The Study Of A Chinese Herb Medicine, Lanyuzan Granules, On Airway Remodelling In A Murine Model Of Chronic Asthma

Backgroud:Asthma is a chronic inflammatory disease involving many different cell types and cellular elements,Evidence suggests that,in the long term,this inflammation leads to remodeling of the airways。Airway remodelling is a central feature of asthma,which refers to the structural changes that occur in the airway wall in asthma。These include epithelial hyperplasia and metaplasia, subepithelial fibrosis,muscle cell hyperplasia and angiogenesis。These structural changes result in thickening of the airway wall, airway hyperresponsiveness(AHR), and a progressive irreversible loss of lung function。The degree of structural changes seems to be correlated with asthma severity。Glucocorticoids act primarily as anti-inflammatory agents in asthma,are the most effective therapy currently available for the treatment of asthma。 Unfortunately,patients with chronic asthma who take these drugs for a long and indefinite period of time may develop serious side effects,particularly over the long term。Traditional herbal medicines may be viable alternatives to corticosteroid therapy for treatment of asthma。The G.veitchiorum Hemsl, a Traditional Chinese herb medicine,has been used to treat acute and chronic respiratory system diseases,such as asthma, chronic bronchitis,cough and lung infections in China for centuries。The Lanyuzan granules,contains the G.veitchiorum Hemsl and panax notoginside(PNS)two species of medicinal plants elements, Previous clinical findings and Pharmacology studys indicated that Lanyuzan granules has a bright future in preventing and treating pulmonary fobrosis (PF), can enhance the activity of SOD, improve the oxidation/deoxidation balance, restrain the accumulation of collagen and protect the lungs from Bleomycin(BL)-induced experimental pulmonary fibrosis。However, the therapeutic mechanisms of the Lanyuzan granules on airway remodeling of chronic asthma are still far from clear。Therefore,we compared the effects of the Lanyuzan granules and dexamethasone (DXM) on airway inflammation and remodeling in a murine model of chronic asthma。This study was performed to evaluate the protective effect of Lanyuzan granules on airway remodelling in a murine model of chronic asthma and its mechanism。METHODS:1.Female BABL/C Mice were sensitized on days 0,7 and 14 of the experiment by three intraperitoneal injections of 100μg chicken egg ovalbumin(OVA,gradeⅢ,Sigma,USA)emulsified in 2.6 mg of aluminum hydroxide in a total volume of 0.2 mL,after the last sensitization,mice were exposed to 3% OVA aerosolized for 30 min/day,one times/day for 6 weeks,to establish the chronic asthmatic model。2. To evaluate the protective effect of Lan-Yu-Zan granules and Dex on the airway inflammation,airway subepithelial fibrosis and collagen deposition in asthmatic mouse。Female BABL/C mice were randomly divided into 4 groups(n =8 mice/group):the normal group(A),the model group(B),the Lanyuzan granules group(C,1.0 g/3.0 kg body mass)and the dexamethasone group(D,2.0 mg/kg body mass)。Lung tissue sections were stained with hematoxylin and eosin(HE)for general morphology and airway inflammation scores,Sirius Red stain and hydroxyproline content for evaluation of airway fibrosis and immunostained for assessment of TGF-β1。We calculated the area of positive staining of SR and TGF-β1 content in the airway wall with the computerized image analysis system。We calculated the changes of airway remodelling dimensions,such as the airway outer perimeter(Po), the internal perimeter(Pi),airway wall thickness (Awt),airway wall area (Aaw)and the area of positive staining of TGF-β1 content in the airway wall with the computerized image analysis system(Image Pro-Plus 6.0,Media Cybernetics,USA)。Finally,all dimensions were referenced to perimeter of the basement membrane(Pbm)。3. To evaluate the protective effect of Lanyuzan granules and DXM on the epithelial hypertrophy and metaplasia,airways mucus hypersecretion and Bcl-2 expression in a murine model of chronic asthma。Lung tissue sections were stained with Alician Blue-Periodic Acid-Schiff(AB-PAS)for identification of goblet cells and immunostained for assessment of Bcl-2。We calculated the area of airway epithelium layer(Aep),the positive staining of AB-PAS and Bcl-2 content in the airway wall with the computerized image analysis system。Finally,all dimensions were referenced to the perimeter of the basement membrane(Pbm)(e.g. Aep?Pbm)。 RESULTS:1. As previously described by others,Mice exposed to recurrent airway challenge with ovalbumin had characteristics of airway inflammation and airway remodeling,including marked peribronchial and perivascular inflammatory infiltrates,subepithelial fibrosis,goblet cell hyperplasia and mucus hypersecretion,and airway wall thicken。Computer image analysis find that the airway remodelling dimensions of chronic asthma mice,including Aaw/ Pbm,Awt/Pbm and Po/Pi,all were significant higher than the normal control group。2. Sirius Red staining showed that B group mice had characteristics of airway subepithelial fibrosis and collagen deposition。The diference of these dimensions,such as the positive staining of SR/ Pbm,the positive staining of TGF-β1/Pbm , the hydroxyproline content and the scores of airways inflammation between B and C group all were significant(P all <0.01);diferences were also significant when C group was compared with A group and D group(P<0.05)。A close correlation between the positive staining of TGF-β1 and the hydroxyproline content was demonstrated (rs=0.669,P<0.01),and the rs=0.709 between the hydroxyproline content and the positive staining of SR is significant too(P<0.01)。3. B group mice showed that epithelial hypertrophy and metaplasia,goblet cell hyperplasia。The diference of those dimensions,such as the positive staining of AB-PAS/ Pbm,the positive staining of Bcl-2/ Pbm between B and C group all were significant(P all <0.01);the Aep/ Pbm in B group was significant higher than the C group(P<0.01),but no differences with D group(P>0.05)。A close correlation between the positive staining of AB-PAS and the Bcl-2 content(rs=0.671,P<0.001),and the rs=0.784 between the Aep/ Pbm and the positive staining of Bcl-2 was significant too(P<0.001)。CONCLUSION:1.We have developed a chronic asthmatic murine model of airway remodeling under the approach as above。2.Lanyuzan granules could suppress the infiltration of inflammatory cells to lung tissues and decrease the airway inflammation scores in a murine model of chronic asthma,In addition,Lanyuzan granules might reduce these airway remodelling dimensions of chronic asthma,including Aaw/Pbm,Awt/Pbm and Po/Pi。3.Lan-Yu-Zan granules might suppress airway subepithelial fibrosis by decreasing the expression of TGF-β1 and reducing deposition of collagen in a murine model of chronic asthma.。4.Lanyuzan granules might reduces airways mucus hypersecretion and suppress airway epithelium metaplasia moderately in this chronic asthma model by downregulating Bcl-2 expression。5.In spite of Lanyuzan granules,unlike Dex,has not the same efficacy on inhibiting airway inflammation and remodeling in chronic asthma resulting from chronic allergen exposure。These results demonstrate that Lanyuzan granules might effectively delay the progression of airway inflammation and remodeling, Consequently,Lanyuzan granules may be a useful treatment of asthma。Additional studies are needed to characterize the precise mechanism of action of it in the treatment of asthma.