| Ding Gaofeng,Duan Qingke,Wang Wen,et al.Role of PINK1/Parkin mediated mitochondrial autophagy in radiation-induced mesenchymal transition of lung epithelial cells[J].Chinese Journal of Radiological Medicine and Protection,2025,45(7):629-636 |
| Role of PINK1/Parkin mediated mitochondrial autophagy in radiation-induced mesenchymal transition of lung epithelial cells |
| Received:July 18, 2024 |
| DOI：10.3760/cma.j.cn112271-20240718-00266 |
| KeyWords:Mitophagy X-ray irradiation Epithelial-mesenchymal transition (EMT) Radiation-induced pulmonary fibrosis (RIPF) |
| FundProject:国家自然科学基金(82004090);河南省中原青年拔尖人才项目(ZYQR201912189) |
| Author Name | Affiliation | E-mail | | Ding Gaofeng | Department of Radiation Oncology, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450003, China | | | Duan Qingke | Department of Radiation Oncology, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450003, China | | | Wang Wen | Department of Radiation Oncology, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450003, China | | | Lu Yufei | Department of Radiation Oncology, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, Zhengzhou 450003, China | yufeilu501@163.com |
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| Abstract:: |
| Objective To investigate the role of ionizing radiation in regulating mitochondrial autophagy and epithelial mesenchymal transaction (EMT) in lung, in order to provide experimental evidence for further elucidating the pathogenesis and clinical treatment of radiation-induced pulmonary fibrosis (RIPF). Methods Beas-2B cells were irradiated with 6 Gy X-rays, and their morphological changes were observed at 0, 12, 24 and 48 h after irradiation. The changes of mitochondrial autophagy and EMT-related proteins in PINK1/Parkin pathway were detected by Western blot assay. The changes of mitochondrial membrane potential were detected by JC-1 staining. TEM was used to observe the changes of cell ultrastructure 48h after radiation. Beas-2B cells were then divided into control group, irradiation group (RI), RI + vector plasmid group (RI+oeNC), RI+PINK1 overexpression group (RI+oePINK1), and the protein changes of FN1 and LC3 were detected by immunofluorescence. Flow cytometry was used to detect the change of reactive oxygen species (ROS) in each group. The changes of mitochondrial autophagy and EMT-related protein contents were detected by immunofluorescence, flow cytometry and Western blot, respectively. Results After X-ray irradiation, the cell morphology of human epithelial cells Beas-2B was changed from irregular polygon to spindle shape along with the time increase after irradiation, showing EMT appearance. JC-1 staining showed that, along with the time after irradiation, the red fluorescence was weakened, and the green fluorescence was enhanced, so that the red/green fluorescence ratio was decreased. TEM observation indicated that the cell morphology changed to spindle shape and the number of autophagic lysosomes decreased significantly at 48 h after irradiation. Western blot assay showed that the protein expression levels of PINK1, Parkin and Beclin1 were significantly decreased, while the expression of p62 protein was significantly increased after irradiation. Moreover, the expressions of E-cad and CK19 were significantly decreased, while the expressions of N-cad and Vim were significantly increased (t = 6.48, 3.72, 6.06, -18.71, P＜0.05). Immunofluorescence assay showed that LC3 expression was increased and FN1 expression was decreased in the oePINK1 group (t = 6.06, -21.49, -9.58, 3.58, P < 0.05). Flow cytometry assay showed that ROS in the oePINK1 group was significantly decreased (t = -342.54, 88.01, 25.48, P＜0.05). After PINK1 overexpression, the expression levels of PINK1, Parkin and Beclin1 were significantly increased, while the expression of p62 protein was significantly decreased (t = -25.57, -8.76, -11.24, 34.81, P＜0.05); meanwhile, the expressions of E-cad and CK19 were significantly increased, while the expressions of N-cad and Vim were significantly decreased (t =-7.12, 12.04, 67.92, -7.64, P＜0.05). Conclusions X-ray irradiation promoted EMT and impaired mitochondrial function of Beas-2B cells, and weakened mitochondrial autophagy mediated by PINK1/Parkin pathway. Overexpression of PINK1 promoted mitochondrial autophagy, which improved mitochondrial function and effectively inhibited cell EMT, thus alleviating pulmonary fibrosis. |
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