| The interstitial lung diseases (ILDs) represent a large number of conditionsthat involve the parenchyma of the lung. This group of disorders is classifiedtogether because of similar clinical manifestations of exertional dyspnea,roentgenographic diffuse infiltration, restrictive pulmonary function，decreasedDLCO and hypoxemia, and pathologic manifestations of alveolitis and fibrosis.It is not clarified clearly about the pathogenesis of ILD. Usually theoccurrence is divided into three phases, initiation stage, progression stage and endstage. Pathogenic factors are usually toxins and antigens. Exposure to thepathogenic factors can lead to alveolitis which is the most important part ofILD.The activation of inflammatory cells and immunocytes can release oxygenradicals and other toxins and injure typeⅠalveolar epithelial cells and capillaryendothelial cells. Alveolitis also can lead to injury of interstitium, collagen tissueand basement membrane by releasing proteinase.When alveolitis progress andlead to severe injury in the lungs, there may be fibroblasts proliferations, collagendeposit and formation of fibrosis.When the fibrosis produce, the injury ofalveolar wall can not be repaired.The injury of alveolar epithelial cells can lead apoptosis of large number ofalveolar epithelial cells, and Fas/FasL signal pathway is an important reason forthe apoptosis. While DcR3can bind to FasL and block the Fas/FasL pathway,thenreduce the apoptosis. We suppose that the administration of DcR3to the ILDpatient in the early stage may reduce the apoptosis of alveolar epithelial cells andprevent the progression of pulmonary fibrosis.In the first part of the study, the CCL-149cells were cultured in vitro. Threegroups were designed: control group, FasL group and DcR3group.The cellswere incubated with FasL to induce apoptosis in FasL group, and in DcR3group the cells were incubated with FasL and DcR3together. The cells were gated andanalyzed by MTT and flow cytometry for apoptosis. The expression of Fas andcaspase3were analyzed by RT-PCR and Western blot. MTT and flow cytometryresults showed: apoptosis percentage of DcR3group were lower than FasL groupand there was significant difference (p<0.05). RT-PCR and Western blot resultsshowed: the expression of Fas of DcR3group is lower than FasL group. Theresults suggest that DcR3can inhibit the apoptosis of CCL-149cells induced byFasL.In the second part, we established pulmonary fibrosis model in rat byinstillation with bleomycin into the tracheal. The rats are divided into threegroups: control group, bleomycin group, and DcR3group.The rats in controlgroup and bleomycin group were injected intravenously with normal saline0.5mlevery other day and the rats in DcR3group were injected intravenously withDcR3protein(3.33μg/kg) every other day from the beginning of the model. Therats were sacrificed at the time of7d,21d and28d respectively. The right lungswere submerged in10%paraformaldehyde solution, paraffin-embedded, stainedsections in order to organize staining. The left lungs were saved for the detectionof Fas、FasL and caspase3by Western blot. The serum were collected for thedetection of hydroxyproline by ELLISA.Histopathology results showed: control group: the structure of lungs arenormal at all the time of7d,14d and28d and Masson staining showed no greencollagen deposit. Bleomycin group: At7d,the structure of alveolar was clear andinfiltrated with large number of neutrophils and macrophages. At14d, alveolarseptum were thick and infiltrated with neutrophils,there were small amount offibroblasts in the interstitium.Masson staining showed small amount of greencollagen deposit. At28d, the inflammatory cells reduced and the structure ofalveolar were destroyed.The alveolar septum were significantly thickened,andthere were large amount of fibroblasts proliferation and fibrosis produced.Masson staining showed large amount of green collagen deposit.DcR3groups: the degrees the alveolitis and fibrosis were lower than bleomycin group andhigher than control group. Pathological assessment: the extent of alveolitis ofDcR3group manifested as a gradual reducing, the degree of inflammation werelower than the bleomycin group, there was significant difference (p<0.05); theextent of pulmonary fibrosis of DcR3group were lower than the bleomycingroup and there was significantly different (p<0.05).The result of hydroxyproline content in serum by ELISA showed: thehydroxyproline content of DcR3group was lower than bleomycin group buthigher than control group, there were significantly different (p<0.05). Westernblot results showed： At28d,the expression of Fas,FasL and caspase3inbleomycin group were higher than control group, there was significantly different(p<0.05), the expression of Fas, FasL and caspase3in DcR3group were lowerthan bleomycin group, and there were significantly different (p<0.05). TheImmunohistochemistry results showed: the expression of Fas, FasL and caspase3in the lungs of DcR3group are lower than bleomycin group but higher thancontrol group, and there were significantly different (p<0.05). The TUNELresults showed:the cell apoptosis index of DcR3group are lower than bleomycingroup but higher than control group, and there were significantly different(p<0.05).Combining the results of experiments, the following conclusions can bedrawn: DcR3can reduce the apoptosis of alveolar epithelial cells induced byFas/FasL pathway in CCL-149in vitro. In the pulmonary fibrosis animalmodel,the degree of alveolitis and the pulmonary fibrosis in DcR3group werelower than bleomycin group.Thus,in the pulmonary fibrosis animal modelinduced by bleomycin,the administration of DcR3may reduce the progression ofpulmonary fibrosis through inhibiting the apoptosis of alveolar epithelial cellsinduced by Fas/FasL signal pathway. The study improved the pathogenesis ofpulmonary fibrosis, and also has great significance to the clinical treatment. |
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