimulating both the herbs and the acupoint,further to regulate internal Zangfu system to cure the general diseases.Massive empirical study confirmed that crude herb moxibustion can adjust and enhance organism immune function mainly based on adjusting cytokine,WBC and T homeocyte.As a kind of traditional Chinese medicine treament,Crude herb moxibustion palys a remarkable clinical therapeutic effect in treating asthma."TDD application of crude herb moxibustion",a new dosage from the original crude herb moxibustion medication,was developed in this study based on the combination of TDD and traditional crude herb moxibustion,and its mechanism on treating asthma was studied,too.Guinea pigs were made as asthma model,and received the treatment of acupoint application.The eclipse period(the time from atomization to nutation breath),symptom and severity degree(such as frequency and argument of nod respiratory,convulsion and shock,etc.)of the outbreak,and the serum levels of TNF-αand ET-1,EOS infiltration count in bronchus and lung alveolustissue of guinea pig with asthma were observed.By the comparing to that of the modle group,negative and positive control groups,the fucntion of TDD application was studied,such as the anti-inflammatory,reducing airway spasm and airway hyperreactivity,hence to provide an objective and reproducible basis for pre-clinical experiment,also provide a theoretical basis for the clinical application of TDD application.Method and content 1.Experimental animals and preparation of asthma modelGuinea pigs were selected as the experimental animal.48 guinea pigs were randomly divided into four groups equaly,control group,model group,TDD group and dexamethasone injection group.Guinea pig model of allergic asthma was made by sensitizing OVA,peritoneal injection 1ml 10%(10g/100ml) of normal saline solution of OVA on the first day.2.Induced asthma and intervention treatment after making the asthma model2 weeks after sensitization,guinea pigs were under the sensitization state.Then ultrasonic atomizing inhalation with 1%(1g/100ml)of normal saline solution of OVA was given,until the guinea pigs showed restlessness,cough,hyperpnea and nodded motion.The provocation had been given for two weeks withl time 2 days.The guinea pigs in the TDD group and dexamethasone group were treated with TDD application(acupoints included DU14,BL13,BL23) and intraperitoneal injection of 0.5 mg.kg of dexamethasone respectivly for 7 times totally(1 time 2 days).Animals in the control group and model group did not receive any intervention or therapy.3.Symptoms observation and specimen preparation3.1 Symptoms observation:The symptoms were observed from the succeed sensitization to the sacrifice of animal,including asthmatic eclipse period of each group guinea pigs(between the beginning of atomization to the presentation of nod breath)and the severity degree of the outbreak symptoms(for example nod respiratory rate,scope,faint and so on).3.2 Specimen preparation:2～4h after the last asthma induction,animals were rapidly beheaded and the blood was obtained,which further received centrifugation,clear supernatant liquid seperation,and contained in the refrigerator for being tested.After that,the chest cavity of the animals were immediately opened with the operating scissor to exposure the lung.Then 4 pieces(1mm×1mm) of the right bronchi and the lung hilum were taken respectively,being fixed with 10%of formaldehyde solution,and being paraffin-embedded conventionally and coronal sliced 24 hours later.4.The examination of the serum levels of TNF-αand ET-1,and EOS count in bronchus and lung alveolus tissueThe serum levels of TNF-αand ET-1 were examed by enzyme linked immunosorbent assay(ELISA).EOS infiltration degree in bronchial and lung alveolus tissue were observed by the pathology inspection(10×40,HE dyeing).5.Statistical methodThe data were processed by SPSS13.0 software.Count data were expressed as MEAN±S.The examination results of the serum levels of TNF-αand ET-1 and EOS count in bronchial and lung alveolus tissue of each group were analyzed with one-factor analysis of variance(One-way ANOVA),LSD for homogeneous variances or Welch or Brown-Forsythe were used,with P＜0.01 or P＜0.05 as the standards of statistics significance.Results1.Asthmatic eclipse period and the severity degree of the symptomsFrom asthma induction after sensitization to the end of the intervention and treatment,we observed that,besides normal group,asthmatic eclipse period of the other 3 groups and the severity degree of the symptoms of asthma outbreak were similar.The asthmatic eclipse period of the model group was the shortest,the time of asthma outbreak was the longest,and the symptoms were the most serious,compared to that of TDD group and dexamethasone group,which with an obvious longer asthmatic eclipse period,shorter time of asthma outbreak and light symptoms.2.The effect of TDD therapy on the serum levels of TNF-αand the ET-1 of guinea pig with asthmaThe serum levels of TNF-αand ET-1 detected by ELISA showed that,the serum levels of TNF-αand ET-1 of model group were obviously higher than that of normal groups(P＜0.01),which illustrated that the model preparation was successful,and also proved that serum levels of TNF-αand ET-1 were closely related to asthma.The serum levels of TNF-αand ET-1 of TDD group and dexamethasone group were obviously lower than that of model group(P＜0.01),and the serum levels of TNF-αof dexamethasone group had not any difference with that of TDD group(P＞0.05),which illustrated that both treatment could decrease serum levels of TNF-αand ET-1,and the influence of TDD application on serum level of ET-1 was not obvious compared to that of dexamethasone.The serum levels of ET-1 of dexamethasone group had not any difference with that of the normal group(P＞0.05).But compared to that of dexamethasone group and normal group,the serum levels of ET-1 of TDD group was significant different(P＜0.01),which indicated that the influence of TDD application on serum level of ET-1 was not obvious compared to that of dexamethasone.3.The effect of TDD therapy on EOS count in bronchi and lung alveolus tissues of guinea pig with asthmaEOS count in bronchi and lung alveolus tissues were observed under the optical microscope.EOS count of model group was obvious higher than that of the normal group(P＜0.01),which indicated that EOS infiltration of guinea pig with asthma was very obvious.EOS count in bronchi and lung alveolus tissues of TDD group and dexamethasone group were obviously lower than that of the model group(P＜0.01), and were higher than that of the normal group(P＜0.01),which indicated that both of TDD application and dexamethasone could relieve EOS infiltration but could not completely diminish EOS in bronchi and lung alveolus tissues.ConclusionThis experimental study discovered that TDD application to acupoints could prolong asthmatic eclipse period,reducd times of asthma outbreak and relieve asthma symptoms,and even reduce EOS count in bronchial and lung alveolus tissues, decrease serum levels of TNF-αand ET-1 of guinea pig with asthma,which indicated that TDD application to acupoints played its roles by stimulating acupoints and simultaneously through skin entering blood circulation to inhibit the occurrence and development of airway inflammation,reduce bronchi spasm,decrease airway hyperreactivity,and might affecte the airway remodeling.
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