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Radiation-induced pulmonary fibrosis (RIPF) is one of the most serious late complications after nuclear radiation accident, bone marrow transplantation pretreatment and thoracic tumor radiotherapy. The formation process of RIPF is complicated and the pathogenesis has not been fully elucidated. Recent studies have shown that radiation-induced epithelial-mesenchymal transition (EMT) of lung epithelial cells is an indispensable segment of RIPF. This article reviews the role of radiation-induced lung EMT in the occurrence and development of RIPF and related drugs with EMT as a potential therapeutic target, providing ideas for the development of therapeutic drugs for RIPF in the future.
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Radiation induced lung injury (RILI) is a common complication after radiation therapy of breast tumors and bone marrow transplantation pretreatment, and it is a critical limiting factor of radiotherapy doses in patients. Once RILI progresses to the radiation-induced pulmonary fibrosis stage, it seriously reduces the patient’s quality of life, while causing the patient’s respiratory failure and eventually leading to death. Ionizing radiation (IR) can induce cell injuries, including apoptosis, epithelial-mesenchymal transition, senescence, pyroptosis and ferroptosis, and these injuries can play an important role in the occurrence and development of radioactive lung injury. Starting from discussion of the occurrence of different forms of injury in different cells after IR stimulation, this review summarizes the pathogenesis of RILI and its clinical prevention and treatment.
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Objective:To establish the mouse model with radiation-induced pulmonary fibrosis, and to identify and analyze it from the aspects of function, imaging and pathology.Methods:Thirty C57BL/6 mice were randomly divided into the control group, 16 Gy irradiation group and 20Gy irradiation group. The mice in the irradiation groups received a single 16 Gy or 20 Gy chest X-ray irradiation, and underwent functional examination, imaging examination and pathological examination at 3 and 6 months after irradiation.Results:At 6 months after irradiation, hair on the chest and back of the mice turned white and fell off, and the airway resistance was increased significantly. CT images showed extensive patch shadows and consolidation in the lung. Three dimensional reconstruction suggested that the lung of mice was distorted and deformed, and the volume was decreased significantly. Pathological examination confirmed that there was extensive pulmonary fibrosis.Conclusions:Significant pulmonary fibrosis occurs after 6 months of chest irradiation in mice. The animal model of radiation-induced pulmonary fibrosis in C57BL/6 mice was successfully established.
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Objective:To investigate whether transplantation of gingival mesenchymal stem cells (GMSCs) can inhibit radiation-induced senescence of alveolar epithelial cells type Ⅱ (AECⅡ) and its role in the prevention of radiation-induced pulmonary fibrosis (RIPF).Methods:Mouse type Ⅱ alveolar epithelial cells (MLE12) were irradiated with 6 Gy X-rays and then co-cultured with GMSCs. The extent of cellular senescence of MLE12 cells was assessed by cell morphology, β-Gal staining, and senescence secretion-associated phenotype (SASP) assay. RIPF model was constructed by unilaterally irradiating the right chest of C57BL/6 mice with 17 Gy X-rays. GMSCs were transplanted 1 d after irradiation. At 180 d after irradiation, the pulmonary organ ratio, HE staining, and Masson staining were used to assess intra-pulmonary structure and interstitial collagen deposition in the lung. β-Gal immunohistochemistry and immunofluorescence co-localization with AECⅡ were measured to assess the degree of cellular senescence in the lung. The SASP expression changes in lung tissue were detected by qRT-PCR. The protein expressions in P53-P21 and P16 pathways were detected by Western blot assay. P21 expression in AECⅡ was detected by immunofluorescence co-localization assay.Results:GMSCs effectively inhibited radiation-induced senescence of MLE12 cells, reduced the ratio of radiation-elevated β-Gal positive cells by 11.8% ( t=6.72, P<0.05), and decreased the expressions of SASP (IL-6, IL-8, IL-1β) ( t=28.43, 28.43, 4.82, P<0.05). GMSCs transplantation improved the survival rate of irradiated mice, prevented radiation-induced alveolar structural collapse thickening and collagen deposition, reduced the number of senescent cells in the irradiated lung tissues by 23.9% ( t=21.83, P<0.05), and inhibited the expressions of SASP ( t=8.86, 20.63, P<0.05). GMSCs also inhibited the expression of P53-P21, P16-related proteins in MLE12 cells and lung tissues of mice after irradiation. Conclusions:GMSCs inhibit senescence-related P53-P21 and P16 pathways, prevent radiation-induced AECⅡ senescence, as well as the development of RIPF.
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Radiation-induced pulmonary fibrosis (RIPF) is a common complication of thoracic tumor radiotherapy. The main manifestation of radiation-induced pulmonary fibrosis is chronic progressive consolidation of pulmonary interstitium, which may cause the lung physiology function reduced or even lost. Furthermore, it can be lethal forrespiratory failure in severe cases. Recent studies have found that mesenchymal stem cells (MSC) play an important role in the modulation of proliferation and the activation of immune cells in lung inflammation. In addition, MSC can also play a part in the treatment of RIPF by differentiating into functional cells and secreting cytokines. Therefore, MSC has a good application prospect in RIPF as a cell therapy method. This article reviews the molecular mechanisms, influencing factors and current status of MSC therapy in RIPF.
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Objective: To observe the effect of neuroopilin-1 (NRP1) gene on the process of radiation-induced pulmonary fibrosis (RIPF), and to explore its roles in the occurrence and development of epithelial-mesenchymal transition (EMT) mediated by Wnt/fi-catenin pathway tail identification was performed in and TGF-β1/Smads pathway, and extracellular matrix (ECM) deposition. Methods: The Cre-LoxP recombinase system was used to construct the transgenic C57BL/6J mice with NRP1 gene specific knockout in alveolar type II epithelial cells (AT-II) and the mice. A total of 160 mice were randomly divided into 4-week group, 8-week group, 16-week group and 24-week group. In each group, the mice were randomly divided into wild type (Con) group, wild type+irradiation (IR) group, NRP1 gene-specific knockout (KO-Con) group, NRP1 gene-specific knockout+irradiation (KO+IR) group according to the method of random number table; there were 10 mice per group. In KO-Con and KO + IR groups, the NRP1 gene was specifically knocked out in the AT-II cells by intraperitoneal injection of tamoxifen, and the mouse models of RTPF were established by 20 Gy total thoracic irradiation in IR group and KO+IR group. After the models were constructed, HE staining and Masson staining were used to verify whether the models were successfully constructed. Immunohistochemistry (IHC) method was used to detect the type I collagen (Col I) and crsmooth muscle actin α-SMA) protein expression levels; Western blotting method was performed to detect the NRP1, β-catenin, TGF-β1, and Smad2 protein expression levels in the lung tissue of the mice; Quantitative fluorensence real-time polymerase chain reaction (qRT-PCR) method was used to detect the expression levels of NRP1, Col I, α-SMA, β-catenin, TGF-βl, Smad2, E-cadherin, N-cadherin, and Vimentin mRNA in the lung tissue of the mice. Results: The results of HE and Masson staining showed the RTPF models were successfully established, and the lung tissue of the mice in IR group mainly showed the pathomorphology of radiation pneumonitis. Compared with Con group, the protein and mRNA expression levels of NRP1 in the lung tissue of the mice in IR group were gradually increased with the prolongation of time (P<0.05), and reached the highest at 24 weeks (P<0.01). Compared with Con group, the expression levels of Col I, α-SMA, β-catenin, TGF-β1, and Smad2 proteins and mRNA in the lung tissue of the mice in IR group and KO+IR group were increased gradually with the prolongation of time (P<0.05 or P<0.01). Compared with IR group, the expression levels of Col I, α-SMA, β-catenin, TGF-β1, and Smad2 protein and mRNA in the lung tissue of the mice in KO+IR group were significantly decreased (P<0.05 or P<0.01), but they were higher than those in Con group (P<0.05 or P<0.01). Compared with Con group, the expression levels of the epithelial cell marker E-Cadherin mRNA in the lung tissue of the mice in IR group and KO+IR group were gradually decreased with the prolongation of time (P< 0.01), and the expression levels of the interstitial cell markers N-Cadherin and Vimentin were increased (P<0.05 or P<0.01), but the expression levels of E-cadhern mRNA in the lung tissue of the mice in KO-IR group were significantly higher than those in IR group (P<0.05 or P<0.01), and the expression levels of N-Cadherin and Vimentin mRNA in the lung tissue of the mice in KO + IR group at each time point were lower than those in IR group (P<0.05 or P<0.01). Conclusion: Knockout of NRP1 gene can inhibit the occurrence and development of RTPF, and its mechanism may be involved in regulating the expressions of Wnt/fi-catenin and TGF-β1/Smads signaling pathways in the lung tissue and inhibiting the EMT process in the mice.
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Aim To observe the correlation between the TPL's suppression on myofibrolbasts(MFBs)activation and TGF-β1/ERK/Smad3 pathway by performing in vivo and in vitro experiments.Methods In vitro model of MFBs activation was set up by stimulating fibroblasts with TGF-β1,and in vivo model of MFBs activation in radiated lung tissue was built by thoracic radiation on C57BL/6 mice.MFBs activation was analyzed by detecting the expression of α-SMA(using RT-PCR and Western blot)and Col Ⅰ(using RT-PCR and ELISA methods).The levels of p-ERK,p-Smad3(Ser208)and p-Smad3(Ser423)were measured by Western blot.ERK siRNA and Smad3 siRNA were used to observe the status of ERK and Smad3 in MFBs activation.Results TGF-β1 activated p-ERK/p-Smad3(Ser208)and p-Smad3(Ser423),increased the expression of α-SMA and synthesis of Col Ⅰ,which indicated MFBs activation.siRNA knockdown assay showed that both ERK and Smad3 were involved in regulating the levels of α-SMA and Col Ⅰ,and ERK influenced MFBs transformation possibly through its phosphorylation of Smad3(Ser208).TPL treatment inhibited the phosphorylation activation of ERK,Smad3(Ser208),Smad3(Ser423)in vitro and in vivo,therefore significantly reduced the level of α-SMA and Col Ⅰ,and the number of activated MFBs was decreased.Conclusion TPL mitigates radiation-induced pulmonary fibrosis by inhibiting the activation of MFBs,which is partly through suppressing TGF-β1/ERK/Smad3 pathway.
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Normal lung tissues are inevitably exposed to X-ray in thoracic radiotherapy,causing radiation-induced lung injury (RILI).The main pathological manifestations include the accumulation of inflammatory cells,release of cytokines,accumulation and proliferation of fibroblasts,and excessive deposition of alveolar interstitial collagen in the irradiated region.RILI severely affects the treatment compliance and quality of life and even threatens the life in the patients receiving radiotherapy.In recent years,numerous studies have found that Th1/Th2 imbalance is closely associated with the development and progression of RILI,and the cytokine network plays an executive role in the progression of RILI.Therefore,restoring the Th1/Th2 balance in vivo may provide a new way to prevent and treat RILI.
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Objective To investigate the protective effects and mechanism of LY2109761 in radiation-induced pulmonary fibrosis (RIPF), and to provide a clue for the clinical treatment of RIPF. Methods A total of 40 healthy female C57BL/6 mice were enrolled and were divided into 4 groups with 10 mice in each group randomly, including blank control group, single irradiation group, irradiation +LY50 group and irradiation + LY100 group which were given 50 and 100 mg/kg of LY2109761 respectively, twice per day. The whole lungs were irradiated by 6 MV X-rays of 12 Gy. At 2 weeks post-irradiation, pathological analyses were performed for each group to evaluate the histological changes in the irradiated lungs, including hematoxylin and eosin staining ( HE ) to observe morphologic changes, the Masson staining to estimate the degree of fibrosis and a combination of immunohistochemistry to analyze the expression levels of TGF-β1 and Smad2. Results Compared with control group, the degree of fibrosis of the lung including alveolar structure changes, fibroblast proliferation and alveolar spacing was significantly enhanced at 14 d post-irradiation. Compared with irradiation group, the fibrosis degree in irradiation +LY100 group was significantly reduced(t=4. 668,P<0. 05). The degree of inflammation and fibrosis of the lung in irradiation+LY50 didn′t obviously reduce compared with irradiation group ( P>0. 05 ) . The result of immunohistochemical assay showed that, the level of protein expressions of TGF-β1 and Smad2 was increased significantly after irradiation(t =13. 545, 6. 418, P <0. 05), and obviously decreased in irradiation+LY100 group (t=15. 263, 6. 855, P<0. 05), but just slightly reduced in irradiation+LY50 group (P>0. 05). Conclusions LY2109761 has protective effect in radiation pulmonary fibrosis possibly by down regulating the expressions of TGF-β1/Smad2.
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Objective To investigate the protective effects and mechanism of LY2109761 in radiation-induced pulmonary fibrosis (RIPF), and to provide a clue for the clinical treatment of RIPF. Methods A total of 40 healthy female C57BL/6 mice were enrolled and were divided into 4 groups with 10 mice in each group randomly, including blank control group, single irradiation group, irradiation +LY50 group and irradiation + LY100 group which were given 50 and 100 mg/kg of LY2109761 respectively, twice per day. The whole lungs were irradiated by 6 MV X-rays of 12 Gy. At 2 weeks post-irradiation, pathological analyses were performed for each group to evaluate the histological changes in the irradiated lungs, including hematoxylin and eosin staining ( HE ) to observe morphologic changes, the Masson staining to estimate the degree of fibrosis and a combination of immunohistochemistry to analyze the expression levels of TGF-β1 and Smad2. Results Compared with control group, the degree of fibrosis of the lung including alveolar structure changes, fibroblast proliferation and alveolar spacing was significantly enhanced at 14 d post-irradiation. Compared with irradiation group, the fibrosis degree in irradiation +LY100 group was significantly reduced(t=4. 668,P<0. 05). The degree of inflammation and fibrosis of the lung in irradiation+LY50 didn′t obviously reduce compared with irradiation group ( P>0. 05 ) . The result of immunohistochemical assay showed that, the level of protein expressions of TGF-β1 and Smad2 was increased significantly after irradiation(t =13. 545, 6. 418, P <0. 05), and obviously decreased in irradiation+LY100 group (t=15. 263, 6. 855, P<0. 05), but just slightly reduced in irradiation+LY50 group (P>0. 05). Conclusions LY2109761 has protective effect in radiation pulmonary fibrosis possibly by down regulating the expressions of TGF-β1/Smad2.
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External beam radiotherapy is a major treatment in the management of cancer .However radia-tion induced pulmonary fibrosis is common .It not only leads to quality -of-life issues in survivors and compro-mise treatment ,but also may even be lethal in outcome .Transforming growth factor β( TGF-β) seems to be a key mediator of fibrosis .TGF-βpromotes the differentiation of fibroblasts into myofibroblasts while the differentiation and activation of fibroblasts and myofibroblastsmay produce the components of scar tissue, which is one of the key pathogenic processes in lung fibrosis .Peroxisome proliferator activated receptors ( PPARs ) are ligand activated transcription factors that belong to the nuclear hormone receptor superfamily .PPARγis also a key regulator of fi-broblast differentiation .Many studies have shown that both natural ligand and synthetic ligand of PPARγcan sup-press fibrosis by interfering with TGF -βsignaling and some other approaches .The antifibrotic effects of PPARγhave been reported to be involved in both PPARγdependent and PPARγindependent mechanisms .PPARγago-nists may have potential for the therapy of radiation induced pulmonary fibrosis .
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PURPOSE: Radiation-induced pulmonary fibrosis (RIF) is a significant complication of radiotherapy for lung cancer. Despite the large number of studies, the molecular mechanisms of RIF are poorly understood. Therefore, the complex protein expression pattern in RIF was characterized by identifying the proteins with an altered expression level after thorax irradiation using two-dimensional electrophoresis (2-DE) and mass spectrometry. MATERIALS AND METHODS: A mouse model of RIF was used to examine the alteration of the lung proteome because of availability of murine data related to human cases and the abundance of murine fibrotic lung samples. A mouse model of RIF was induced in radiosensitive C57BL/6 mice. Twenty-one weeks after 25 Gy irradiation, hematoxylin-eosin staining and hydroxyproline assay confirmed the early-phase pulmonary fibrosis. RESULTS: Lung samples from the irradiated and age-matched control mice were used to generate 16 high quality 2-DE gels containing approximately 1,000 spots. Of the 31 significantly up- or down-regulated protein spots, 17 were identified by MALDI-TOF/MS. CONCLUSIONS: Two important upregulated proteins were found, the alpha-protease inhibitor and galectin-1, which might be used as potential markers for the early phase of RIF.
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Animals , Humans , Mice , Electrophoresis , Galectin 1 , Gels , Hydroxyproline , Lung , Lung Neoplasms , Mass Spectrometry , Proteome , Proteomics , Pulmonary Fibrosis , Radiotherapy , ThoraxABSTRACT
Purpose: To evaluate the HPQP as a modifier of radiation-induced pulmonary fibrosis in rats sacrificed 4 months and 6 months separately after a single dose of 0, 15, 20, 25 Gy60Co r rays to the right hemithorax. Methods: HPQP was administered by a stomach tube 2 times a week after irradiation at a regimen of 100mg/kg. Pulmonary fibrosis was evaluated by lung hydroxyproline( HP) content and histological observation( LM and EM). Results: The content of lung HP increased with increasing radiation dose. The differences between control group and 20,25 Gy group are significant P
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Objective To study whether the expression Smad 7 protein by the recombinant adenovirus with Egr-1 promoter and Smad 7 cDNA in fibroblast cell can block the signal transduction pathway of transforming growth factor-beta1 (TGF-?1) under irradiation thereby inhibiting collagen synthesis in vitro. Methods The location of endogenous Smad 7 and exogenous Smad 7 protein in recombinant adenovirus infected fibroblast cells(3T6) were determined by immunocytochemical method. The infected 3T6 cells were irradiated and then cultured with TGF-?1 4 hours after irradiation. The activity of preventing radiation-induced fibrosis by expression Smad 7 protein was evaluated by the amount of collagen synthesis and proliferation of 3T6 cells. The amount of collagen synthesis was shown by the coruscant per minute (cmp) through the 3?H-Proline incorporation technique. Results The endogenous Smad 7 and exogenous Smad 7 protein both were located in the cytoplasm. When cultured with TGF-?1 4 hours after irradiation, the amount of collagen synthesis in the 3T6 cells infected with the recombinant adenovirus was significantly less than that in the cells without infecting adenovirus after irradiation(P=0.001), But, there was no difference in the proliferation of 3T6 cells between those with and without adenovirus infection (P= 0.312 ). Conclusions The Egr-1 promoter in the recombinant adenovirus can regulate the expression of downstream Smad 7 cDNA in 3T6 cells. The expression Smad 7 protein could block the TGF-?1 signal transduction pathway thereby inhibiting the collagen synthesis. The mechanism of inhibiting the collagen synthesis may be accomplished at the transcription level.