Effects Of Dilong On Airway Remodeling In The Murine Model Of Chronic Allergen-induced Asthma

Objective: Clinical application indicated that Dilong has certain active cure in asthma, butthe effective working parts of Dilong and the mechanism underlying Dilong interveningasthma airway restructuring is unclear. This study was to explore Dilong and itscomponents to intervene restructuring the airways of the asthma in mice.Methods:1. The preliminary separation of DilongFresh earthworm dried under50℃constant temperature, powder, as Dilong sample1.Fresh earthworm ground into a paste with PH6.8phosphate buffer (PB) under4℃,centrifuged15min with rotating speed of10000r/min, centrifugal sedimentation driedunder50℃, as Dilong sample2; Centrifugal supernatant were freeze-dried used assample3.The selected sample3was used to extract the Dilong total protein by using ammoniumsulfate method, freeze-dried as Dilong sample4, preserved in-20℃.2. Establishing the murine model of chronic allergen-induced asthma64Balb/c mice were randomized into8groups (8/group): control group, asthmatic modelgroup, lumbrukinase group, positive group (Montelukast Soudium tablets), Dilong sample1, Dilong sample2, Dilong sample3and Dilong sample4. Except control group, Balb/c mice of other groups were sensitized on0,7,14d by means of celiac injection of20μgOVA+2mg aluminum hydroxide gel, and from21d, all murine were stimulated with2.5%atomized OVA, with amount of0.5ml per minute and30minute per time. The stimulationwas taken three times per week and continued six weeks. Sensitized and stimulated controlgroup murine with phosphate buffer.3. Observe the survival quality of the murine; Extracted bronchoalveolar lavage fluid(BALF), and counted all kinds of inflammatory cells as well as the total number of cells,respectively; Haematoxylin and Eosin (HE) dyeing to observe lung tissue pathologicalchanges, and inflammatory score to preliminarily quantify the infiltration degree ofinflammatory cell; Masson dyeing to observe lung organization subcutaneous fibrosisdegree and the Image-ProPlus6.0system to quantitatively analyze the degree of collagendeposition; Periodic acid-schiff stain (PAS) to observe goblet cells and mucus glandsecretion and the Image-ProPlus6.0system to quantify PAS positive cells.Results:1. Yield for Dilong sample1is14.7%; Yield for Dilong sample2is7.1%; Yield for Dilongsample3is6.7%; Using ammonium sulfate precipitation method to extract Dilongwater-soluble total protein led to a Yield for Dilong sample4not lower than2.62%, inwhich the active protein content not less than48.6%.2. Compared with the control group, Number of inflammatory cells in BALF, collagendeposition and glycogen mucin secretion expression in a asthmatic model group weresignificantly increased (P <0.05), and there are obvious symptoms of dyspnea and poormental states in the model group; Compared with asthmatic model group, the positivegroup and Dilong sample1group had a significant decrease in number of inflammatorycells in BALF (P <0.05), especially the eosnophils (EOS)(P <0.001), but no significantlydifferent (P>0.05) for the other Dilong intervention groups though number also decreased;Airway and vascular inflammatory infiltrates score of each dose group relative to theasthmatic model group, to some extent decreased, but with no significant difference thus nostatistical significance. In terms of airway collagen deposition, compared with asthmaticmodel group, lumbrukinase group, positive group, Dilong sample1and Dilong sample2 had declined significantly (P <0.05), but the other two groups had no statisticalsignificance. Compared with asthmatic model group, lumbrukinase group (P <0.05),positive group (P <0.001) and Dilong sample1group (P <0.01) had significantly decreasein the number of PAS positive cells, but no significant difference for the other drug groups.Conclusion:1. Use of ammonium sulfate precipitation method can extract most water-soluble proteinsof Dilong, and this method is stable and reliable.2. Dilong sample1can improve the mental status of mice, decrease the number ofinflammatory cells in BALF, especially in the number of eosinophils, and besides, it canalso reduce collagen deposition and inhibit glycogen mucin secretion and goblet cellhyperplasia; Dilong sample2and lumbrukinase can reduce collagen deposition, andlumbrukinase can also reduce the high secrete of mucin glycogen, while other dosagegroups have no significant effect.The above experimental results show that Dilong can improve the quality of asthma micelife, mitigate asthma symptoms, inhibit or reverse airway remodeling caused by asthma,and the effective components may be the whole body of Dilong rather than just some of theparts or a single component, and lumbrukinase may play an important role in the process ofinhibiting or reversing airway restructuring in the model murine of chronicallergen-induced asthma.