| Objective:The radiation-induced lung injury(RILI)is an inevitable complication of radiotherapy for thoracic malignant tumors,which seriously affects the therapeutic effect of tumors.The main clinical strategy for this disease is steroid pulse therapy,though,with poor treatment effect.With the ability of self-renewal and multi-directional differentiation,mesenchymal stem cells are involved in the repair of tissue damage,and become the"seed cells"for the treatment of a variety of clinical diseases.Adipose-derived mesenchymal stem cells(Ad-MSCs)have the advantages of sufficient source and autologous transplantation,and have been widely used in basic scientific research and clinical trial.The aim of this study is to observe the repair effect of Ad-MSCs on radiation-induced pulmonary fibrosis and explore the related regulatory mechanism to provide a theoretical basis for clinical prevention and intervention of RILI.Method:(1)In vivo and in vitro models of RILI were established.First,male C57BL/6mice were selected to establish RILI model,then H&E staining and Masson staining were used to analyze the pathomorphological changes of irradiated lung tissue to verify whether the RILI mouse model was successfully established,and immunohistochemistry was used to detect the expression of fibrosis markersα-SMA and TGF-β.The concentration of TGF-βin peripheral blood was measured by ELISA.Then,A549,BEAS-2B and HLF-1 cells were irradiated with different doses,and the expressions of epithelial-mesenchymal transition markers(Vimentin and E-cadherin),fibroblast proliferation mediators(α-SMA and DKK-1)were detected by Western blot at different time points.The conditions for establishing RILI cell model were optimized.(2)The anti-fibrotic effect of Ad-MSCs in RILI cell model was evaluated.Firstly,Western blot and q PCR were used to compare the expression of DKK-1 in different tissue sources,different donors and different passages of MSCs to optimize the conditions for stable high expression of DKK-1 in mesenchymal stem cells.Then DKK-1 reagent was used to intervene RILI cell model,and the expression of p-β-catenin,Vimentin and E-cadherin was detected by Western blot.The RILI cell model was further intervened with Ad-MSCs supernatant.Western blot,immunofluorescence staining and q PCR were used to detect the expression of Wnt signaling pathway markers,fibrosis markers and target genes related to epithelial-mesenchymal transition.Finally,DKK-1 monoclonal antibody was added to detect the changes of the above indicators again to further clarify whether the regulatory effect of Ad-MSCs on Wnt signaling pathway depends on DKK-1.(3)The effect of the infused Ad-MSCs via tail vein on inhibiting pulmonary fibrosis in an animal model.The Ad-MSCs marked with luciferase were injected into RILI mice through the tail vein.The distribution of Ad-MSCs were tracked by a biological imaging system to determine the specific aggregation trend of Ad-MSCs in the irradiated lung.The Ad-MSCs were then infused back into RILI mice via tail vein.The repair effect of Ad-MSCs on radiation-induced pulmonary fibrosis in mice was evaluated from the aspects of gross lung specimens,lung tissue pathology,pulmonary fibrosis-related proteins and inflammatory factors.Results:(1)Firstly,a mouse model of RILI was established to evaluate the degree of injury and the progression of fibrosis.At 6 months after whole-chest irradiation with a single dose of 20 Gy,the histopathological features of lung tissue in mice showed typical fibrosis:The alveoli collapsed,the alveolar septum thickened,and there were obvious inflammatory cell infiltration and a large number of blue collagen fibers deposited around the bronchioles.Immunohistochemistry showed that the expression ofα-SMA and TGF-βprotein was increased.The concentration of TGF-βin peripheral blood gradually increased with time after irradiation,and reached the highest level at 6 months after irradiation.Secondly,the RILI cell model was established to optimize the modeling conditions.At 72h after 6 Gy-X-ray irradiation,the expressions of Vimentin andα-SMA in A549,BEAS-2B and HLF-1 cells reached the peak,and the expression of E-cadherin was the lowest,which indicated that the in vitro model of radiation-induced pulmonary fibrosis was successfully established.At the same time,it was found that ionizing radiation significantly reduced the protein level of DKK-1 in A549,BEAS-2B and HLF-1 cells.It is suggested that the decrease of DKK-1 expression is related to the progression of pulmonary fibrosis in mice.(2)The anti-fibrotic effect of Ad-MSCs in cell model of pulmonary fibrosis was evaluated.The stably and highly expressed DKK-1 in Ad-MSCs were significantly higher than those of BM-MSCs and UC-MSCs in terms of its protein and m RNA levels.Its expression level was not affected by the source of donor and the number of cell passages.The DKK-1 reagent-intervened in vitro RILI model indicated that DKK-1 inhibited epithelial-mesenchymal transition by inhibitingβ-catenin phosphorylation.The results of in vitro RILI model treated with Ad-MSCs supernatant showed that Ad-MSCs supernatant reduced the expression of p-β-catenin and TCF4 protein,and could reverse the effect of ionizing radiation on the expression of Vimentin,E-cadherin andα-SMA.Apart from that,it inhibited the migration of p-β-catenin to the nucleus and down-regulated the expression of Snail,Twist and c-Myc.All these effects were reversed when DKK-1 was neutralized in Ad-MSCs supernatant.(3)The effect of the infused Ad-MSCs via tail vein on inhibiting pulmonary fibrosis in an animal model.The fluorescein-marked Ad-MSCs infused via tail vein efficiently accumulated in the irradiated lungs of mice.The lung tissue of the irradiated group showed consolidation,with a dark red surface,scattered bleeding points,and poor elasticity.However,the lung tissue recovered elastic and pink,and the bleeding spots almost disappeared in the mesenchymal stem cell treatment group.In the treatment group,the pathological damage of lung tissue was significantly reduced,the collagen deposition was significantly reduced,and the alveolar structure was repaired to close to normal lung tissue.The changes in the expression of pulmonary fibrosis markers Vimentin,E-cadherin,α-SMA,p-β-catenin and TCF4were consistent with the in vitro study.Immunohistochemistry showed that the expressions ofα-SMA and TGF-βin the treatment group were significantly lower than those in the irradiation group.After X-ray irradiation,the levels of TGF-βand IL-10 in serum were increased.After the treatment of mesenchymal stem cells,TGF-βwas decreased,while IL-10 was further increased.Conclusion:(1)Whole chest irradiation with a single dose of 20 Gy resulted in up-regulated expression of Wnt/β-catenin signaling pathway markers p-β-catenin and TCF4,up-regulated expression of Vimentin,and decreased expression of E-cadherin in lung tissue of mice.The expression of fibroblast proliferation mediatorα-SMA and epithelial-mesenchymal transition related target genes Snail,Twist and c-Myc were up-regulated.The occurrence of epithelial-mesenchymal transition and the activation of fibroblasts are the key events in radiation-induced pulmonary fibrosis,which accelerate the process of pulmonary fibrosis.(2)Wnt signaling pathway was activated by ionizing radiation,which promoted the phosphorylation and translocation ofβ-catenin to the nucleus,up-regulated the expression of Snail,Twist and c-Myc genes,and induced epithelial-mesenchymal transition in A549 and BEAS-2B cells and activation of HLF-1 cells.(3)In RILI cell model,Ad-MSCs supernatant reduced the expression of Wnt/β-catenin signaling pathway markers,and reversed the effects of the ionizing radiation on the expression of proteins and genes related to epithelial-mesenchymal transition and fibroblast activation.All these effects were reversed when DKK-1 was neutralized in Ad-MSCs supernatant.In RILI mouse model,CXCR4~+Ad-MSCs were preferentially enriched in the irradiated injured parts of lung.With this property,the repair of radiation-induced pulmonary fibrosis can ameliorate the process of pulmonary fibrosis through DKK-1 mediated Wnt/β-catenin signaling pathway.This research project focuses on the prevention and treatment of radiation-induced pulmonary fibrosis,a worldwide challenge.Considering the limited effectiveness of current treatments,and a need to explore more efficient drug targets or therapeutic approaches,we utilize the chemotactic properties of Ad-MSCs towards the injured site,inhibiting the Wnt/β-catenin signaling pathway through DKK-1,intervening in the early stages of pulmonary fibrosis progression,and providing a theoretical basis for the clinical prevention and treatment of pulmonary fibrosis. |
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