| Bleomycin-induced pulmonary fibrosis is one of the animal models widely used in the study of pulmonary fibrosis,and it is recognized as a pharmacodynamic evaluation model in lung fibrosis disease research.Purpose-1:To establish unilateral pulmonary fibrosis model in SD rats induced by different doses of bleomycin(BLM),and to establish an animal model suitable for the research of idiopathic pulmonary fibrosis.Methods:Fifty male SD rats were randomly divided into sham model group(group-1,n=5),low dose of BLM group(1.5mg/kg,100ul/rat)(group-2,n=15),medium dose of BLM group(3.0mg/kg,100ul/rat)(group-3,n=15),high dose of BLM group(5.0mg/kg,100ul/rat)(group-4,n=15).After intraperitoneal anesthesia with pentobarbital sodium(50mg/kg),the skin of the neck was prepared,the skin was cut,the exposed trachea was separated in layers,and the BLM was injected into the left bronchus through intubation between tracheal rings.Close the neck wound in layers.After the operation,keep the animals in 37 C electric blanket until they fully recover.The animals were confirmed to be able to feed and drink freely and then returned to the cage for normal feeding.Five animals in each BLM model group were randomly selected to euthanize on the 7th,14th and 21st day after modeling,and the animals in the sham model group were euthanized on the 21st day.The left lungs of the animals were collected and perfused with formalin.HE and Masson Trichrome staining were performed.Pathological analysis of end bronchioles and pulmonary arterioles injury and inflammation were scored;semi-quantitative evaluation of pulmonary fibrosis(ashcraft score)were performed.Conclusions:By analyzing the pathological changes of left pulmonary fibrosis induced by different doses of BLM,the results showed that the model of BLM-induced pulmonary fibrosis at dose of 3.0 mg/kg provides a reliable pathological window for pharmacological and pharmacodynamic studies.It provides a reliable animal model for further effective pharmacological and pharmacodynamic studies.Purpose-2:This study was to evaluate the Prophylactic effect and therapeutic effect of pirfenidone(Compound-X)on BLM induced unilateral lung fibrosis model on SD rats.Methods:Fourty-seven male SD rats were used and randomly divided into six groups:Sham-1 group(group-1,n=4),Model-1 group(group-2,n=9),positive control Compound-X-1(100mg/kg/d,n=9),sham-2 group(group-3,n=5),Model-2 group(group-2,n=10),positive control Compound-X-2(100mg/kg/d)group(group-3,n=10).All animals of rophylactic effect study received an oral dosage of compound-X two hours before animal modeling and continually for 14 days after modeling;saline was dosed for sham and model group;Compound-X as BID,All animals of therapeutic effect study received an oral dosage of saline or compound-X at day 8 of modeling and totally for 14 days.On modeling day all rats were anesthetized with sodium pentobarbital(50mg/kg,ip),trachea was exposed and received a directly intra-tracheal injection of BLM at dose of 3mg/kg.For sham group rats were administrated to trachea as equal volume of saline.At the end of the study all animals were euthanized and left lungs were harvested,perfused with 10%formalin and then processed for histology.Semi-quantitative scoring of bronchial and parallel pulmonary arteriole damage and inflammatory infiltration were conducted.Left lung tissue fibrosis(semi-quantitative Ashcraft scoring)was also evaluated.The ratio of α-SMA positive staining and collagen I staining in fibrosis core were assayed with IHC method,the Ki-67 positive cell numbers in fibrosis core were calculated also with IHC method to show the status of cell proliferation.Conclusions:Histological examination revealed that the left lungs in model group animals showed the bronchial and alveolar ducts epithelial cell hyperplasia,different quantity of mucus mass in bronchial lumen,inflammatory cell infiltration especially in the adventitial area as compared to sham group animals.Alveoli in fibrosis core were damaged as alveolar epithelial cell shedding and regeneration;alveolar wall inflammatory cell infiltration and fibrosis;alveolar cavity inflammatory cell infiltration with fibrosis mass.IHC staining indicated a significant increase in α-SMA and collagen I positive staining in model group.But Ki-67 positive cell numbers did not show a significant difference between model and sham group.Animals treated with Compound-X showed a great therapeutic effect on limitation of lung fibrosis progress compared to model group both in bronchial/arteriole damages and lung fibrosis score.Ashcraft fibrosis score data indicated animals with Compound-X treatment significantly inhibited the progress of lung fibrosis.α-SMA IHC data indicated that with the Compound-X treatment α-SMA expression in fibrosis core was significantly reduced.Ki-67 positive cells were observed as mainly inflammatory cells in severe fibrosis core area,and as mainly alveolar epithelial cells in slight fibrosis core area.Treatment with Compound-X did not show a significant decrease in collagen I expression.Conclusion:The Compound-X is currently used as a clinical drug.It is usually used as a positive control drug in the development of new drugs on lung fibrosis disease.The results show that Compound-X has a significant effect on unilateral pulmonary fibrosis in rats.It can be concluded that the unilateral pulmonary fibrosis model established by intratracheal injection of BLM into the left lung of rats can be used as a preclinical pharmacological and pharmacodynamic study for the development of new drugs for idiopathic pulmonary fibrosis. |
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