| 丁高峰,段青可,王雯,等.PINK1/Parkin通路介导线粒体自噬在辐射诱导的肺上皮细胞间充质转换中的作用机制研究[J].中华放射医学与防护杂志,2025,45(7):629-636.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].Chin J Radiol Med Prot,2025,45(7):629-636 |
| PINK1/Parkin通路介导线粒体自噬在辐射诱导的肺上皮细胞间充质转换中的作用机制研究 |
| Role of PINK1/Parkin mediated mitochondrial autophagy in radiation-induced mesenchymal transition of lung epithelial cells |
| 投稿时间：2024-07-18 |
| DOI：10.3760/cma.j.cn112271-20240718-00266 |
| 中文关键词: 线粒体 自噬 X射线 上皮间质转化 放射性肺纤维化 |
| 英文关键词:Mitophagy X-ray irradiation Epithelial-mesenchymal transition (EMT) Radiation-induced pulmonary fibrosis (RIPF) |
| 基金项目:国家自然科学基金(82004090);河南省中原青年拔尖人才项目(ZYQR201912189) |
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| 中文摘要: |
| 目的 探讨电离辐射调控线粒体自噬在辐射诱导的上皮细胞间质转化(EMT)中的作用及其机制,为放射性肺纤维化(RIPF)的发病机制及临床治疗提供实验依据。方法 用6 Gy X射线照射Beas-2B细胞,分别在照射后0、12、24和48 h观察细胞的形态变化。Western blot法检测线粒体自噬和EMT相关蛋白含量的变化;JC-1染色检测线粒体膜电位(MMP)的变化;透射电镜观测照射后48 h细胞内超微结构的变化。将Beas-2B细胞随机分为对照组、照射组(RI组)、照射+空载组(RI+oeNC组)、照射+PINK1过表达组(RI+oePINK1组)。利用免疫荧光法、流式细胞仪和Western blot检测线粒体自噬和EMT相关蛋白含量的变化。结果 随着照射后时间的延长,照射组细胞形态由不规则多边形转变为梭形,呈现出EMT表象;JC-1染色结果显示,红色荧光减弱,绿色荧光增强,红/绿荧光比值下降。照射后48 h,细胞内自噬溶酶体数量显著减少;随着时间延长,PINK1、Parkin和Beclin1蛋白表达水平均显著下降,p62蛋白表达显著上升(t=6.48、3.72、6.06、-18.71,P＜0.05);同时E-cad和CK19表达显著降低,N-cad和Vim表达显著升高(t=6.06、-21.49、-9.58、3.58,P＜0.05)。过表达PINK1组LC3表达增强,FN1表达减弱,活性氧(ROS)显著减低(t=-342.54、88.01、25.48,P＜0.05)。Western blot结果显示,过表达PINK1后,PINK1、Parkin和Beclin1蛋白表达水平均显著增加,p62蛋白表达显著降低(t=-25.57、-8.76、-11.24、34.81,P＜0.05);E-cad和CK19表达显著增加,N-cad和Vim表达显著降低(t=-7.12、12.04、67.92、-7.64,P＜0.05)。结论 X射线照射可促进Beas-2B细胞EMT并损害其线粒体功能,减弱PINK1/Parkin介导的线粒体自噬;过表达PINK1促进其线粒体自噬,可以改善其线粒体功能,有效抑制细胞EMT。 |
| 英文摘要: |
| 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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