Radiation-induced Pulmonary Fibrosis In Mice Research Articles

DNA-PKcs/AKT1 inhibits epithelial-mesenchymal transition during radiation-induced pulmonary fibrosis by inducing ubiquitination and degradation of Twist1.

Radiation-induced pulmonary fibrosis (RIPF) is a chronic, progressive, irreversible lung interstitial disease that develops after radiotherapy. Although several previous studies have focused on the mechanism of epithelial-mesenchymal transition (EMT) in lung epithelial cells, the essential factors involved in this process remain poorly understood. The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exhibits strong repair capacity when cells undergo radiation-induced damage; whether DNA-PKcs regulates EMT during RIPF remains unclear. To investigate the role and molecular mechanism of DNA-PKcs in RIPF and provide an important theoretical basis for utilising DNA-PKcs-targeted drugs for preventing RIPF. DNA-PKcs knockout (DPK-/-) mice were generated via the Cas9/sgRNA technique and subjected to whole chest ionizing radiation (IR) at a 20Gy dose. Before whole chest IR, the mice were intragastrically administered the DNA-PKcs-targeted drug VND3207. Lung tissues were collected at 1 and 5 months after IR. The expression of DNA-PKcs is low in pulmonary fibrosis (PF) patients. DNA-PKcs deficiency significantly exacerbated RIPF by promoting EMT in lung epithelial cells. Mechanistically, DNA-PKcs deletion by shRNA or inhibitor NU7441 maintained the protein stability of Twist1. Furthermore, AKT1 mediated the interaction between DNA-PKcs and Twist1. High Twist1 expression and EMT-associated changes caused by DNA-PKcs deletion were blocked by insulin-like growth factor-1 (IGF-1), an AKT1 agonist. The radioprotective drug VND3207 prevented IR-induced EMT and alleviated RIPF in mice by stimulating the kinase activity of DNA-PKcs. Our study clarified the critical role and mechanism of DNA-PKcs in RIPF and showed that it could be a potential target for preventing RIPF.

Rosmarinic Acid Alleviates Radiation-Induced Pulmonary Fibrosis by Downregulating the tRNA N7-Methylguanosine Modification-Regulated Fibroblast-to-Myofibroblast Transition Through the Exosome Pathway.

Radiation-induced pulmonary fibrosis (RIPF) is a common complication after radiotherapy in thoracic cancer patients, and effective treatment methods are lacking. The purpose of this study was to investigate the protective effect of rosmarinic acid (RA) on RIPF in mice as well as the mechanism involved. m7G-tRNA-seq and tRNA-seq analyses were conducted to identify m7G-modified tRNAs. Western blotting, immunohistochemistry, northwestern blotting, northern blotting, immunofluorescence, wound-healing assays and EdU experiments were performed to explore the molecular mechanism by which RA regulates fibroblast-to-myofibroblast transformation (FMT) by affecting the exosomes of lung epithelial cells. Ribo-seq and mRNA-seq analyses were used to explore the underlying target mRNAs. Seahorse assays and immunoprecipitation were carried out to elucidate the effects of RA on glycolysis and FMT processes via the regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) acetylation. We found that RA had an antifibrotic effect on the lung tissues of RIPF model mice and inhibited the progression of FMT through exosomes derived from lung epithelial cells. Mechanistically, RA reduced the transcription and translation efficiency of sphingosine kinase 1 in lung fibroblasts by decreasing N7-methylguanosine modification of tRNA, downregulating the expression of tRNAs in irradiated lung epithelial cell-derived exosomes, and inhibiting the interaction between sphingosine kinase 1 and the N-acetyltransferase 10 protein in fibroblasts. Furthermore, the acetylation and cytoplasmic translocation of PFKFB3 were reduced by exosomes derived from irradiated lung epithelial cells, which following RA intervention. This suppression of the FMT process, which is triggered by glycolysis, and ultimately decelerating the progression of RIPF. These findings suggest that RA is a potential therapeutic agent for RIPF.

Inhibition of TGFβ1 activation prevents radiation-induced lung fibrosis.

Radiotherapy is the main treatment modality for thoracic tumours, but it may induce pulmonary fibrosis. Currently, the pathogenesis of radiation-induced pulmonary fibrosis (RIPF) is unclear, and effective treatments are lacking. Transforming growth factor beta 1 (TGFβ1) plays a central role in RIPF. We found that activated TGFβ1 had better performance for radiation pneumonitis (RP) risk prediction by detecting activated and total TGFβ1 levels in patient serum. αv integrin plays key roles in TGFβ1 activation, but the role of αv integrin-mediated TGFβ1 activation in RIPF is unclear. Here, we investigated the role of αv integrin-mediated TGFβ1 activation in RIPF and the application of the integrin antagonist cilengitide to prevent RIPF. ItgavloxP/loxP ;Pdgfrb-Cre mice were generated by conditionally knocking out Itgav in myofibroblasts, and wild-type mice were treated with cilengitide or placebo. All mice received 16Gy of radiation or underwent a sham radiation procedure. Lung fibrosis was measured by a modified Ashcroft score and microcomputed tomography (CT). An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum TGFβ1 concentration, and total Smad2/3 and p-Smad2/3 levels were determined via Western blotting. Conditional Itgav knockout significantly attenuated RIPF (p<.01). Hounsfield units (HUs) in the lungs were reduced in the knockout mice compared with the control mice (p<.001). Conditional Itgav knockout decreased active TGFβ1 secretion and inhibited fibroblast p-Smad2/3 expression. Exogenous active TGFβ1, but not latent TGFβ1, reversed these reductions. Furthermore, cilengitide treatment elicited similar results and prevented RIPF. The present study revealed that conditional Itgav knockout and cilengitide treatment both significantly attenuated RIPF in mice by inhibiting αv integrin-mediated TGFβ1 activation. Activated TGFβ1 has a superior capacity in predicting radiation pneumonitis (RP) risk and plays a vital role in the development of radiation-induced pulmonary fibrosis (RIPF). Conditional knock out Itgav in myofibroblasts prevented mice from developing RIPF. Cilengitide alleviated the development of RIPF by inhibiting αv integrin-mediated TGFβ1 activation and may be used in targeted approaches for preventing RIPF.

Glycyrrhetinic Acid Mitigates Radiation-Induced Pulmonary Fibrosis via Inhibiting the Secretion of TGF-β1 by Treg Cells

Radiation-induced pulmonary fibrosis (RIPF) is a common side effect of radiation therapy for thoracic tumors without effective prevention and treatment methods at present. The aim of this study was to explore whether glycyrrhetinic acid (GA) has a protective effect on RIPF and the underlying mechanism. A RIPF mouse model administered GA was used to determine the effect of GA on RIPF. The cocultivation of regulatory T (Treg) cells with mouse lung epithelial-12 cells or mouse embryonic fibroblasts and intervention with GA or transforming growth factor-β1 (TGF-β1) inhibitor to block TGF-β1 was conducted to study the mechanism by which GA alleviates RIPF. Furthermore, injection of Treg cells into GA-treated RIPF mice to upregulate TGF-β1 levels was performed to verify the roles of TGF-β1 and Treg cells. GA intervention improved the damage to lung tissue structure and collagen deposition and inhibited Treg cell infiltration, TGF-β1 levels, epithelial mesenchymal transition (EMT), and myofibroblast (MFB) transformation in mice after irradiation. Treg cell-induced EMT and MFB transformation in vitro were prevented by GA, as well as a TGF-β1 inhibitor, by decreasing TGF-β1. Furthermore, reinfusion of Treg cells upregulated TGF-β1 levels and exacerbated RIPF in GA-treated RIPF mice. GA can improve RIPF in mice, and the corresponding mechanisms may be related to the inhibition of TGF-β1 secreted by Treg cells to induce EMT and MFB transformation. Therefore, GA may be a promising therapeutic candidate for the clinical treatment of RIPF.

Transcriptional inhibition of miR-486-3p by BCL6 upregulates Snail and induces epithelial\u2013mesenchymal transition during radiation-induced pulmonary fibrosis

BackgroundIonizing radiation (IR) can induce pulmonary fibrosis by causing epithelial mesenchymal transition (EMT), but the exact mechanism has not been elucidated. To investigate the molecular mechanism of how radiation induces pulmonary fibrosis by altering miR-486-3p content and thus inducing EMT.MethodsThe changes of miR-486-3p in cells after irradiation were detected by RT-qPCR. Western blot was used to detect the changes of cellular epithelial marker protein E-cadherin, mesenchymal marker N-cadherin, Vimentin and other proteins. The target gene of miR-486-3p was predicted by bioinformatics method and the binding site was verified by dual luciferase reporter system. In vivo experiments, adeno-associated virus (AAV) was used to carry miR-486-3p mimic to lung. Radiation-induced pulmonary fibrosis (RIPF) model was constructed by 25Gy60Co γ-rays. The structural changes of mouse lung were observed by HE and Masson staining. The expression of relevant proteins in mice was detected by immunohistochemistry.ResultsIR could decrease the miR-486-3p levels in vitro and in vivo, and that effect was closely correlated to the occurrence of RIPF. The expression of Snail, which induces EMT, was shown to be restrained by miR-486-3p. Therefore, knockdown of Snail blocked the EMT process induced by radiation or knockdown of miR-486-3p. In addition, the molecular mechanism underlying the IR-induced miRNA level reduction was explored. The increased in BCL6 could inhibit the formation of pri-miR-486-3p, thereby reducing the levels of miR-486-3p in the alveolar epithelial cells, which would otherwise promote EMT and contribute to RIPF by targeting Snail.ConclusionIR can exacerbate RIPF in mice by activating the transcription factor BCL6, which inhibits the transcription of miR-486-3p and decreases its content, which in turn increases the content of the target gene slug and triggers EMT.

Irradiation Activates MZF1 to Inhibit miR-541-5p Expression and Promote Epithelial-Mesenchymal Transition (EMT) in Radiation-Induced Pulmonary Fibrosis (RIPF) by Upregulating Slug.

Understanding miRNAs regulatory roles in epithelial-mesenchymal transition (EMT) would help establish new avenues for further uncovering the mechanisms underlying radiation-induced pulmonary fibrosis (RIPF) and identifying preventative and therapeutic targets. Here, we demonstrated that miR-541-5p repression by Myeloid Zinc Finger 1 (MZF1) promotes radiation-induced EMT and RIPF. Irradiation could decrease miR-541-5p expression in vitro and in vivo and inversely correlated to RIPF development. Ectopic miR-541-5p expression suppressed radiation-induced-EMT in vitro and in vivo. Knockdown of Slug, the functional target of miR-541-5p, inhibited EMT induction by irradiation. The upregulation of transcription factor MZF1 upon irradiation inhibited the expression of endogenous miR-541-5p and its primary precursor (pri-miR-541-5p), which regulated the effect of the Slug on the EMT process. Our finding showed that ectopic miR-541-5p expression mitigated RIPF in mice by targeting Slug. Thus, irradiation activates MZF1 to downregulate miR-541-5p in alveolar epithelial cells, promoting EMT and contributing to RIPF by targeting Slug. Our observation provides further understanding of the development of RIPF and determines potential preventative and therapeutic targets.

MiRNA-155–5p inhibits epithelium-to-mesenchymal transition (EMT) by targeting GSK-3β during radiation-induced pulmonary fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a major lung complication in using radiotherapy to treat thoracic diseases. MicroRNAs (miRNAs) are reported to be the therapeutic targets for many diseases. However, the miRNAs involved in the pathogenesis of RIPF are rarely studied as potential therapeutic targets. Alveolar epithelial cells participate in RIPF formation by undergoing epithelial-mesenchymal transition (EMT). Here we demonstrated the critical role of miR-155–5p in radiation-induced EMT and RIPF. Using the previously established EMT cell model, we found that miR-155–5p was significantly down-regulated through high-throughput sequencing. Irradiation could decrease the expression of miR-155–5p in intro and in vivo, and it was inversely correlated to RIPF formation. Ectopic miR-155–5p expression inhibited radiation-induced-EMT in vitro and in vivo. Knockdown of glycogen synthase kinase-3β (GSK-3β), the functional target of miR-155–5p, reversed the induction of EMT and enhanced the phosphorylation of p65, a subunit of NF-κB, which were mediated by the down-regulation of miR-155–5p. Moreover, our finding demonstrated that ectopic miR-155–5p expression alleviated RIPF in mice by the GSK-3β/NF-κB pathway. Thus, radiation downregulates miR-155–5p in alveolar epithelial cells that induces EMT, which contributes to RIPF using GSK-3β/NF-κB pathway. Our observation provides further understanding on the regulation of RIPF and identifies potential therapeutic targets.

Haplodeletion of Follistatin-Like 1 Attenuates Radiation-Induced Pulmonary Fibrosis in Mice

Radiation-induced pulmonary fibrosis (RIPF) is a severe and life-threatening complication of radiation therapy in patients with thoracic cancer; however, the exact molecular mechanisms remain unknown, and there is no effective treatment method in clinic. Here, we assessed the role of follistatin-like 1 (Fstl1) in RIPF. Protein and messenger RNA levels of Fstl1 in lung tissues from symptomatic RIPF patients, Rhesus macaques, and mice were assessed. Fibrotic and inflammatory responses to radiation-induced lung injury and accumulation of myofibroblasts in Fstl1 haplodeficient (Fstl1+/-) mice were determined. Finally, radiation-induced differentiation and activation of fibroblasts in primary Fstl1+/- lung fibroblasts were evaluated. FSTL1 amounts were significantly increased in serum and/or radiation-injured lung specimens from symptomatic RIPF patients, Rhesus macaques, and mice. Haplodeletion of Fstl1 in Fstl1+/- mice was protective against x-ray-induced lung injury in mice invivo, as well as myofibroblast activation invitro. These findings suggest that Fstl1 plays an important role in lung fibrosis and may offer a potential approach to attenuate RIPF in radiation therapy of patients with thoracic cancer.

Mechanism of MEN1 gene in radiation-induced pulmonary fibrosis in mice

ObjectiveTo investigate the regulatory mechanism of MEN1 gene in radiation-induced lung fibrosis in mice and provide a new theoretical basis for the clinical treatment of radiation pulmonary fibrosis. MethodsFirst, 80 C57BL/6 mice aged 8 weeks and weighing 18–22 g were selected, half of them were male and the other half were female. The mice were divided into control group and irradiation group (40 mice in each group) according to the method of the random number table. A radiation-induced lung fibrosis mouse model was established in which a single X-ray irradiation of 20 Gy was applied to the right lung in the irradiation group; H&E and Masson staining were used to verify whether the model was successful at 4, 8, 16 and 24 weeks after irradiation. The expression of MEN1, smooth muscle actin (α-SMA), Collagen-1 and transforming growth factor (TGF-β) in lung tissue were detected by Western blot and qPCR. Secondly, in the mouse embryonic fibroblast cell line (MEF) and mouse lung epithelial cell line (MLE-12), we constructed cell models of MEN1 knockout and interference separately with the irradiation of 10 Gy X-rays. The expression of α-SMA, Collagen-1, and TGF-β/Smads signaling pathway molecules was detected by qPCR. Finally, using the immunoprecipitation (IP) method, we can detect the interaction between Smad2 and the protein menin encoded by the MEN1 gene. ResultsThe results of the radiation pulmonary fibrosis model in mice showed that compared with the control group, the alveolar septum widens, the alveolar integrity decreases, the lung tissue slightly thickens, and a small amount of collagen deposits appear after 4–8 weeks in the model group. At twenty-fourth weeks, a large number of cells in the interstitial space of the lung tissue and a localized focal fibrosis area were observed. Further study found that radiation induced fibrogenic inflammatory cytokines TGF-β up-regulation, down-regulation of MEN1 gene expression, and then enhanced the expression of α-SMA and promotes the transformation of fibroblasts to myofibroblasts; At the same time, the expression of Collagen-1 was enhanced, which suggested that the extracellular matrix was overconcentrated and eventually promoted the formation of pulmonary fibrosis. In vitro, we found that knockout and interference of MEN1 gene can significantly enhance radiation-induced fibrosis, and up-regulate the expression of downstream molecules Smad2 and Smad3 of TGF-β signaling pathway, and down-regulate the expression of Smad7. Furthermore, it played an important role in regulating the process of radionuclide fibrosis. ConclusionMEN1 plays a key role in the formation of pulmonary fibrosis by regulating the secretion of TGF-β and the activation of TGF-β/Smads signaling pathway.

Effects of CpG Oligodeoxynucleotide 1826 on transforming growth factor-beta 1 and radiation-induced pulmonary fibrosis in mice.

BackgroundCytosine-phosphate-guanine (CpG) oligodeoxyribonucleotides (ODNs) are synthetic DNA fragments containing unmethylated cytosine-guanine motifs with potential immune modulatory effects and have recently been suggested to enhance sensitivity to traditional therapies in lung cancer. This study aimed to examine the effects of CpG ODN1826 on transforming growth factor-beta 1(TGF-β1) and radiation-induced pulmonary fibrosis in mice.MethodsThe radiation-induced pulmonary fibrosis mouse model was established by a single dose of 20 Gy, 6 MV X-rays exposure to the left lung. ICR mice were evenly randomized into four groups, comprising: a control group, a radiation group (RT group), a CpG group and a radiation combined with CpG ODN1826 group (RT + CpG group), with 40 mice in each group. CpG ODN1826 was intraperitoneally injected into mice at 1, 3, 5, 7 and 9 d post-irradiation. The mice were sacrificed at 1, 5, 15, 30 and 90 d post-irradiation. Paraffin sections of the radiated lung were subjected to H&E staining and Masson staining. The Ashcroft scale was used for quantitative histological analysis of fibrotic changes induced by irradiation. Concentrations of serum TGF-β1 were determined by ELISA, and concentrations of Hydroxyproline(Hyp) in the lung were determined with the alkaline hydrolysis method. Relative gene expression of FoxP3 was determined by real-time PCR.ResultsThe radiation-induced pulmonary fibrosis mouse model was successfully established. The serum concentrations of TGF -β1 of RT group were higher than those of the RT + CpG group (t = 5.212, 7.126, 7.972 and 3.785, P < 0.05). The Hyp in the lung of RT group was higher than that of RT + CpG group (t = 4.606, P < 0.05). The relative expressions of FoxP3 gene in the lung of the RT group were higher than those of RT + CpG group (t = 8.395, 5.099 and 6.147, P < 0.05).ConclusionsCpG ODN1826 could reduce the serum concentrations of TGF-β1 and the lung content of Hyp in radiation-induced pulmonary fibrosis, which might be related to the possibility that CpG ODN1826 can reduce expression of the FoxP3 gene.

Acute adaptive immune response correlates with late radiation-induced pulmonary fibrosis in mice.

BackgroundThe lung response to radiation exposure can involve an immediate or early reaction to the radiation challenge, including cell death and an initial immune reaction, and can be followed by a tissue injury response, of pneumonitis or fibrosis, to this acute reaction. Herein, we aimed to determine whether markers of the initial immune response, measured within days of radiation exposure, are correlated with the lung tissue injury responses occurring weeks later.MethodsInbred strains of mice known to be susceptible (KK/HIJ, C57BL/6J, 129S1/SvImJ) or resistant (C3H/HeJ, A/J, AKR/J) to radiation-induced pulmonary fibrosis and to vary in time to onset of respiratory distress post thoracic irradiation (from 10–23 weeks) were studied. Mice were untreated (controls) or received 18 Gy whole thorax irradiation and were euthanized at 6 h, 1d or 7 d after radiation treatment. Pulmonary CD4+ lymphocytes, bronchoalveolar cell profile & cytokine level, and serum cytokine levels were assayed.ResultsThoracic irradiation and inbred strain background significantly affected the numbers of CD4+ cells in the lungs and the bronchoalveolar lavage cell differential of exposed mice. At the 7 day timepoint greater numbers of pulmonary Th1 and Th17 lymphocytes and reduced lavage interleukin17 and interferonγ levels were significant predictors of late stage fibrosis. Lavage levels of interleukin-10, measured at the 7 day timepoint, were inversely correlated with fibrosis score (R = −0.80, p = 0.05), while serum levels of interleukin-17 in control mice significantly correlated with post irradiation survival time (R = 0.81, p = 0.04). Lavage macrophage, lymphocyte or neutrophil counts were not significantly correlated with either of fibrosis score or time to respiratory distress in the six mouse strains.ConclusionSpecific cytokine and lymphocyte levels, but not strain dependent lavage cell profiles, were predictive of later radiation-induced lung injury in this panel of inbred strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0359-y) contains supplementary material, which is available to authorized users.

Transforming growth factor β3 attenuates the development of radiation-induced pulmonary fibrosis in mice by decreasing fibrocyte recruitment and regulating IFN-γ/IL-4 balance

Radiation-induced pulmonary fibrosis is a frequently occurred complication from radiotherapy of thoracic tumors. The transforming growth factor-β (TGF-β) superfamily plays a key regulatory role in pulmonary fibrosis. As TGF-β3 showed the potential anti-fibrotic properties especially in scar-less wound healing as opposed to the fibrotic function of TGF-β1, we sought to explore the role of TGF-β3 in radiation-induced pulmonary fibrosis. A single thoracic irradiation of 20Gy was applied in mice to establish the model of radiation-induced pulmonary fibrosis and the mice were treated by intraperitoneal injections of recombinant TGF-β3 weekly after irradiation. We found that TGF-β3 decelerated the progress of radiation-induced pulmonary fibrosis and hindered the recruitment of fibrocytes to lung. In addition, Th1 response was suppressed as shown by diminished IFN-γ in bronchoalveolar lavage fluid (BALF) after irradiation, and enhancement of Th2 response was marked by increased IL-4 in BALF. TGF-β3 administration significantly attenuated these effects and increased the percentage of Tregs in lung during the progression of pulmonary fibrosis. Taken together, these data suggest that TGF-β3 might be involved in the regulatory mechanism for attenuation of radiation-induced pulmonary fibrosis.

Abstract 3849: Triptolide reduces activity of RNA polymerase II

Abstract Triptolide (TPL) possesses potent anticancer and anti-inflammatory effects. Our previous study demonstrated that radiation-induced pulmonary fibrosis in mice can be significantly reduced by TPL treatment (0.25 mg/kg/every 3 days for 1-3 months via intravenous injection). Treatment was associated with the reduction of a panel of inflammation molecules, as evidenced by a membrane array of 62 inflammatory molecules. We found that TPL has a high level of biointeraction with RNA polymerase II (Pol II). Pol II is a major enzyme complex responsible for the production of mRNA and could explain improved tumor response and reduced normal tissue toxicity. The mRNA of interleukin-1 beta (IL-1α), tumor necrosis factor-alpha (TNFα), IL-6, and Cox-2 were used as downstream products of Pol II. Alpha-amanitin (a specific inhibitor of Pol II) and actinomycin D (a pan Pol inhibitor) were used as positive controls. The results showed that: 1) Pol II was suppressed as early as 1 hour after TPL was added to cells; 2) TPL (effective at ng/ml levels) is more potent than α-amanitin and actinomycin D (effective at µg/ml levels) at reducing housekeeping genes L32 and S14; 3) the suppression of Pol II by TPL was dose dependent; and 4) TPL inhibited radiation-induced mRNA of IL-1α and TNFα at 3 hours after irradiation and IL-6 and Cox-2 at 8 hours after irradiation. It is unknown whether these early effects of TPL on Pol II impact either tumor response or normal tissue complications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3849. doi:1538-7445.AM2012-3849

Combined Tlr2 and Tlr4 Deficiency Increases Radiation-Induced Pulmonary Fibrosis in Mice

To determine whether Toll-like receptor 2 or 4 genotype alters the lung response to irradiation in C57BL/6 mice using a model developing a phenotype that resembles radiotherapy-induced fibrosis in both histological characteristics and onset post-treatment. The pulmonary phenotype of C57BL/6 mice deficient in each or both of these genes was assessed after an 18-Gy single dose to the thoracic cavity by survival time postirradiation, bronchoalveolar lavage cell differential, histological evidence of alveolitis and fibrosis, and gene expression levels, and compared with that of wild-type mice. The lung phenotype of Tlr4-deficient and Tlr2-deficient mice did not differ from that of wild-type mice in terms of survival time postirradiation, or by histological evidence of alveolitis or fibrosis. In contrast, mice deficient in both receptors developed respiratory distress at an earlier time than did wild-type mice and presented an enhanced fibrotic response (13.5% vs. 5.8% of the lung by image analysis of histological sections, p < 0.001). No differences in bronchoalveolar cell differential counts, nor in numbers of apoptotic cells in the lung as detected through active caspase-3 staining, were evident among the irradiated mice grouped by Tlr genotype. Gene expression analysis of lung tissue revealed that Tlr2,4-deficient mice have increased levels of hyaluronidase 2 compared with both wild-type mice and mice lacking either Tlr2 or Tlr4. We conclude that a combined deficiency in both Tlr2 and Tlr4, but not Tlr2 or Tlr4 alone, leads to enhanced radiation-induced fibrosis in the C57BL/6 mouse model.

Protection of Angelica sinensis against radiation-induced pulmonary fibrosis in mice

To evaluate the Angelica Sinensis as a protecting agent affecting the radiation-induced pulmonary fibrosis in an animal model, The thoraces of C57BL/6 mice were exposed to either sham irradiation or single fraction of 12 Gy. Four groups were defined: that received neither irradiation nor Angelica Sinensis (N group), that received Angelica Sinensis but no irradiation (A group), that underwent irradiation without Angelica Sinensis (NX group) and that received both Angelica Sinensis and irradiation (AX group). Mice were sacrificed at 1, 24, 72 hours and 1, 2, 4, 8, 16, 24 weeks post-irradiation. The lungs tissue were removed and processed for definitive analysis, including hydroxyproline content, HE and Masson staining, and the TGF-beta1, (Transforming Growth Factor beta1, TGF-beta1) mRNA expressions. Compared with N and A group, there was some differences in the AX group, but a significant histological and pathologic changes in NX group. Non-irradiated groups (N and A group) exhibited low levels of hydroxyproline (0.775 +/- 0.024) microg/mg and (0.751 +/- 0.034) microg/mg, and there was a significantly elevated level of hydroxyproline in NX group (0.875 +/- 0.009) microg/mg (P < 0.05). AX group (0.782 +/- 0.010) microg/mg was in between the non-irradiated groups (N and A group) and the radiation-only group (NX group), and the difference between AX group and NX group was significant (P < 0.01). The results of real-time quantitative RT-PCR showed that the relative mRNA expressions of cytokine TGF-beta1 in NX group(249.655 +/- 16.320) was significantly higher than that in group A (1.254 +/- 0.061) and N (1.324 +/- 0.057) (P < 0.01), and that in AX group (108.076 +/- 9.870) decreased than that of NX group (P < 0.01). An animal model of mice with radiation-induced lung injure was established successfully. This study has demonstrated that Angelica sinensis in Hibits the progress of radiation-induced pulmonary fibrosis, Possibly by down-regulating the expression of the proinflammatory cytokine Tgfb1. These data suggest that Angelica sinensis maybe useful in preventing and/or treating radiation-induced pulmonary fibrosis in the clinic.

Effects of intercellular adhesion molecule 1 (ICAM-1) null mutation on radiation-induced pulmonary fibrosis and respiratory insufficiency in mice.

Therapy-induced inflammation and progressive fibrosis limit the efficacy of thoracic radiotherapy for lung neoplasms. However, mice bearing a null mutation of intercellular adhesion molecule 1 (ICAM-1) have previously been found to display no inflammatory cell infiltration into the lung following thoracic irradiation. We investigated the role of ICAM-1-mediated inflammation in the pathogenesis of radiation-induced pulmonary fibrosis in mice with a homozygous null mutation in the ICAM-1 gene (ICAM-1(-/-)) and in wild-type mice (ICAM-1(+/+)). Groups of 10 mice were each irradiated with total doses of 12.5, 14, 16, 17, or 18 Gy to the thorax or were mock irradiated. Inflammatory cell infiltration was measured by immunohistochemical staining of lung sections for leukocyte-common antigen (LCA). Dynamic pulmonary compliance was determined by plethysmography. Pulmonary fibrosis was evaluated by measuring alveolar septal wall thickness and the hydroxyproline content of lungs by immunohistochemical staining of lung sections for collagen III and by Masson's trichrome staining of lung sections. All statistical tests were two-sided. Lungs of irradiated ICAM-1(-/-) mice had statistically significantly fewer LCA-positive cells than the lungs of irradiated ICAM-1(+/+) mice at all radiation doses (P<.001). ICAM-1(-/-) mice had a higher mean dynamic pulmonary compliance than ICAM-1(+/+) mice following irradiation. The incidence of respiratory insufficiency 18 months after thoracic irradiation was statistically significantly lower in ICAM-1(-/-) mice than in ICAM-1(+/+) mice (odds ratio = 0.19 [95% confidence interval = 0.06 to 0.58], overall P =.0036). ICAM-1(-/-) mice had less pulmonary fibrosis and reduced thickening of the alveolar septum following thoracic irradiation than did ICAM-1(+/+) mice. The lungs of irradiated ICAM-1(-/-) mice had less hydroxyproline than did the lungs of irradiated ICAM-1(+/+) mice (P =.04). ICAM-1 and inflammation contribute to pulmonary fibrosis and impaired pulmonary compliance following thoracic irradiation. Agents that block ICAM-1 function or expression should be studied for their effects on the prevention of radiation-induced pulmonary fibrosis.

Whole-Thorax Radiation Lethality in Penicillamine-Treated Mice

weight was recorded biweekly for 1 year after irradiation. The LD50/60-180 daysOf untreated and penicillamine-treated mice was 15.9 ? 0.7 and 16.5 ? 0.9 Gy, respectively, yielding a drug DRF of 1.04 (P > 0.05). In contrast, LD50/181-360 days Of the two groups was 12.0 ? 1.3 and 14.4 ? 0.9 Gy, respectively, yielding a penicillamine DRF of 1.20 (P < 0.05). Thus it appears that penicillamine is ineffective against fatal radiation pneumonitis but significantly reduces mortality resulting from radiation-induced pulmonary fibrosis in mice irradiated to the whole thorax. While penicillamine did not reduce the incidence of early (<6 months) lung deaths, drug-treated mice weighed significantly (P < 0.05) more than their untreated counterparts during that time.