| PrefaceInterstitial lung disease consists of more than 200 kinds of diseases with a potentially and prognosis and unclear pathogenic mechanism.There are some common characterstics of the clinical manifestation,laboratory and pathological changes in them and respective characters in each one.The pathologic features of the pulmonary fibrosis are characteristic of alveolar epithelium injury and excessive accumulation of inflamematory cells,fibroblast overproliferation,exrtacellular matrix deposition, fibroproliferation foci and honeycomb lung.The Neomycin-induced may lead to acute lung injury and pulmonary fibrosis in rodents and this is a mature model of pulmonary fibrosis research.NF-κB is an important transcription factor and may regulate the transcripton of many inflammatory cytokines.P65 is an important function subunit in NF-κB.Researches showed that the expression of NF-κB significantly augmented in pathological process by bleomycin-introduced pulmonary fibrosis.Fibroblasts differentiate to myofibroblasts which expressα-smooth muscle actin play an important role in the lung fibrosis pathological processese.This experiment use the immunohistochemistry mathod to investigate the dynamic expression and distribotion of NF-κBp65 andα-SMA in mice with bleomycin-introduced interstitial pulmonary fibrosis,and study the role of NF-κB activation and fibroblast phenotype transdifferentiation in pulmonary fibrosis. Material and Methods1.Experimental material8 weeks old C57BL/6,female(weight:17～20g)were maintained in specific pathogen-free conditions(Beijing Weitong Lihua experimental animal technology company).Bleomycin,Mouse NF-κBp65 momoclonal antibody against human being, Mouseα-SMA momoclonal antibody against human being(Santa Cruz Biotechnolngy,lnc),Histostain-Plus Kits(Beijing Zhongshan goldengridge biotechnology co,ltd).2.Animal model,experimental groups and tissue preparation(1)Animal model establishing and experimental groupingSixty female C57BL/6 were randomly divided into model group and control group,Mice were anesthetized with 10%Chloralhydrate by intraperitoneal ingestion and operated to expose the trachea in sterile condition.①Model group:fifty mice were treated with Bleomycin A5(BLM,5mg/kg weight in 20μl sterile isotonic saline)by intertratracheal ingection on day 0.②Control group:ten mice were treated with sterile isotonic saline(20μl)by intertratracheal ingection on day 0.(2)Sampling and preparation of lung tissue specimens(At 1,3,7,14,28 days postinstillation,selected ten mice from model group randomly and at 7,14 days selected five mice from control group randomly.)Animals were euthanized by exsanguination from abdominal aorta.The lung tissues were collected and stored in 4%paraformaldehyde and embedded in paraffin,then were cut into 4μm -thick serial sections.3.Histopathological assessment(1)Routine pathological manifestationLung tissue were dehydrated in a graded series of ethanols,stained with haematoxylin-eosin and Masson' s trichrome.(2)Histopathological grading of extent of alveolar inflammation and fibrosis in lung parenchyma. According to Szapiel' s method,grade 0 normal tissue,grades 1～4 for prsence of pulmonary inflatmmation and fibrosis,the severity of lesions was graded as 1(mild),2(moderate),3(severe)and 4(severe inflammation accompanied by total distortion of structura).4.Experimental methods and index determinedImmunohistochemistry(SP method)was used to detect the location and expression of NF-κBp65 andα-SMA protein in lung tissue.5.Statistical analysisAll data wre expressed as the means±SD.The T test and one-way ANOVA were carried out using SPSS 13.0 statistical software.A value of less than 0.05 was considered statistically significant.Results1.Results of histopathological analysis in mice.On day 3 postbleeomycin administration,lung from model group showed marked alveolar infiltration with inflammatory cells and effusion.Thickening of interalveolar septa,inflammatory cells infiltration and focal solidation,and excessive collagen sediment were observed on day 7.On day 14 and 28 extensive solidation of lung,lossof normal alveolar structure,obvious fibrosis presence and abundant collagen sediment with Masson trichrome staining appeared.Compared to mice in model group,there were no remarkable inflammatory and fibrosis presence in control group.2.Histopathological grading of extent of alveolar inflammation and fibrosis in lung parenchyma(1)Comparison of alveolar inflammation grading in miceOn each day postbleomycin administration,alveolar inflammation grading in mice from model group were respectively high compare to mice in control group which being 1.0±0.00(P＜0.01).The grading was highest on 7 day in which alveolar inflammation was severest.(2)Comparison of lung parenchyma fibrosis in miceAfter bleomycin administration,lung parenchyma fibrosis acured on 7 day and the extent of lung parenchyma fibrosis gradually aggravated.(Compara to control group,P＜0.01).On day 28 postbleomycin,the extent of parenchyma fibrosis was severest and the grading was 3.8±0.42.3.Results of NF-κBp65 andα-SMA immunohistochemical(IHC) staining in lung.Immunohistochemical shows that the positive expression of NF-κBp65 in model group were higher than that in control group all the time especially on day 1 first. The positive expression ofα-SMA in model group were higher than that in control group all the time and the extent was gradually highed.4.Correlation analysis inα-SMA and NF-κBp65After bleomycin administration,on early days(from 0 day to 3 day),there was a positive correlation between NF-κBp65 andα-SMA(r=0.529,P＜0.01).Conclusion1.NF-κB in pathological process by bleomycin-induced pulmonary fibrosis is continuallt active from the first day to the last.2.The phenotypic transformation of fibroblasts to myofibroblasts exist in Animal model of pulmonary fibrosis,nuclear factor-kappa B activation can promote fibroblast phenotype transdifferentiation and play an important role in pulmonary fibrosis.
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