| Background and purpose:Radiation-induced pulmonary fibrosis(RIPF)is a common complication of chest tumor radiotherapy and nuclear radiation accidents,with an average incidence rate of 16%to 28%after radiotherapy.RIPF is a process that progresses from early interstitial pneumonia to latestage pulmonary fibrosis.It is mainly characterized by worsening dyspnea,deteriorating lung function,and ultimately respiratory failure and death,posing a considerable threat to human health.However,the pathogenesis of RIPF is still unclear,and the clinical treatment with drugs such as nintedanib and pirfenidone has limited efficacy.Therefore,it is urgent to elucidate the pathogenesis of RIPF,laying the foundation for its prevention and treatment.Studies have shown that the epithelial-mesenchymal transition(EMT)of alveolar epithelial cells(AECs)is one of the fibroblast sources involved in RIPF,and the damage and EMT of AECs play a crucial role in the pathogenesis of pulmonary fibrosis(PF).Multiple factors influence EMT,and recent studies have shown that thrombospondin-1(TSP-1)is involved in PF and EMT.TSP-1 is an extracellular matrix glycoprotein with multiple functions and plays biological roles in cell adhesion,migration,proliferation,senescence,apoptosis,angiogenesis,oxidative stress,inflammation,and immune regulation.It has been shown that TSP-1 is involved in transforming growth factor-β(TGF-β1)-induced EMT and bleomycininduced PF,but its role in radiation-induced EMT and PF has not been reported.Therefore,exploring the relationship between TSP-1 and radiation-induced EMT will provide experimental evidence for clarifying the pathogenesis of RIPF.In this study,AECs and mice were used to evaluate the role of TSP-1 in RIPF and the regulation of EMT,providing a new target for the prevention and treatment of RIPF.Methods:1.Evaluation of TSP-1 expression changes in RIPF(1)In vivo experiment:C57BL/6 mice were divided into the Control group(Control)and the irradiation group(IR).PF model was established by whole lung irradiation with 60Co γ-rays at 16 Gy.Micro-CT and lung coefficient were used to evaluate lung injury and lung consolidation.H&E staining was performed to observe the morphological changes in lung tissues,and Masson staining was performed to observe the collagen deposition in lung tissues.The protein and mRNA expression of TSP-1 in lung tissues were detected by immunohistochemistry,Western blot,and qRT-PCR.2.Investigating the effect of TSP-1 inhibition on radiation-induced EMT and PF(1)In vivo experiment:C57BL/6 mice and TSP-1 knockout mice were divided into the irradiation group(IR)and TSP-1 knockout group(IR+TSP-1-/-).PF models were established by whole lung irradiation with 60Co γ-rays at 16 Gy.Relevant experiments were conducted 12,16,and 24 weeks after modeling.Micro-CT and lung coefficient were used to evaluate lung injury and consolidation,H&E staining was performed to observe morphological changes,Masson staining and Sirius Red staining were performed to observe collagen deposition in lung tissue,and FlexiVent was used to evaluate lung function in mice.(2)In vitro experiment:RLE-6TN cells were used as research subjects and divided into four groups:negative control group(siTSP-1NC),TSP-1 transfection group(siTSP-1),negative control irradiation group(siTSP-1NC+IR),and TSP-1 transfection irradiation group(siTSP1+IR).The protein levels of TSP-1,E-cadherin,α-SMA,and N-cadherin in each group were assessed using Western blot.Additionally,the cells were observed for morphological changes under a light microscope.3.Exploring the mechanism of TSP-1-mediated cellular senescence in radiation-induced EMT3.1 Investigating the effect of TSP-1 inhibition on cellular senescence and oxidative stress(1)In vivo experiment:C57BL/6 mice and TSP-1 knockout mice were used as research subjects.Lung tissue senescence was examined by β-galactosidase(SA-β-Gal)staining,and the proteins of lung tissue senescence markers p21 and p16 were detected by immunofluorescence.ELISA was used for detecting lung tissue oxidative stress markers SOD,MDA,and GSH.(2)In vitro experiment:RLE-6TN cells were divided into four groups:control group(Con),transfection group(siTSP-1),irradiation group(IR),and TSP-1 transfection irradiation group(IR+siTSP-1).Flow cytometry was used to examine ROS levels,and cellular senescence was detected by SA-β-Gal staining.3.2 Exploring the mechanism of TSP-1-mediated cellular senescence in radiation-induced EMT(1)Investigating the reiationship between oxiaative stress and senescenceRLE-6TN cells were divided into four groups:control(Con),N-acetylcysteine(NAC),irradiation(IR),and irradiation with NAC(IR+NAC).ROS levels were measured by flow cytometry,and cellular senescence was detected using SA-β-Gal staining.(2)Evaluating the effect of senescence on radiation-induced EMT①RLE-6TN cells were induced to undergo cellular senescence with different concentrations of D-galactose(D-Gal).Cellular viability and senescence were measured using CCK-8 cell proliferation assay and SA-β-Gal staining,and the optimal concentration was selected to establish the cellular senescence model.②RLE-6TN cells were divided into four groups:control(Con),D-Gal,irradiation(IR),and irradiation with D-Gal(IR+D-Gal).Senescence markers p21 and p16 and EMT markers Ecadherin and N-cadherin were detected using Western blot.Results:1.TSP-1 is highly expressed in RIPFThe degree of lung injury and lung coefficient was higher in the IR group than in the Control group,as shown by micro-CT and lung coefficient.H&E and Masson’s staining revealed worse alveolar structure damage and increased collagen deposition in lung tissue in the IR group than in the Control group.Immunohistochemical,Western blot,and qRT-PCR results demonstrated that the expression of TSP-1 protein and mRNA was higher in the IR group than in the Control group.2.Inhibiting TSP-1 alleviates radiation-induced PF and EMT(1)The IR+TSP-1-/-group exhibited a decreased lung coefficient,reduced lung tissue injury,decreased collagen deposition,and improved lung function compared to the IR group,as indicated by H&E,Masson,and Sirius Red staining,and FlexiVent function,respectively.(2)Western blot revealed that E-cadherin protein increased and α-SMA and N-cadherin protein decreased in the siTSP-1+IR group compared with the siTSP-1NC+IR group.As observed under the light microscope,the number of mesenchymal cells was reduced in the si TSP-1+IR group.3.TSP-1-mediated cellular senescence promotes radiation-induced EMT3.1 TSP-1 inhibition alleviates lung tissue senescence and oxidative stress.(1)SA-β-Gal staining showed that the IR group had accelerated lung tissue aging while the IR+TSP-1-/-group had reduced lung tissue senescence.ELISA measurements indicated that SOD and GSH decreased,and MAD increased in the IR group.Compared with the IR group,lung tissue senescence and MAD decreased,whereas SOD and GSH increased in the IR+TSP1-/-group.(2)Flow cytometry and SA-β-Gal staining showed that the IR group had increased ROS and accelerated cellular senescence compared with the Con group.TSP-1 inhibition alleviated ROS and cellular senescence after radiation.3.2 Oxidative stress mediates cellular senescenceFlow cytometry and SA-β-Gal staining showed that ROS and cellular senescence were significantly increased in the IR group compared with the Con group.Compared with the IR group,the IR+NAC group had reduced levels of ROS and cellular senescence.3.3 Accelerated senescence promotes radiation-induced EMTThe cell senescence model was established using 20 mg/mL D-Gal based on SA-β-Gal staining and by CCK-8 cell proliferation assay.Western blot showed that compared with the IR group,the IR+D-Gal group had increased p21 and p16 proteins,decreased E-cadherin,and increased N-cadherin.As observed under the light microscope,The IR+D-Gal group had more mesenchymal cells than the IR group.Conclusion:1.A PF animal model was established by 60Co γ-rays irradiation of 16 Gy,and it was used to confirm the high expression of TSP-1 in RIPF.2.TSP-1 is involved in RIPF by regulating EMT,and inhibiting TSP-1 can effectively alleviate RIPF.3.This study is the first to demonstrate that TSP-1 influences RIPF and regulates radiationinduced EMT through oxidative stress-mediated cellular senescence. |
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