The Differences Of The Process Of Radiation Pulmonary Injuries Between C57BL/6J And C3H/HeN Mice And The Underlying Mechanisms: A Comparative Study

Objective: Pulmonary fibrosis which may be caused by physical, chemical and biological factors, is characterized by the excessive deposition of collagen, resulting in respiratory failure. Although inter-individual variations in the development of pulmonary fibrosis have been reported, the pathogenesis of RPF at the molecular level is still unclear. This study was to investigate the differences of the process of RPI between C57BL/6J and C3H/HeN mice and the underlying molecular mechanisms by antibody microarray and tissue microarray, so as to find the molecules related with the fibrosis-prone. On the basis of that, some of these molecules will serve as new therapeutic target for this disease.Methods:(1) The thoraces of C57BL/6J and C3H/HeN mice were irradiated with 20Gy ⁶⁰Co gamma rays. Treated and sham-irradiated control mice were sacrificed at 1, 3 and 6 months after irradiation. Collagen deposition in the lung was determined by hydroxyproline measurement and sirius red staining. Cell proliferative activity in the lung was investigated by the histochemical staining of AgNORs. Expressions of FN, LN andα-SMA were determined by tissue microarray and immunohistochemical analysis. (2) Differences of protein expression in the lung in C57BL/6J and C3H/HeN mice in control group and 3 month postirradiation group were detected by antibody microarrays. (3) LFs in C57BL/6J and C3H/HeN mice were isolated, cultured and irradiated by 2, 4, 6 and 8Gy ⁶⁰Co gamma rays and then treated by 10 and 30nmol/L Raf1 kinase inhibitor I. The proliferative activity and cell cycle were detected by MTT colorimetry, AgNOR staining and FACS. Then the expressions ofα-SMA, MMP-1 and TIMP-1 in LFs were observed by immunocytochemical staining.Results: (1) The changes of the lung in C57BL/6J mice were as follows: the pathological changes undergone interstitial pneumonia, proliferation and fibrosis; significant increase of collagen deposition could be found; Besides that, FN was increased rapidly to a statistically significant higher level on 1 and 3 months after irradiation than that in the control, and then decreased gradually on 6 months after irradiation; LN was elevated gradually after irradiation; the expression ofα-SMA was intenser than that in C3H/HeN mice. The changes of the lung in C3H/HeN mice were as follows: the pathological changes mainly developed institial pneumonitis; no significant changes of FN expression were observed after irradiation compared to that in the control; LN was increased significantly on 1 and 3 months after irradiation, and then decreased gradually. (2) RPI tissue microarrays were established, of which HE and IHC staining effects were identified with that of common sections. (3) Overexpression of DLP1 were detected in the lung in the C3H/HeN mice in control group. At 3 months after irradiation, overexpression of Raf1, TRF2 and HO1 were detected in the lung in C57BL/6J mice, whereas overexpression of MST3 and GSPT2 were detected in the lung in C3H/HeN mice. (4) The proliferative activity of LFs of C57BL/6J mice was not promoted by 2, 4, 6 and 8Gy Co gamma rays, but the number of aneuploid cells wereincreased. MMP-1 in LFs of C57BL/6J mice was weak positive, while the expressions ofα-SMA and TIMP-1 in LFs of C57BL/6J mice were enhanced by ⁶⁰Co gamma rays. The proliferative activity of LFs of C3H/HeN mice was not promoted by 2, 4, 6 and 8Gy ⁶⁰Co gamma rays either, but the number of cells in G₂/M phase increased. The expressions of MMP-1 and TIMP-1 in LFs of C3H/HeN mice were enhanced, while the expression ofα-SMA was decreased by ⁶⁰Co gamma rays. (5) As for the LFs in C57BL/6J mice were treated by Raf1 kinase inhibitor I, the expression ofα-SMA was decreased and the number of aneupoid cells was not increased.Conclusion: (1) The models of prone-RPF and resistant-RPF were satisfactorily established by irradiated with gamma rays, C57BL/6J mice developed RPF characterized by the significant accumulation of collagen. C3H/HeN mice did not develop RPF. (2) RPI-related tissue microarray was established. (3) Overexpression of Raf1, TRF2 and HO1 in the lung may be positively associated with the RPF, while overexpression of MST3 and GSPT2 in the lung may be negatively associated with the RPF. (4) Different phenotype and radiosensitivity of LFs in C57BL/6J and C3H/HeN mice may be related with prone-RPF or resistant-RPF. (5) Raf1 kinase inhibitor I could inhibit the activation of radiated LFs of C57BL/6J mice, which may be a potentially feasible and effective target for the prevention of RPF.