| Objective1. To establish rat model of semi-thorax radiation-induced lung injury with high dose irradiation. The histological changes of radiation-induced lung injury were dynamically observed.2. To observe the changes of lung tissue apoptosis, Bax, Bcl-2 and Transforming growth factor-betal (TGF-β1) caused by radiation and investigate the association between the apoptosis and radiation-induced lung injury, for finding out a new way to treat radiation-induced lung injury.3. To observe the protective effectiveness of interferon-gamma (IFN-γ) with histology and expression of Bax, Bcl-2 and TGF-β1, to provide experiment evidence of treatment with IFN-γfor radiation-induced lung injury.Methods1. Establishment of rat model for semi-thorax radiation-induced lung injury.The rats were anaesthetized by 3% pentobarbital(30ml/kg), and fixed on the board. Then put the rats into sterilized box, and exposed it under a X-ray simulator. The radiation field was 3.0cm×5.0cm and compassed right lung. SSD was 98cm, total dose 30Gy, energy 6MV, and dose rate 300cGy/min.2. Observation of the changes in cytokines and histology.The rats were randomly divided into 6 groups, including control (N) and radiation group (A, B, C, D and E). After radiation, the rats were killed at 1d, 7d, 14d, 28d and 56d. The lung tissue was stained with H. E. The expression of Bcl-2, Bax and TGF-β1 were examined using immunohistochemical SP method. Cell apoptosis was counted with flow cytometry.3. The Intervention role of IFN-γin radiation-induced lung injury.The rats were randomly divided into 4 groups, including 2 control groups and 2 treatment groups. The treatment groups were administrated with IFN-γ50,000IU per day and the control groups were administrated with sodium chloride 3ml per day. The rats were killed at 14d and 28d. The lung tissue was stained with H. E. The expression of Bcl-2, Bax and TGF-β1 were examined using immunohistochemical SP method.4. Statistical analysis.The outcome was analyzed with SPSS 13.0 software.Results1. Experimental study of cytokine and histology in radiation-induced lung injury.(1) Histological changes and apoptosis in radiation-induced lung injury.Histological changes in radiation-induced lung injury were divided into 3 stages, acute inflammatory stage, proliferative phase and fibrosis phase. TheⅡpulmonary alveoli epithelial cell of the apoptosis was clear at each stage in super-micro structural observation. The cell apoptosis index was obviously higher than control group on the first day after irradiation, and the difference was significant (P＜0.001). It indicated early radiation-induced lung injury, whereⅡalveoli epithelial cell began to apoptosis. And in the process the apoptosis ofⅡalveoli epithelial cell was still obvious. (2) The expression of Bcl-2 and Bax.In radiation groups, the expression of the thin bronchus endothelial cell and pulmonary alveoli endothelial cell Bcl-2 attenuated in a lung injury area, while the expression of mesenchymal cell enhanced obviously, particularly in the bronchus surroundings. The enhanced expression of Bax in bronchus epithelial cell and pulmonary alveoli epithelial cell in the lung injury area were also observed in the mesenchymal cell. The expression of Bcl-2 in mesenchymal cell and Bax in epithelial cell in radiation groups were higher than control group. The expression of Bax in epithelial cell of radiation group was higher than control group at 7, 14, 28 and 56d (P＜0.01). The expression of Bcl-2 in mesenchymal cell of radiation group was higher than control group at 14 and 28d (P＜0.01). However, compare to control group, the expression of Bcl-2 in epithelial cell and Bax in the mesenchymal cell didn't show the significant difference in radiation groups.(3) The expression of TGF-β1.In radiation groups, the expression of TGF-β1 in pulmonary alveoli epithelial cell, bronchus epithelial cell, endothelium cell and mesenchymal cell were obviously, and the expression of TGF-β1 was higher than control group at 14d, 28d and 56d (P＜0.01).2. The intervention role of IFN-γin radiation-induced lung injury.(1) In treatment groups, the pulmonary fibrosis was significantly lower than in control groups.(2) In treatment groups, the expression of Bcl-2 in mesenchymal cell attenuated, while the expression of Bax in mesenchymal cell enhanced. The expression of Bcl-2 in mesenchymal cell in treatment groups were lower than in control groups (P＜0.05).(3) The TGF-β1 expression in the treatment group was lower than in the control group at 28d (P＜0.05). Conclusion1. The histological changes of radiation-induced lung injury in rats were a dynamic course, while we could observed all histological changes at different time points. The apoptosis ofⅡalveolar epithelium cell happened at the early radiation-induced lung injury, and could be observed at different time points. It indicated that the apoptosis ofⅡalveolar epithelium cell played a important role in the development of radiation-induced lung injury.2. During the course of radiation-induced lung injury, the expression of Bcl-2 significantly increased and Bax decreased in mesenchymal cell, whereas the expression of Bcl-2 significantly decreased and Bax increased in epithelial cell. These changes suggested that the unbalance between apoptosis promoting factor and apoptosis inhibition factor might play a very important role in the development of radiation-induced lung injury.3. During the course of radiation-induced lung injury, the expression of TGF-β1 significantly increased. It suggested that TGF-β1 might play an important role in the development of cell apoptosis and pulmonary fibrosis.4. From the histological observation, we found that IFN-γmight inhibit pulmonary fibrosis. IFN-γcould inhibit the expression of Bcl-2 in mesenchymal cell, which resulted in increasing the ratio of Bax/Bcl-2. It suggested that IFN-γpromoted the apoptosis of mesenchymal cell. In addition, IFN-γcould inhibit the expression of TGF-β1 and the development of pulmonary fibrosis.
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