ABSTRACT
NF-E2-related factor 2 (Nrf2) is a key transcription factor recently implicated in the control of radiation-induced lung fibrosis (RILF). However, the molecular mechanism of Nrf2 in the pathogenesis of RILF is still unclear. The purpose of this study was to evaluate the regulatory effect and mechanism of Nrf2 in the pathogenesis of RILF. The effects of different Nrf2 expression levels on RILF were explored in vitro and in vivo. The RILF model of Nrf2 knockout mice was established for in vivo study. In the study of the mechanism of action, ChIP-seq assay and metabolomics analysis were performed. The discovered mechanism of Nrf2-mediated RILF alleviation was further validated in vitro and in vivo. We found that overexpression of Nrf2 significantly alleviated the fibrosis caused by irradiation in vivo and in vitro. Conversely, Nrf2 silencing strongly aggravated the development of RILF. Mechanistically, Nrf2 signaling increased the expression of piwi-like RNA-mediated gene silencing 2 (PIWIL2), leading to the alteration of purine metabolism and contributing to the relief of RILF. These results suggest that Nrf2 promotes the attenuation of RILF in vivo and in vitro by directly targeting PIWIL2 and activating purine metabolism.
ABSTRACT
Radiation-induced lung fibrosis (RILF) is a complication of radiotherapy in thoracic cancer patients. Thalidomide (THD) has a therapeutic effect on fibrotic and inflammatory disorders. The purpose of the current study was to investigate the therapeutic effect of THD on RILF in mice and better understand the underlying regulatory mechanisms of the therapeutic effect. We found that THD mitigated the fibrosis caused by irradiation in mice. The action of THD on RILF was related to the elevation of low levels reactive oxygen species (ROS), which inhibited the transforming growth factorß (TGFß)/Smad3 signaling pathway through activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Analysis of the therapeutic effect of THD using Nrf2-/- mouse model confirmed the role of Nrf2 in vivo. In addition, no radioprotective effect of THD on thoracic cancer cell lines was observed. In conclusion, these data showed that THD attenuated RILF in mice, which was mediated by Nrf2-dependent down-regulation of the TGF-ß/Smad3 pathway, suggesting THD as a potential novel agent for RILF prevention.
Subject(s)
Pulmonary Fibrosis/prevention & control , Radiation Injuries, Experimental/prevention & control , Radiation-Protective Agents/pharmacology , Smad3 Protein/metabolism , Thalidomide/pharmacology , Transforming Growth Factor beta/metabolism , X-Rays/adverse effects , A549 Cells , Animals , Cell Line, Tumor , Epithelial Cells , Female , Gene Expression Regulation , Humans , Lung/drug effects , Lung/metabolism , Lung/pathology , Lung/radiation effects , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Pulmonary Fibrosis/etiology , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Radiation Injuries, Experimental/etiology , Radiation Injuries, Experimental/metabolism , Radiation Injuries, Experimental/pathology , Reactive Oxygen Species/agonists , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Signal Transduction , Smad3 Protein/antagonists & inhibitors , Smad3 Protein/genetics , THP-1 Cells , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/geneticsABSTRACT
AIM: To obtain fetal mesenchymal stem cells isolated from human placental tissue (hfPMSCs) and study the effects of IFN-γ on immunomodulatory property of hfPMSCs in vitro. METHODS: hfPMSCs were isolated from the fetal side of human placentas by enzyme digestion. Expressions of cell surface antigens CD73, CD90, CD105, CD14, CD34, CD45 and HLA-DR were tested by flow cytometry. The fetal origin of the placental cells was verified by DNA identity test using four known short tandem repeat (STR) polymorphic markers D2S1399, D10S2325, D18S535 and GATA198B05. After hfPMSCs were treated with 10 ng/mL IFN-γ, the expressions of IL-6, IL-8, IL-10 and HGF at both mRNA and protein levels were measured using quantitative real-time PCR (q-PCR) and ELISA. The expressions in controls without IFN-γ treatment were examined in the same way. RESULTS: The isolated cells expressed MSCs surface markers CD73, CD90 and CD105, but did not express CD14, CD34, CD45 and HLA-DR. For all the STR markers tested, the MSCs from the fetal side of the placenta carried the alleles of both maternal and paternal origin, indicative of the fetal genome. q-PCR indicated that the expression of IL6 mRNA in IFN-γ treated group was significantly higher than those in the control group (P<0.05), whereas the levels of IL-10 and HGF mRNA were down-regulated by IFN-γ treatment (P<0.05). ELISA revealed that IFN-γ treated group expressed strikingly the higher level of IL-6 protein compared with the control group (P<0.05), and the lower levels of IL-8, IL-10 and HGF (P<0.05). CONCLUSION: The method used in the study can obtain hfPMSCs from human placenta tissues, and IFN-γ has a negative effect on immune suppression function of the hfPMSCs.