| Cough variant asthma(CVA)is a special type of asthma,and chronic cough is its main clinical symptom without obvious wheeze.It also known as cough type asthma,hidden asthma or allergic cough.In essence,CVA is a kind of chronic airway inflammation with bronchial hyperresponsive which many inflammatory cytokines are involved.Research shows that cough patients of variant asthma are more depressed and anxious than classic asthma patients,so CVA have affected people’s work and life seriously.Traditional Chinese medicine which treats CVA by expelling wind,ventilating lung,removing phlegm,promoting blood circulation and invigorating spleen and kidney warmLy has specialties of long-term efficacy of stable,concurrent treatment of biao-ben and low recurrence rate,so it is great significance to treatment of CVA.Zihepochuan Particles is the clinical experience of the effect side,and it is composed of astragalus,Guizhi,licorice,aster,Magnolia,medicine Terminalia fruit and other Chinese herbal medicine,which has the efficacy of strengthening spleen,tonifying lung and relieving cough and asthma.Preliminary studies have shown that the compound can significantly improve the clinical CVA symptoms.This study which is based on the functions of Zihepochuan Particles and the pathogensis of CVA established guinea pig model with cough variant asthma and animal model with cough to observe pharmacodynamics of Zihepochuan Particles and investigate the mechanism.1 Effect of Zihepochuan Particles on guinea pigs with cough variant asthma induced by OVAObjective:To observe the effects of Zihepochuan Particles on number of cough,expression of cytokines and lung tissue pathological histology of guinea pig model with cough variant asthma.Methods:SPF guinea pigs were randomLy divided into six groups,including normal group,model group,Montelukast group,low dose group,middle dose group,high dose group of Zihepochuan Particles.All guinea pigs were given 4%OVA by intramuscular injection and 2%Al(OH)3 by intraperitoneal injection except the guinea pigs of normal group on the first day.The normal group used normal saline instead of OVA and Al(OH)3 at the same time.Two weeks later,the guinea pigs who were given OVA and Al(OH)3 were challenged with aerosolized 1%OVA for 7 times on alternate days,and the guinea pigs of normal group used saline instead of OVA.The same day low dose group,middle dose group,high dose group of Zihepochuan Particles and Montelukast group were given medicine by intragastric administration once a day for 2 weeks 30 minutes before the atomization.The normal group and model group were given same volume of water.The tests of cough sensitivity to capsaicin were performed 30min after the last administration and the cough times were recorded.24 hours later,all the guinea pigs were anesthetized with 10%chloral hydrate by intraperitoneal injection,then blood plasma were collected by abdominal aortic method.The content of IL-4,IL-5,IL-6,IL-10,IL-13,IL-17,TNF-α,IFN-γ in serum and IL-4,IL-10,IL-17,TNF-α,IFN-γ in BALF were determined by enzyme-linked immune sorbent assay.Observed the pathologic change by HE staining.Results:After the stimulus of capsaicin,the cough frequency of model group was significantly higher than normal group(P<0.05).Compared with model group,the cough numbers of middle dose group and high dose group of Zihepochuan Particle were obviously reduced(P<0.05).Compared with normal group,the levels of serum IL-10 and INF-γ in model group were significantly lower(P<0.05),and the level of serum IL-6 was significantly increased(P<0.05).Compared with model group,the levels of serum IL-5 and IL-6 in low dose group of Zihepochuan Particles were significantly lower(P<0.01),and the levels of serum IL-10、INF-y were significantly increased(P<0.05).The levels of serum IL-5、IL-6 in middle dose group of Zihepochuan Particles were significantly lower(P<0.01),and the levels of serum IL-10、INF-γ were significantly increased(P<0.05 or P<0.01).The levels of serum IL-4、IL-10、IL-17、INF-γ in high dose group of Zihepochuan Particles were significantly increased(P<0.05).The levels of serum IL-4、IL-10、INF-γ in Montelukast group were significantly increased(P<0.05),and the level of serum IL-6 was significantly lower(P<0.05).Compared with normal group,the levels of BALF IL-4,IL-17,INF-γ in model group were significantly lower(P<0.05).Compared with model group,the levels of BALF IL-4 and IL-17 in low dose group of Zihepochuan Particles were significantly increased(P<0.01 or P<0.05),and the levels of BALF IL-10 and TNF-α were significantly lower(P<0.05 or P<0.01).The levels of BALF IL-4 and IL-17 in middle dose group of Zihepochuan Particles were significantly increased(P<0.01 or P<0.05),and the levels of BALF IL-10、TNF-αwere significantly lower(P<0.05).The levels of BALF IL-4,IL-17 and INF-y in Montelukast group were significantly increased(P<0.01 or P<0.05),and the level of BALF IL-10 were significantly lower(P<0.05).Compared with normal group,the histopathological score of model group was significantly increased(P<0.01),and there were lots of inflammatory cells around the bronchia,mucosal and vessel.There also were more epithelial exfoliation and secreta in bronchia,and mucous plug has formed.The bronchial smooth muscle were much thicker.Most alveoli were damaged.Compared with model group,the histopathological scores of low,middle and high dose group of Zihepochuan Particles and Montelukast group were significantly reduced(P<0.01).Conclusion:Compared with normal group,the cough and pulmonary inflammation of model group were more serious,and the secretion of cytokines was abnormal.So the guinea pig model with cough variant asthma was established successfully.The results showed that Zihepochuan Particles could treat the cough,regulate the cytokine secretion and improve airway inflammation.2 Effect of Zihepochuan Particles on guinea pigs with cough induced by citric acid and mice with cough induced by ammonia waterObjective:To observe the antitussive effect of Zihepochuan Particles on guinea pigs and mice.Methods:After 1 week for acclimation,SPF guinea pigs were placed in closed box one by one,then passed into 17.5%aerosolized citric acid.Record the times of cough,then the guinea pigs whose cough number were more than 10 times were positive.The else were obsolete.The positive animals were divided into five groups,including model group,Jizhi syrup group,low dose group,middle dose group,high dose group of Zihepochuan particles.All the groups were given medicine by intragastric administration for 3 days except model group who was replaced normal saline.Record the incubation period and times of cough by the same method 1 hour after the last administration.After 1 week for acclimation,ICR mice were divided into five groups,including model group,Jizhi syrup group,low dose group,middle dose group,high dose group of Zihepochuan particles.All the groups were given medicine by intragastric administration for 3 days except model group who was replaced normal saline.One hour after the last administration,all animals were placed in closed box one by one,injecting 0.3mL of strong ammonia water into 50mg cotton ball which was put into the closed box.Record the incubation period and times of cough.Results:After the stimulus of strong ammonia water or citric acid,compared with model group,the cough in Jizhi syrup group,low dose group,middle dose group and high dose group of Zihepochuan particle obviously reduced(P<0.05 or P<0.01).Conclusion:Zihepochuan Particles had certain antitussive effects.3 Effect of Zihepochuan Particles on expression of NF-κB,IκB,RANK and TGF-β1 of guinea pigs with cough variant asthmaObjective:To explore the mechanism of Zihepochuan Particles by NF-κB signaling pathway.Methods:SPF guinea pigs were randomLy divided into six groups,including normal group,model group,Montelukast group,low dose group,middle dose group,high dose group of Zihepochuan Particles.All guinea pigs were given 4%OVA by intramuscular injection and 2%Al(OH)3 by intraperitoneal injection except the guinea pigs of normal group on the first day.The normal group used normal saline instead of OVA and Al(OH)3 at the same time.Two weeks later,the guinea pigs who were given OVA and Al(OH)3 were challenged with aerosolized 1%OVA for 7 times on alternate days,and the guinea pigs of normal group used saline instead of OVA.The same day low dose group,middle dose group,high dose group of Zihepochuan Particles and Montelukast group were given medicine by intragastric administration once a day for 2 weeks 30 minutes before the atomization.The normal group and model group were given same volume of water.On the 29th day,observed the expression of IκB,NF-κBand RANK by IHC.Detected the expression of mRNA and prot of NF-κB and TGF-β1 by Real-time PCR and Western blot technology.Results:The content of mRNA of NF-κB,TGF-β1 and protein of TGF-β1 in model group was significantly more than normal group(P<0.05),and protein of IκB was significantly lower(P<0.05).In the result of WB,the content of protein of NF-κB in model group was significantly more than normal group(P<0.05).Compared with model group,the contents of protein of NF-κB and RANK in low dose group were significantly reduced(P<0.05).The contents of protein and mRNA of NF-κB and mRNA of TGF-β1 in middle dose group were reduced significantly(P<0.05),and the expression of IκB in middle dose group was significantly increased(P<0.05).The contents of protein of RANK,protein and mRNA of NF-κB and TGF-β1 in high dose group were reduced significantly(P<0.05),and the expression of IκB in high dose group was significantly increased(P<0.05).The contents of protein and mRNA of NF-κB,protein of TGF-β1 and RANK in Montelukast group were reduced significantly(P<0.05 or P<0.01),and the expression of IκB in Montelukast group was significantly increased(P<0.05).Conclusion:Zihepochuan Particles could regulate the expression of NF-κB,TGF-β1,RANK and IκB,so the mechanism of Zihepochuan Particles was in connection with NF-κB signaling pathway. |
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