Establishment And Possible Mechanism Of Radiation-induced Pulmonary Fibrosis Mouse Model

Purpose: Radiation-induced pulmonary fibrosis is a serious and irreversible complication after radiotherapy of clinicalthorcic tumors.This study is to investigate the possible mechanism of radiation-induced pulmonary fibrosis by establishing a mouse model of radiation-induced pulmonary fibrosis and exploring the role of type Ⅱ alveolar epithelial stem cells（AEC Ⅱ）in the injury repair process and the expression levels of the factors associated with fibrosis.Method: Part Ⅰ: Establishment of a mouse lung fibrosis model: A total of 112 female C57 BL / 6j mice at 8 weeks of age which have been shown to be sensitive to radiation,were randomly divided into an irradiated group（n=71）and a control group（n=41）.After the mice were anesthetized,they were fixed on a plate allowing only chest of irradiated mice to be exposed to X-rays Irradiated mice were given a total single dose of 20 Gy of X-rays,and the control group was received sham irradiation（0 Gy）.Accidental dead mice were recorded after irradiation,Mice were euthanized in 100% CO₂ and the lungs were collected at 24 hours,1 week,and 1 to 6 months after the irradiation.The right lungs were first put in liquid nitrogen and then stored at-80℃ for m RNA or protein analysis.The left lungs were fixed in neutral formalin for histopathological analysis.Hematoxylin / eosin（H & E）,Masson staining and Picro-Sirius Red staining were used to evaluate pathological changes of different lung injury stages.Part Ⅱ: Explore the dynamic phenotypes of AEC Ⅱ and the expression level of the factors related to fibrosis post-irradiation: Surface active protein C（Pro SP-C）is used as a marker of ACE Ⅱ cells,homeobox only protein X（HOPX）is applied as marker of type Ⅰ alveolar epithelial cells（ACE I）,Vimentin is a marker of mesenchymal cells.While TGF-β1 and β-catenin（β-catenin）are closely related to pulmonary fibrosis.The phenotypes of AEC Ⅱ were determined by immunofluorescence staining.Western blot and RT-PCR were used to quantify the protein and m RNA expressions.Result: Part Ⅰ: A single thoracic irradiation with 20 Gy X-ray was able to cause different lung damages.The histopathological results showed that bronchioli had a mucosal inflammation with epithelial hyperplasia and some alveolar spaces were filled with blood and inflammatory cells at 24 h and 1 month after irradiation.Many dilated capillaries with thickened walls were congested with erythrocyte and interstitial tissue was hyperplasia at 2 months post-irradiation.Fibrotic tissue was present at 3 months after irradiation,but it was more obvious at 5 months.Part Ⅱ 1.Pro SP-C: immunofluorescent staining showed the numbers of Pro SP-C positive（Pro SP-C+）cells decreased in irradiated mice at 3 months after irradiation compared to control group and several Pro SP-C+ cells scattered in the lungs after 5 or 6 months of irradiation（Fig.4a）.The expressions of Pro SP-C protein and m RNA in lung tissue were significantly decreased in irradiated groups compared with corresponding control group at 2-6 months after irradiation（Table 2-1/ 2-2）.2.HOPX: Double-color immunofluorescent staining showed that co-expression of pro SP-C / HOPX cells（Pro SP-C + /HOPX +）scattered at 3 months after irradiation,but no significant colocalization was seen at 5 months compared with the control group,and in the experimental group,HOPX positive cells（HOPX +）decreased significantly at 5 months after irradiation（Fig.4b）.In addition,it points that expression of HOPX protein and m RNA in the experimental group were significantly reduced compared with the control group at 4-6 months after irradiation（Table 2-1 / 2-2）.3.Vimentin: Double-color immunofluorescent staining showed that co-localized cells of pro SP-C and Vimentin（pro SP-C + /Vimentin +）in the experimental group Appeared at 3 months after irradiation,increased significantly after 5 months,but there were no co-localized cells in the control group（Fig.4c1/c2）.Moreover,the expression of Vimentin protein was increased at 2-6 months after irradiation compared with the control group（Table 2-1）.4.TGF-β1: The immunofluorescence showed that the TGF-β1 positive cells（TGF-β1 +）in the experimental group increased significantly at 3 months after irradiation,and a large number of TGF-β1 + cells were seen after 5 or 6 months.There are a small number of this cells in the control group（Fig.4d）.However,the expression of TGF-β1 protein and m RNA were lessen than the control group at 2 months after irradiation,while increased at 3-6 months after irradiation（Table 2-1 / 2-2）.5.β-catenin: The immunofluorescence showed that β-catenin positive cells（β-catenin +）increased significantly in the experimental group at 3 months after irradiation,and a large number of such cell were seen after 5 or 6 months.There was a small number of such cells in the control group.However,the results of β-catenin protein and m RNA in experimental group increased than the control group after 3-6 months（Table 2-1 / 2）.Conclusion: 8 weeks of C57 BL / 6j female mice with full-chest 20 GY irradiation,a mouse model of pulmonary fibrosis was successfully established.The change that decrease of pro SP-C + cells and pro-SP-C + / HOPX + cells,and increased of pro SP-C + /Vimentin + cell and TGF-β1 + cells,which were used to further confirm with Western blot and RT-PCR.It suggests that AEC Ⅱ are reduced differentiate into type I cells,but into mesenchymal cells.In addition,the immunofluorescent,Western blot and RT-PCR also showed that TGF-β1 and β-catenin increased simultaneously in pulmonary fibrosis tissue.Moreover.domestic and foreign literatures confirmed that there are multiple pathways between TGF-β1 and β-catenin.According to this,we speculate that TGF-β1 and β-catenin may be involved in the regulation of type Ⅱ cell differentiation.